title	PMID	abstracts
0	SARS-CoV-2-Induced Multisystem Inflammatory Syndrome in a Young Adult: Case Report.	34179694	We describe a case of a previous healthy 20-year-old male athlete who presented with an atypical clinical profile with multiorgan involvement within five weeks after confirmed SARS-CoV-2 infection, suggestive for multisystem inflammatory syndrome (MIS); MIS is a rare, potentially life-threatening complication associated with SARS-CoV-2. MIS shares similar clinical features compatible with several overlapping lifethreatening hyperinflammatory syndromes, such as incomplete Kawasaki Disease (KD) and toxic shock syndrome (TSS) associated to a cytokine storm suggestive of a macrophage activation syndrome (MAS) without fulfilling the criteria for hemophagocytic lymphohistiocytosis (HLH), that may create a great challenge to distinguish between them. MIS should promptly be considered and treated, as uncontrolled MIS has a high mortality. In MIS cardiac involvement, heart failure may present as an additional problem, especially because volume loading is advised in accordance with proposed therapy. Carefully monitoring of the respiratory and cardiac status in response of resuscitation is therefore warranted.
1	Pancytopenia Resulting From Low-Dose Methotrexate Use: A Diagnostic Challenge.	34178513	Rheumatoid arthritis (RA) is a common autoimmune disease primarily affecting small joints which leads to crippling erosion of the articular cartilage and bone. It is associated with complications related to both its disease course and treatment. Methotrexate (MTX) is a folate antagonist responsible for modulating cell-specific signaling pathways and inhibiting the proinflammatory properties of major cell lineages involved in the pathogenesis of RA. It is considered to be the first-line agent in RA because of its disease-modifying ability and safety profile at low doses. This case report discusses how a middle-aged female presented with severe bone marrow suppression secondary to MTX toxicity, an unusual presentation at the usual low-dose regimen. Her presentation overlapped with several other conditions, especially with Felty's syndrome, a rare complication of RA, characterized by the triad of splenomegaly, neutropenia, and RA. Other differentials included hemophagocytic lymphohistiocytosis, hematologic neoplasms, drug reaction, and infection. Therefore, it was essential to exclude all possible differentials before initiating therapy. We found the corrected reticulocyte count coupled with a good response to leucovorin to be an effective way to differentiate MTX-induced pancytopenia from other possible hematologic diagnoses without the use of a bone marrow biopsy. Additionally, our case incidentally demonstrated a potential interaction between piperacillin/tazobactam and MTX.
2	Disseminated Tuberculosis Associated Hemophagocytic Lymphohistiocytosis in a Pregnant Woman With Evans syndrome: A Case Report and Literature Review.	34177917	Tuberculosis (TB) is a leading cause of morbidity and mortality in underdeveloped and developing countries. Disseminated TB may induce uncommon and potentially fatal secondary hemophagocytic lymphohistiocytosis (HLH). Timely treatment with anti-tuberculosis therapy (ATT) and downmodulation of the immune response is critical. However, corticosteroid treatment for TB-associated HLH remains controversial. Herein, we report a successful case of disseminated TB-associated HLH in a pregnant woman with Evans syndrome accompanied by a literature review.A 26-year-old pregnant woman with Evans syndrome was transferred to the Third Affiliated Hospital of Sun Yat-Sen University because of severe pneumonia. She presented with cough, fever, and aggravated dyspnea. Nested polymerase chain reaction for Mycobacterium tuberculosis (M. tuberculosis) complex in sputum was positive. Sputum smear sample for acid-fast bacilli was also positive. Metagenome next-generation sequencing (mNGS) of the bronchoalveolar lavage fluid identified 926 DNA sequence reads and 195 RNA sequence reads corresponding to M. tuberculosis complex, respectively. mNGS of blood identified 48 DNA sequence reads corresponding to M. tuberculosis. There was no sequence read corresponding to other potential pathogens. She was initially administered standard ATT together with a low dose of methylprednisolone (40 mg/day). However, her condition deteriorated rapidly with high fever, acute respiratory distress syndrome, pancytopenia, and hyperferritinemia. Bone marrow smears showed hemophagocytosis. And caseating tuberculous granulomas were found in the placenta. A diagnosis of disseminated TB-associated HLH was made. Along with the continuation of four drug ATT regimen, therapy with a higher dose of methylprednisolone (160 mg/day) combined with immunoglobulin and plasma exchange was managed. The patient's condition improved, and she was discharged on day 19. Her condition was good at follow-up with the continuation of the ATT.Clinicians encountering patients with suspected TB accompanied by unexplainable inflammation not responding to ATT should consider complications with HLH. Timely administration of ATT combined with corticosteroids may result in a favorable outcome.
3	Multifocal Pyoderma Gangrenosum with an Underlying Hemophagocytic Lymphohistiocytosis: Case Report and the Review of the Literature.	34176093	Pyoderma gangrenosum (PG) is an uncommon, serious, ulcerating skin disease of uncertain etiology. It manifests as a noninfectious, progressive necrosis of the skin characterized by sterile neutrophilic infiltrates. It seems to be a disorder of the immune system. PG is associated with certain underlying conditions in at least 50% of cases. Therefore, it is important to look carefully for comorbidities in every patient with PG and treat them adequately to improve the prognosis. Here, we demonstrate a 35-year-old man diagnosed with multifocal PG and hemophagocytic lymphohistiocytosis (HLH) with fatal outcome, despite combined, long-term, intensive dermatological and hematological treatment with high doses of steroids, cyclosporin, intravenous immunoglobulins (IVIG), HLH-2004 protocol with intravenously administered etoposide, and anakinra. This case is presented owing to the extremely rare coexistence of PG and HLH and the related diagnostic and therapeutic difficulties. It is also worth underlying that the diagnosis of HLH should perhaps be considered in the presence of a high percentage of double-negative T lymphocytes (DNTs) in flow cytometry, after excluding the diagnosis of lymphoma and leukemia. In this article we have also performed and present the critical literature review of local and systemic options in the management of PG lesions based on a detailed search of the PubMed database.
4	Prevalence of Epstein-Barr Viral DNA among children at a single hospital in Suzhou, China.	34174213	This study aimed to describe the prevalence of Epstein-Barr virus (EBV)-DNA among children in Suzhou, and to explore the association between plasma EBV load and disease diagnosis.All children admitted to the Children's Hospital of Soochow University between January 2018 and September 2020 and subjected to the plasma EBV-DNA assay were included. The authors retrospectively collected demographic and discharge diagnostic information of the participants, and ascribed the disease distribution characteristics of children with positive plasma EBV-DNA by age and viral load.A total of 38,175 patients underwent plasma EBV-DNA PCR assay, of which 2786 (7.3%) had EBV-DNA in their plasma. Children aged 3-4 years had a high prevalence of EBV infection. Plasma EBV positivity was common with infectious mononucleosis (IM, 40.0%), respiratory infection (20.1%), atypical EBV infection (14.2%), acute leukemia (6.4%), hemophagocytic lymphohistiocytosis (HLH, 4.8%), and idiopathic thrombocytopenic purpura (ITP, 2.9%). With increasing age, plasma EBV positivity was more common in children with IM and atypical EBV infection. However, an inverse correlation was observed in children with respiratory infections and ITP. High levels of EBV loads were more likely to occur in HLH, IM, and atypical EBV infection, especially in HLH. However, lower viral loads were found in respiratory infection and acute leukemia.This is a large sample study that revealed the prevalence of plasma EBV-DNA levels in children of various ages and presenting illnesses.
5	T Cell-Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis (HLH) Occurs in Non-Asians and Is Associated with a T Cell Activation State that Is Comparable to Primary HLH.	34173902	T cell-Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (T cell-EBV-HLH) is prevalent in East Asia and has poor prognosis. Understanding of this disease is limited, and literature regarding prevalence in North America is scarce. Herein, we summarize our experience.A retrospective analysis of T cell-EBV-HLH patients admitted to Children's Healthcare of Atlanta (GA, USA) from 2010 to 2020 was conducted. Additional immune studies were completed in a subset of patients.We report 15 patients (10 months-19 years of age) diagnosed with T cell-EBV-HLH. Nine patients were Hispanic, and the majority did not have primary HLH (p-HLH) gene defects. Soluble interleukin-2 receptor levels in T cell-EBV-HLH were significantly higher than other forms of secondary-HLH but comparable to p-HLH, and it correlated with disease severity at presentation. Natural killer cell function was decreased in most patients despite a negative workup for p-HLH. Depending on disease severity, initial therapy included dexamethasone or dexamethasone and etoposide. Refractory patients were managed with blended regimens that included one or more of the following therapies: combination chemotherapy, alemtuzumab, emapalumab, and nivolumab. Rituximab did not appreciably decrease EBV viremia in most patients. Non-critically ill patients responded well to immunosuppressive therapy and are long-term survivors without undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Alemtuzumab resulted in inflammation flare in two of the three patients. Three patients underwent allogeneic HSCT, with disease relapse noted in one. At a median follow-up of 3 years, 10 of the 15 patients are alive.T cell-EBV-HLH occurs in the USA among the non-Asian populations, especially in those who are Hispanic.
6	Inherited Genetic Susceptibility to Nonimmunosuppressed Epstein-Barr Virus-associated T/NK-cell Lymphoproliferative Diseases in Chinese Patients.	34170459	Epstein-Barr virus (EBV) T/NK-cell lymphoproliferative diseases are characterized by clonal expansion of EBV-infected T or NK cells, including chronic active EBV infection of T/NK-cell type (CAEBV+T/NK), EBV-associated hemophagocytic lymphohistiocytosis (EBV+HLH), extranodal NK/T-cell lymphoma of nasal type (ENKTL), and aggressive NK-cell leukemia (ANKL). However, the role of inherited genetic variants to EBV+T/NK-LPDs susceptibility is still unknown. A total of 171 nonimmunosuppressed patients with EBV+T/NK-LPDs and 104 healthy donors were retrospectively collected and a targeted sequencing study covering 15 genes associated with lymphocyte cytotoxicity was performed. The 94 gene variants, mostly located in UNC13D, LYST, ITK, and PRF1 genes were detected, and mutations covered 28/50 (56.00%) of CAEBV-T/NK, 31/51 (60.78%) of EBV+HLH, 13/28 (46.42%) of ENKTL, and 13/48 (27.09%) of ANKL. Most mutations represented monoallelic and missense. Three-year overall survival rate of patients with CAEBV-T/NK and EBV+HLH was significantly lower in patients with germline mutations than in those without germline mutations (P=0.0284, P=0.0137). Our study provided novel insights into understanding a spectrum of nonimmunosuppressed EBV+T/NK-LPDs with respect to genetic defects associated with lymphocyte cytotoxicity and reminded us that the gene sequencing may be an auxiliary test for diagnosis and risk stratification of EBV+T/NK-LPDs.
7	Haemophagocytic lymphohistiocytosis and Epstein-Barr virus: a complex relationship with diverse origins, expression and outcomes.	34169507	Epstein-Barr virus (EBV) is a ubiquitous herpesvirus with rare but severe potential for lymphoproliferative complications. EBV is associated with a variety of presentations of haemophagocytic lymphohistiocytosis (HLH). HLH is a life-threatening hyperinflammatory syndrome that can occur in patients with genetic defects associated with dysregulation of the immune response (familial HLH) or arise in patients with underlying infection or malignancy (non-familial or secondary HLH). EBV can both serve as the incidental trigger of familial HLH or as the driving factor in patients with selective inherited vulnerability (e.g. X-linked lymphoproliferative disease). Alternatively, acute infection can idiosyncratically cause non-neoplastic HLH in patients without inherited predisposition (i.e. secondary HLH), while EBV-associated T/natural killer (NK)-cell lymphoproliferative disorders and lymphomas can cause neoplasia-associated HLH. The present review will discern between EBV-associated familial and non-familial HLH and highlight diagnostic and therapeutic considerations. Non-familial EBV-associated HLH is a major diagnostic dilemma, as it represents a diverse spectrum of disease ranging from highly curable (non-neoplastic EBV-HLH) to indolent but incurable (chronic active EBV) to acutely fatal (systemic EBV-positive T-cell lymphoma of childhood). Increased clinical awareness and understanding of this rare and potentially devastating subset of EBV-related complications is desperately needed to improve survival for patients with neoplasia-associated HLH.
8	Hemophagocytic lymphohistiocytosis occurring after liver transplantation: A case series and review of the literature.	34159642	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease characterized by excessive inflammation and tissue destruction due to a dysregulated immune response. Its secondary form is most commonly triggered by viral infection or malignancy. There have previously been 11 cases of acquired HLH described following liver transplantation in adult transplant recipients, most occurring within the first year following transplantation. Herein we describe 2 cases of HLH in liver transplant recipients that both occurred remotely following transplantation. In the first case, HLH was thought to be triggered by the development of a post-transplant lymphoproliferative disorder in a patient who was initially diagnosed with recurrent autoimmune hepatitis. In the second, it was thought to be triggered by a newly acquired human herpesvirus-8 infection. In both cases, the syndrome was not recognized until treatment for the initial putative diagnoses was unsuccessful. Despite treatment, both patients unfortunately died from multiorgan failure. HLH in the post-liver transplant setting is likely under-recognized and has a high mortality; early diagnosis and intervention may lead to improved outcomes. This article is protected by copyright. All rights reserved.
9	A novel gene LbHLH from the halophyte Limonium bicolor enhances salt tolerance via reducing root hair development and enhancing osmotic resistance.	34157974	Identifying genes involved in salt tolerance in the recretohalophyte Limonium bicolor could facilitate the breeding of crops with enhanced salt tolerance. Here we cloned the previously uncharacterized gene LbHLH and explored its role in salt tolerance.The 2,067-bp open reading frame of LbHLH encodes a 688-amino-acid protein with a typical helix-loop-helix (HLH) domain. In situ hybridization showed that LbHLH is expressed in salt glands of L. bicolor. LbHLH localizes to the nucleus, and LbHLH is highly expressed during salt gland development and in response to NaCl treatment. To further explore its function, we heterologously expressed LbHLH in Arabidopsis thaliana under the 35S promoter. The overexpression lines showed significantly increased trichome number and reduced root hair number. LbHLH might interact with GLABRA1 to influence trichome and root hair development, as revealed by yeast two-hybrid analysis. The transgenic lines showed higher germination percentages and longer roots than the wild type under NaCl treatment. Analysis of seedlings grown on medium containing sorbitol with the same osmotic pressure as 100 mM NaCl demonstrated that overexpressing LbHLH enhanced osmotic resistance.These results indicate that LbHLH enhances salt tolerance by reducing root hair development and enhancing osmotic resistance under NaCl stress.
10	Intrapleural infections in patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL) are susceptible to hemophagocytic lymphohistiocytosis (HLH).	34155740	Patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL) are prone to the development of hemophagocytic lymphohistiocytosis (HLH). It is not known whether small infections in SPTCL patients can trigger the development of HLH. The clinical data were collected from 21 SPTCL patients admitted to our hospital from January 2006 to October 2019. Among 21 cases of SPTCL, 6 cases had HLH as the first manifestation (SPTCL / HLH), 7 cases had intrathoracic infection (ITI), 5 cases were SPTCL / HLH, 13 cases had no ITI or HLH (SPTCL / no HLH).Two patients with SPTCL/noHLH healed spontaneously. We found that 28.6% of the SPTCL patients had HLH as the first presentation. ITI may cooperate with SPTCL to trigger HLH and a small number of SPTCL/noHLH can fully recover without treatment. This article is protected by copyright. All rights reserved.
11	Recombinant human luteinizing hormone co-treatment in ovarian stimulation for assisted reproductive technology in women of advanced reproductive age: a systematic review and meta-analysis of randomized controlled trials.	34154604	Several studies suggest that luteinizing hormone (LH) could improve IVF outcome in women of advanced reproductive age by optimizing androgen production. In this review, we assessed the role of recombinant-human LH (r-hLH) and recombinant human follicle stimulating hormone (r-hFSH) co-treatment in ovarian stimulation for assisted reproductive technology in women of advanced reproductive age candidates for assisted reproduction.Using a preregistered protocol we systematically searched Medline/PubMed, Scopus and the ISI Web of Science databases to identify randomized controlled trials in which r-hFSH monotherapy protocols were compared with r-hFSH/r-hLH co-treatment in women ≥35 years undergoing fresh IVF cycles. We calculated the pooled odds ratio (OR) for dichotomous data and the weight mean difference (WMD) for continuous data with an associated 95% confidence interval (CI). The meta-analyses were conducted using the random-effect model. P values < 0.05 were considered statistically significant. Subgroup analyses of all primary and secondary outcomes were performed only in women aged 35-40 years.Twelve studies were identified. In women aged between 35 and 40 years, r-hFSH/r-hLH co-treatment was associated with higher clinical pregnancy rates (OR 1.45, CI 95% 1.05-2.00, I2 = 0%, P = 0.03) and implantation rates (OR 1.49, CI 95% 1.10-2.01, I2 = 13%, P = 0.01) versus r-hFSH monotherapy. Fewer oocytes were retrieved in r-hFSH/r-hLH-treated patients than in r-hFSH-treated patients both in women aged ≥35 years (WMD -0.82 CI 95% -1.40 to - 0.24, I2 = 88%, P = 0.005) and in those aged between 35 and 40 years (WMD -1.03, CI - 1.89 to - 0.17, I2 = 0%, P = 0.02). The number of metaphase II oocytes, miscarriage rates and live birth rates did not differ between the two groups of women overall or in subgroup analysis.Although more oocytes were retrieved in patients who underwent r-hFSH monotherapy, this meta-analysis suggests that r-hFSH/r-hLH co-treatment improves clinical pregnancy and implantation rates in women between 35 and 40 years of age undergoing ovarian stimulation for assisted reproduction technology. However, more RCTs using narrower age ranges in advanced age women are warranted to corroborate these findings.
12	Absent XIAP expression in T cell blasts and causal XIAP mutations including non-coding deletion.	34145698	X-linked inhibitor of apoptosis protein (XIAP) deficiency is one of inborn errors of immunity characterized by recurrent hemophagocytic lymphohistiocytosis (HLH) and refractory inflammatory bowel disease (IBD) mimicking Crohn's disease (CD). The aim of this study is to make accurate diagnosis of XIAP deficiency based on genetic and XIAP expression studies and to investigate endoscopic findings shared by patients with this disease.Four male patients with recurrent HLH and long-term refractory IBD were studied for the diagnosis of XIAP deficiency. Parallelly, endoscopic findings of the four patients were also studied.These four patients were diagnosed with XIAP deficiency based on the absent XIAP expression in cultured T-cell blasts. Sequence analysis of the responsible gene, XIAP, demonstrated two novel nonsense mutations of p.Gln114X and p.Glu25X, and a previously reported nonsense mutation of p.Arg381X. Although no mutations in the coding region were detected in the fourth patient, further studies demonstrated a novel 2199 bp deletion encompassing non-coding exon 1, presumably affecting transcription and stability of XIAP mRNA. All of the patients eventually underwent hematopoietic stem cell transplantation, leading to a complete or partial remission of IBD. These four patients shared an endoscopic finding of multiple wide and longitudinal ulcers with straight and non-raised edge in the colon.XIAP expression in T-cell blasts could facilitate the diagnosis of this disease especially with causal mutations in non-coding regions.
13	Anakinra treats fulminant myocarditis from Neisseria meningitidis septicaemia and haemophagocytic lymphohistiocytosis: a case report.	34142010	Fulminant myocarditis is a life-threatening condition characterized by acute cardiac dysfunction requiring pharmacological or mechanical circulatory support. Haemophagocytic lymphohistiocytosis (HLH) is an uncommon state of immune dysregulation and overactivation. Inflammation mediated by interleukin-1 (IL-1) is thought to play a role in the pathogenesis of myocarditis and HLH, and there is some evidence that the IL-1 receptor antagonist Anakinra may play a role in treating both these conditions.A 26-year-old previously healthy male presented to the Emergency Department with a 3-day history of malaise, headache, vomiting, diarrhoea, and fever. He was profoundly hypotensive on arrival, diagnosed with septic shock, and commenced on broad-spectrum antibiotics and vasopressors. Blood tests showed lymphopenia, thrombocytopenia, low fibrinogen and elevated high sensitivity troponin T, ferritin, and C-reactive protein. Echocardiography demonstrated severely impaired biventricular systolic function and a diagnosis of fulminant myocarditis was made. His condition deteriorated and he required intubation and additional inotropic support. A diagnosis of HLH was made and he was commenced on Anakinra and Methylprednisolone. His condition improved rapidly thereafter. Polymerase chain reaction testing subsequently confirmed infection with Neisseria meningitidis.In this case, fulminant myocarditis and HLH were life-threatening manifestations of meningococcal septicaemia, and the patient's condition improved rapidly following administration of the IL-1 receptor antagonist Anakinra. These complications should be borne in mind in septic patients with marked haemodynamic instability and multiorgan dysfunction, and treatment with Anakinra should be considered in those who fail to respond to conventional therapy.
14	Genome-Wide Analysis of the G2-Like Transcription Factor Genes and Their Expression in Different Senescence Stages of Tobacco (Nicotiana tabacum L.).	34135936	The Golden2-like (GLK) transcription factors play important roles in regulating chloroplast growth, development, and senescence in plants. In this study, a total of 89 NtGLK genes (NtGLK1-NtGLK89) were identified in the tobacco genome and were classified into 10 subfamilies with variable numbers of exons and similar structural organizations based on the gene structure and protein motif analyses. Twelve segmental duplication pairs of NtGLK genes were identified in the genome. These NtGLK genes contain two conserved helix regions related to the HLH structure, and the sequences of the first helix region are less conserved than that of the second helix motif. Cis-regulatory elements of the NtGLK promoters were widely involved in light responsiveness, hormone treatment, and physiological stress. Moreover, a total of 206 GLK genes from tomato, tobacco, maize, rice, and Arabidopsis were retrieved and clustered into eight subgroups. Our gene expression analysis indicated that NtGLK genes showed differential expression patterns in tobacco leaves at five senescence stages. The expression levels of six NtGLK genes in group C were reduced, coinciding precisely with the increment of the degree of senescence, which might be associated with the function of leaf senescence of tobacco. Our results have revealed valuable information for further functional characterization of the GLK gene family in tobacco.
15	Malignancy-induced hemophagocytic lymphohistiocytosis in a case of signet ring cell carcinoma of the stomach.	34135144	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially life-threatening condition associated with high mortality and can be classified into primary (familial) and secondary HLH. Primary HLH is an inherited, autosomal recessive disorder associated with defects in perforin function, whereas secondary HLH is associated with infections, especially Epstein-Barr virus infection, malignancies, and autoimmune disorders. Patients with malignancy-associated secondary HLH experience symptoms that overlap with those described for other HLH types, which is associated with an increased incidence of misdiagnosis and mortality. Here, we report the case of a patient with secondary HLH associated with a solid malignancy (signet-ring cell carcinoma of the stomach).
16	A Fatal Case of Dengue-Associated Hemophagocytic Lymphohistiocytosis and Retroperitoneal Hematoma in a Patient With Autoimmune Hemolytic Anemia.	34131542	Hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially life-threatening complication of dengue infection which necessitates early diagnosis and treatment to improve patient outcomes. Severe dengue infection complicated by HLH may require interventions such as systemic corticosteroids, intravenous immunoglobulin, or chemotherapy. Here, we report a case of concurrent dengue-induced HLH and flare of underlying autoimmune hemolytic anemia (AIHA). The disease was refractory to treatment including corticosteroids and intravenous immunoglobulin. The course of illness was later complicated by dengue shock syndrome, severe liver dysfunction, and a large retroperitoneal hematoma. Unfortunately, the patient succumbed on day 10 of illness.
17	Whole exome sequencing reveals a novel LRBA mutation and clonal hematopoiesis in a common variable immunodeficiency patient presented with hemophagocytic lymphohistiocytosis.	34120644	Common variable immunodeficiency (CVID) was a kind of primary immunodeficiency disorders with heterogeneous phenotype and genotype. Lipopolysaccharide-responsive and beige-like anchor (LRBA) mutation was identified as disease associated in CVID, advanced genetic method will help to detect atypical cases. We report a case of adult patient manifested as hemophagocytic lymphohistiocytosis (HLH), bone marrow examination suggested prosperity to MDS, manifested as increased immature myeloid cells and dysplastic hematopoiesis. Whole exome sequencing (WES) identified a novel heterogeneous c.1876T > C (p.W626R) mutation in LRBA and four somatic mutations: ASXL1 (c.1967dupA); PTPN11 (c.226G > A), U2AF1 (c.101C > T and c.470A > G), among which ASXL1 was a high-risk marker of clonal hematopoiesis. Combined with her recurrent severe infections and immune abnormalities such as hypoimmunoglobulinemia, the patient was diagnosed with CVID. Subsequent hematopoietic stem cell transplantation saved her from severe cytopenia and immune deficiency. This case report highlights the great promise of utilization of WES for diagnosing rare disease with atypical manifestations and guiding further treatment.
18	A hungry Histiocyte, altered immunity and myriad of problems: Diagnostic challenges for Pediatric HLH.	34118134	Hemophagocytic lymphohistiocytosis (HLH) is an immune deregulation disorder with varied clinical presentation which clinically overlaps with widespread tropical infections.We conducted a retrospective chart review of children diagnosed with HLH at our center from February-2017 to October-2020.Out of the nine diagnosed patients, genetic predisposition was present in three children; two had identified infectious triggers. The mean age of presentation was 30 months with male predominance. The most common clinical findings were fever, organomegaly, and pancytopenia. The median value of fibrinogen was-156 mg/dL, ferritin-12 957 ng/mL and for triglycerides-349 mg/dL, respectively. In children with identified genetic predisposition, serum ferritin levels were usually more than 10 000 ng/mL. The majority of our patients had evidence of hemophagocytosis on bone marrow examination. In our experience, although nonspecific, very high ferritin and serum triglycerides with low fibrinogen in a patient with bi-cytopenia, pancytopenia was the most suggestive evidence of HLH. Genetic evaluation in our series identified three children, one with primary HLH genetic mutation and two with underlying immune deficiency syndrome. The presence of HLH in the accelerated phase of Chediak-Higashi and AD Hyper IgE syndrome with HLH is extremely rare. Leishmaniasis (in nonendemic area) and Ebstein-Barr virus (EBV) was identified as an infectious trigger in two cases. Most of our cases received treatment as per HLH 2004 protocol. Three children died during the initial diagnosis and treatment. HLH with subcutaneous panniculitis-like T-cell lymphoma recovered well.HLH remains a life-threatening disorder associated with a variety of underlying illnesses as highlighted by our case series.
19	Outcome of L-DEP regimen for treatment of pediatric chronic active Epstein-Barr virus infection.	34112210	We intended to investigate the clinical features of paediatric patients with chronic active Epstein-Barr virus infection (CAEBV) and to examine the effectiveness of the L-DEP regimen before haematopoietic stem cell transplantation (HSCT).A retrospective analysis was performed on 35 patients with CAEBV at Beijing Children's Hospital from January 2016 to January 2020. The efficacy and adverse events of the L-DEP regimen were evaluated.The median age of the 35 patients was 7.0 years old (range 2.5-17.5 years). Twenty-eight patients achieved a clinical response (80.0%, 22 in clinical CR, 6 in clinical PR) after L-DEP. In terms of virological response, 7 patients (20%) were assessed as having virological CR, and 23 patients (65.7%) had virological PR. Finally, 29 patients underwent allo-HSCT. The median survival time was 18 months (2-50 months). The 3-year overall survival rates in patients treated with chemotherapy only (n = 6) and chemotherapy followed by HSCT (n = 25) were 33.3% and 75.4%, respectively. After L-DEP 1st treatment and L-DEP 2nd treatment, the EBV-DNA loads in blood and plasma were significantly reduced compared with those before chemotherapy (median: 4.29 × 105 copies/ml vs. 1.84 × 106 copies/ml, Mann-Whitney U: P = 0.0004; 5.00 × 102 copies/ml vs. 3.17 × 103 copies/ml, Mann-Whitney U; P = 0.003; 2.27 × 105 copies/ml vs. 1.84 × 106 copies/ml, P = 0.0001; 5.00 × 102 copies/ml vs. 3.17 × 103 copies/ml, P = 0.003). Compared with the liver and spleen size before chemotherapy, the size of the liver and spleen shrank significantly after L-DEP 2nd (median 3.8 cm vs. 1.9 cm, P = 0.003; 3.8 cm vs. 0 cm, P < 0.008). In addition, after L-DEP treatment, there was no difference in the clinical or virological response rate regardless of HLH status (clinical response: 77.3% vs. 84.6%, P = 0.689; virological response: 90.9% vs. 76.9%, P = 0.337).The L-DEP regimen is an effective therapy in CAEBV for bridging to allo-HSCT.
20	Imaging of native transcription and transcriptional dynamics in vivo using a tagged Argonaute protein.	34107044	A flexible method to image unmodified transcripts and transcription in vivo would be a valuable tool to understand the regulation and dynamics of transcription. Here, we present a novel approach to follow native transcription, with fluorescence microscopy, in live C. elegans. By using the fluorescently tagged Argonaute protein NRDE-3, programmed by exposure to defined dsRNA to bind to nascent transcripts of the gene of interest, we demonstrate transcript labelling of multiple genes, at the transcription site and in the cytoplasm. This flexible approach does not require genetic manipulation, and can be easily scaled up by relying on whole-genome dsRNA libraries. We apply this method to image the transcriptional dynamics of the heat-shock inducible gene hsp-4 (a member of the hsp70 family), as well as two transcription factors: ttx-3 (a LHX2/9 orthologue) in embryos, and hlh-1 (a MyoD orthologue) in larvae, respectively involved in neuronal and muscle development.
21	[Sucecesfull bone marrow transplantation in a case of familial hemophagocytic lymphohistiocytosis type 3].	34106167	Hemophagocytic lymphohistiocytosis (HLH) is an exaggerated activation of the immune system which can be either primary (familial) or secondary. Familial hemophagocytic lymphohistiocytosis type 3 (FHL-3) is a severe immune disorder, caused by mutations in the UNC13D gene, which codes for a protein crucial to the cytotoxic function of lymphocytes.To describe the diagnostic relevance of next-generation sequencing in the approach of a patient with suspected FHL and to demonstrate the effectiveness of bone marrow transplantation as the only curative measure.4-year-old preschool male, previously healthy, who presented with mononucleosis syndrome and positive IgM for Epstein Barr virus, developing hepatosplenomegaly and progressive clinical de terioration. A lymphoproliferative syndrome was suspected, which was ruled out by bone marrow aspiration, finding evidence of active hemophagocytosis. The patient met the criteria for hemophago cytic syndrome (bone marrow aspiration, pancytopenia, elevated ferritin, and hypertriglyceridemia) and, given the lack of response to first-line management, including antiviral treatment, a possible primary etiology was considered. A molecular study was completed with NGS that was positive for FHL-3. Due to the progressive clinical deterioration, a bone marrow transplantation was performed, presenting successful results after the first year had elapsed.NGS is an indispensable tool in the diagnosis of FHL, mainly when the response to standard treatment is not adequate and facilitates the timely implementation of the necessary therapeutic measures.
22	Natural-killer cell cytotoxicity as a diagnostic and prognostic marker for adult patients with secondary hemophagocytic lymphohistiocytosis: a prospective phase II observational study.	34104375	Hemophagocytic lymphohistiocytosis (HLH) can be life-threatening if not detected and treated appropriately. The diagnosis of HLH can be confusing due to other similar febrile diseases that present with cytopenia. Natural-killer cell (NK)-cytotoxicity is an important diagnostic parameter for primary HLH; however, its role in secondary HLH in adults has not been well-elucidated.We prospectively enrolled 123 adult patients with febrile conditions accompanied by cytopenia or marrow hemophagocytosis. A diagnosis of HLH was based on HLH-2004 criteria and treated based on HLH-94 protocol. NK-cytotoxicity was calculated at the time of diagnosis by K562-cell direct lysis using flow-cytometry.HLH (n = 60) was determined to be caused by Epstein-Barr virus (EBV) (n = 11), infection other than EBV (n = 16), malignancies (n = 19), and unknown (n = 14). Febrile diseases other than HLH (n = 63) were diagnosed as autoimmune disease (n = 22), malignancies (n = 21), infection (n = 12), non-malignant hematological diseases (n = 6), and unknown (n = 2). A lower NK-cytotoxicity level was observed at diagnosis in patients with HLH, compared with other causes of febrile disease (12.1% versus 26.2%, p < 0.001). However, NK-cytotoxicity had a borderline effect on diagnosis of HLH, with an area under receiver operation characteristic curve of 0.689. It also showed no significant role for the prediction of survival outcome. Multivariate analysis revealed that malignant disease and high ferritin level were related with poor survival outcome. In non-malignant disease subgroups, old age, EBV-association, and low NK-cytotoxicity were related with poor survival.Febrile disease with cytopenia was associated with decreased NK-cytotoxicity, especially in adults with HLH; however, its diagnostic role for adult HLH is still arguable. The diagnostic criteria for adult HLH should be further discussed.Clinical Research Information Service [Internet]; Osong (Chungcheongbuk-do), Korea, Centers for Disease Control and Prevention, Ministry of Health and Welfare (Republic of Korea); https://cris.nih.go.kr/cris/index.jsp; Feb, 16th 2016; KCT0001886 (KC15TISE0936).
23	Piecemeal regulation of convergent neuronal lineages by bHLH transcription factors in Caenorhabditis elegans.	34100067	Cells of the same type can be generated by distinct cellular lineages that originate in different parts of the developing embryo ('lineage convergence'). Several Caenorhabditis elegans neuron classes composed of left/right or radially symmetric class members display such lineage convergence. We show here that the C. elegans Atonal homolog lin-32 is differentially expressed in neuronal lineages that give rise to left/right or radially symmetric class members. Loss of lin-32 results in the selective loss of the expression of pan-neuronal markers and terminal selector-type transcription factors that confer neuron class-specific features. Another basic helix-loop-helix (bHLH) gene, the Achaete-Scute homolog hlh-14, is expressed in a mirror image pattern relative to lin-32 and is required to induce neuronal identity and terminal selector expression on the contralateral side of the animal. These findings demonstrate that distinct lineage histories converge via different bHLH factors at the level of induction of terminal selector identity determinants, which thus serve as integrators of distinct lineage histories. We also describe neuron-to-neuron identity transformations in lin-32 mutants, which we propose to also be the result of misregulation of terminal selector gene expression.
24	Virus-triggered secondary hemophagocytic lymphohistiocytosis.	34096649	Primary (familial/hereditary) and secondary (non-familial/hereditary) hemophagocytic lymphohistiocytosis (HLH) are hyperinflammatory and hypercytokinemic syndromes. Secondary HLH includes infection- (eg viral/bacterial/fungal/parasitic) and non-infection- (eg collagen disease or malignancy) related diseases. Viral HLH is the major type among all age groups. Secondary viral HLH and primary HLH must be differentiated carefully because primary HLH can be associated with viral infection(s), and the outcome is dismal without a timely diagnosis and hematopoietic stem cell transplantation (HSCT). Epstein-Barr virus (EBV)-related HLH (EBV-HLH) is the most common type of viral HLH in childhood. For non-EBV-HLH, appropriate treatment of viral infection, followed by immunomodulatory agent(s) such as corticosteroids, intravenous immunoglobulin or cyclosporine A, is usually successful; however, recent SARS-CoV-2-related HLH may become life-threatening. EBV-HLH may occur heterogeneously associated with the primary infection, with chronic active EBV infection or with underlying primary HLH. Although immunomodulatory agent(s) are effective in the majority of EBV-HLH cases, management differs from that of non-EBV-HLH because severe and refractory cases may require etoposide-containing HLH-1994/2004 regimens or other experimental agents. The novel agent, emapalumab (an anti-IFN-γ monoclonal antibody) can be used to treat EBV-HLH cases to avoid the risk of secondary malignancy due to etoposide. Finally, HSCT is required for refractory EBV-HLH cases and can also be curative in some other cases.
25	Mitf Involved in Innate Immunity by Activating Tyrosinase-Mediated Melanin Synthesis in Pteria penguin.	34093521	The microphthalmia-associated transcription factor (MITF) is an important transcription factor that plays a key role in melanogenesis, cell proliferation, survival and immune defense in vertebrate. However, its function and function mechanism in bivalve are still rarely known. In this research, first, a Mitf gene was characterized from Pteria penguin (P. penguin). The PpMitf contained an open reading frame of 1,350 bp, encoding a peptide of 449 deduced amino acids with a highly conserved basic helix-loop-helix-leucine zipper (bHLH-LZ) domain. The PpMITF shared 55.7% identity with amino acid sequence of Crassostrea gigas (C. gigas). Tissue distribution analysis revealed that PpMitf was highly expressed in mantle and hemocytes, which were important tissues for color formation and innate immunity. Second, the functions of PpMitf in melanin synthesis and innate immunity were identified. The PpMitf silencing significantly decreased the tyrosinase activity and melanin content, indicating PpMitf involved in melanin synthesis of P. penguin. Meanwhile, the PpMitf silencing clearly down-regulated the expression of PpBcl2 (B cell lymphoma/leukemia-2 gene) and antibacterial activity of hemolymph supernatant, indicating that PpMitf involved in innate immunity of P. penguin. Third, the function mechanism of PpMitf in immunity was analyzed. The promoter sequence analysis of tyrosinase (Tyr) revealed two highly conserved E-box elements, which were specifically recognized by HLH-LZ of MITF. The luciferase activities analysis showed that Mitf could activate the E-box in Tyr promoter through highly conserved bHLH-LZ domain, and demonstrated that PpMitf involved in melanin synthesis and innate immunity by regulating tyrosinase expression. Finally, melanin from P. penguin, the final production of Mitf-Tyr-melanin pathway, was confirmed to have direct antibacterial activity. The results collectively demonstrated that PpMitf played a key role in innate immunity through activating tyrosinase-mediated melanin synthesis in P. penguin.
26	Hemophagocytic lymphohistiocytosis after COVID-19 vaccination.	34088334	Cases of thrombotic thrombocytopenia induced by coronavirus disease 2019 (COVID-19) vaccines have been reported recently. Herein, we describe the first case of another critical disorder, hemophagocytic lymphohistiocytosis (HLH), in a healthy individual after COVID-19 vaccination. A 43-year-old Chinese farmer developed malaise, vomiting, and persistent high fever (up to 39.7 °C) shortly after receiving the first dose of the inactivated SARS-CoV-2 vaccine. The initial evaluation showed pancytopenia (neutrophil count, 0.70 × 109/L; hemoglobin, 113 g/L; platelet, 27 × 109/L), elevated triglyceride (2.43 mmol/L), and decreased fibrinogen (1.41 g/L). Further tests showed high serum ferritin levels (8140.4 μg/L), low NK cell cytotoxicity (50.13%-60.83%), and positive tests for Epstein-Barr virus (EBV) DNA. Hemophagocytosis was observed in the bone marrow. Therefore, HLH was confirmed, and dexamethasone acetate (10 mg/day) was immediately prescribed without etoposide. Signs and abnormal laboratory results resolved gradually, and the patient was discharged. HLH is a life-threatening hyperinflammatory syndrome caused by aberrantly activated macrophages and cytotoxic T cells, which may rapidly progress to terminal multiple organ failure. In this case, HLH was induced by the COVID-19 vaccination immuno-stimulation on a chronic EBV infection background. This report indicates that it is crucial to exclude the presence of active EBV infection or other common viruses before COVID-19 vaccination.
27	Clinicopathological findings of systemic Epstein-Barr virus-positive T-lymphoproliferative diseases in younger and older adults.	34088321	Systemic Epstein-Barr virus+ T-cell lymphoma (sEBV+ TCL) occurs in childhood and young adults, and is exceptionally rare in older adults.We investigated clinicopathological features in 16 patients of various ages with systemic EBV+ CD8+ T-lymphoproliferative diseases.Eight younger patients and four of eight older adults had sEBV+ CD8+ TCL, with invasion by medium-sized to/or large atypical lymphocytes primarily in bone marrow and lymph nodes, hemophagocytic lymphohistiocytosis (HLH), and progressive clinicopathological course. A further two patients demonstrated EBV+ node-based CD8+ large TCL without HLH, while the remaining two had the systemic form of chronic active EBV infection (sCAEBV) with CD8+ small lymphocytes. Past history of sCAEBV-like lesions was observed in one sEBV+ TCL patient (8.3%). Immunohistologically, in 12 sEBV+ TCL patients, atypical lymphocytes were positive for phosphate signal transducer and activator of transcription 3 (66.7%), CMYC (83.3%), and p53 (75%). Strong reactions of programmed cell death-ligand (PD-L)1+ tumor or non-neoplastic cells were detected in nine sEBV+ TCL patients (75%). Clonal peaks of the T-cell receptor (TCR) γ gene were detected in eight sEBV+ TCL patients by polymerase chain reaction. Four younger patients in sEBV+ TCL (33.3%) are in remission with chemotherapies including etoposide, and three of the four underwent allogeneic stem cell transplantation (SCT).sEBV+ CD8+ TCL was observed in younger and older adults with less history of sCAEBV. HLH, tumor cell atypia, immunohistological findings, and progressive clinical course were characteristic of sEBV+ CD8+ TCL. Prompt chemotherapy and SCT induced tumor regression in sEBV+ CD8+ TCL patients.
28	Hemophagocytic histiocytosis in severe SARS-CoV-2 infection: A bone marrow study.	34086418	The clinical and laboratory features of severe COVID-19 infection overlap with those of hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory disorder often associated with several viral infections. The clinical syndrome of HLH encompasses fever, organomegaly, cytopenias, hyperferritinemia, hypertriglyceridemia, raised transaminases, hypofibrinogenemia, absent natural killer (NK) cell activity, increased soluble CD25 and hemophagocytic lymphohistiocytosis in bone marrow, spleen, and lymph nodes.We analyzed clinicopathological and laboratory features of thirteen patients with severe COVID-19 infection suspected to have HLH and found to have hemophagocytic histiocytosis on bone marrow examination (BME).Five of thirteen (38.46%) patients fulfilled five of eight HLH 2004 criteria and/or had a H-score ≥169. Three (23.08%) satisfied four of eight and remainder five (38.46%) satisfied three of eight HLH 2004 criteria. Fever, raised serum ferritin (13/13, 100%), transaminases (9/13, 69.23%), triglycerides (4/13, 30.76%), cytopenias (5/13, 38.46%), hypofibrinogenemia (2/13, 15.38%), and organomegaly (1/13, 7.69%) were observed in our patients. BME showed hemophagocytic histiocytosis without lymphocytosis in all. Contrary to HLH, lymphocytopenia (11/13, 84.61%), leukocytosis (7/13, 53.84%), neutrophilia (7/13, 53.84%), and hyperfibrinogenemia (7/13, 53.84%) were observed. Serum CRP, LDH, and plasma D-dimer were elevated in all, while serum albumin was decreased in 12 of 13 (92.3%) patients. Five patients recovered with high-dose pulsed corticosteroid therapy.The immune response associated with severe COVID-19 infection is similar to HLH with few differences. HLH should be suspected in severe COVID-19 infection although all patients may not fulfill required HLH diagnostic criteria. BME should be done in suspected cases so that appropriate therapy may be initiated early.
29	Central Nervous System Involvement in Adult-Onset Hemophagocytic Lymphohistiocytosis.	34084687	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease marked by high cytokine levels, uncontrolled lymphocyte, and macrophage proliferation. It is generally a systemic disorder with varying degrees of central nervous system (CNS) involvement, with the vast majority of cases affecting children. We report a case of CNS-HLH in a 51-year-old male who initially presented with fevers, night sweats, fatigue, bilateral arthralgia, and altered mental status. Computed tomography (CT) of the chest, abdomen, and pelvis showed hepatosplenomegaly. Magnetic resonance imaging (MRI) of the brain showed enhancing lesions mainly in the right frontal lobe with a small hemorrhagic focus. An extensive workup for infectious, autoimmune, and neoplastic and genetic etiologies was only significant for cytopenia with markedly elevated C-reactive protein (CRP), ferritin, and lactate dehydrogenase (LDH), in addition to mild triglyceridemia. Bone marrow and liver biopsy revealed hemophagocytosis. Brain biopsy revealed no evidence of malignancy or infection. The patient was treated with high-dose dexamethasone and etoposide and fully recovered with resolution of all of HLH parameters and decrease/resolution of brain lesion. Clinicians should have a high index of suspicion for CNS-HLH in adults who present with sepsis-like illness with fevers, altered mental status, and cytopenia but negative cultures and unusual radiographic cerebral abnormalities so that early diagnosis and treatment can be initiated to prevent end-organ failure and death.
30	Genetic and clinical characteristics of pediatric patients with familial hemophagocytic lymphohistiocytosis.	34083498	Our study was designed to investigate the frequencies and distributions of familial hemophagocytic lymphohistiocytosis (FHL) associated genes in Saudi patients.FHL associated gene screening was performed on 87 Saudi patients who were diagnosed with hemophagocytic lymphohistiocytosis (HLH) between 1995 and 2014. The clinical and biochemical profiles were also retrospectively captured and analyzed.Homozygous mutations and mono-allelic variants were identified in 66 (75.9%) and 3 (3.5%) of the study participants, respectively. STXBP2 was the most frequently mutated gene (36% of patients) and mutations in STXBP2 and STX11 accounted for 58% of all FHL cases and demonstrated a specific geographical pattern. Patients in the FHL group presented at a significantly younger age than those belonging to the unknown-genetics group (median, 3.9 vs. 9.4 mo; P=0.005). The presenting clinical features were similar among the various genetic groups and the 5-year overall survival (OS) was 55.4% with a 5.6 year median follow-up. Patients with PRF1 mutations had a significantly poorer 5-year OS (21.4%, P=0.008) and patients undergoing hematopoietic stem cell transplant (72.4%) had a significantly better 5-year OS (66.5% vs. 0%, P=0.001).Our study revealed the predominance of the STXBP2 mutations in Saudi patients with FHL. A genetic diagnosis was possible in 80% of the cohort and our data showed improved survival in FHL patients who underwent hematopoietic stem cell transplant.
31	An Unusual Case of Hemophagocytic Lymphohistiocytosis Presentation in Acute Human Immunodeficiency Virus.	34078147	Hemophagocytic lymphohistiocytosis (HLH) in acute human immunodeficiency virus (HIV) patients has been scarcely reported in the English literature. To the best of our knowledge, only 12 cases have been described. We present a case of a 27-year-old male with no past medical history who was admitted with a new-onset headache, fever, night sweats, and chills. Further laboratory tests revealed transaminitis, leukopenia, thrombocytopenia, positive HIV antigen/antibody test, and markedly elevated ferritin levels, which promoted our suspicion of HLH. This case demonstrates HLH as an unusual presentation of HIV during its seroconversion stage. This report adds a rare disease process to the available literature, and we emphasize that markedly elevated ferritin levels in acute HIV patients should raise suspicion toward a diagnosis of HLH.
32	Ruxolitinib is an alternative to etoposide for patient with hemophagocytic lymphohistiocytosis complicated by acute renal injury: A case report.	34074166	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by excessive production of inflammatory cytokines and multiple organs injury. Ruxolitinib, an oral selective JAK1/2 inhibitor, has recently shown efficacy and safety in the treatment of secondary HLH, which may be an alternative to intensive chemotherapy.We report a case of a 2-year-old boy who presented to our institution with recurrent fever and acute renal failure. We made the diagnosis of Epstein-Barr virus related HLH based on the HLH-2004 protocol, and gave the treatment of ruxolitinib instead of etoposide.The patient received dexamethasone and continuous renal replacement therapy due to renal failure, but he still had fever and anuria. Given that the use of etoposide may deteriorate renal function, ruxolitinib was administered instead of etoposide. After 5 days of ruxolitinib treatment, the patient's fever was resolved and renal function also gradually recovered 14 days later.Currently, dexamethasone, etoposide and cyclosporine A are the main drugs in HLH treatment. However, cytotoxic chemotherapy can temporally deteriorate organ damage and induce serious myelosuppression, which makes clinicians hesitate to implement these regimens. Ruxolitinib has shown efficacy in treating HLH without much toxicity in clinical trials. Thus, we suggest that ruxolitinib constitutes a treatment option for secondary HLH complicated by severe renal damage which may reduce toxic effects compared with intense chemotherapy.
33	Biophysical and Biochemical Comparison of Extracellular Vesicles Produced by Infective and Non-Infective Stages of Trypanosoma cruzi.	34068436	Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote's EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.
34	The first pediatric case of hemophagocytic lymphohistiocytosis secondary to Crimean-Congo haemorrhagic fever successfully treated with therapeutic plasma exchange accompanying ribavirin and intravenous immunoglobulin.	34061376	Although Crimean-Congo hemorrhagic fever (CCHF) is mild and self-limited in children, some patients may develop excessive bleeding, massive liver necrosis, and multiple organ failure associated with secondary hemophagocytic lymphohistiocytosis (HLH) induced by cytokine storm. Treatment of CCHF is mainly symptomatic and supportive. The efficacy of ribavirin, which is the only antiviral drug in the treatment of CCHF, remains controversial. Although therapeutic plasma exchange (TPE) has been shown to beneficial in small case series with primary and secondary HLH, there is no pediatric patient with HLH secondary to CCHF treated with TPE in the literature. In this report, we describe the first pediatric patient who was successfully recovered from HLH secondary to CCHF with ribavirin, intravenous immunoglobulin, and TPE.
35	Successful rescue of a lethal Griscelli syndrome type 2 presenting with neurological involvement and hemophagocytic lymphohistiocytosis: a case report.	34058999	Griscelli syndrome type 2 (GS2) is a rare autosomal recessive disease caused by mutations in RAB27A gene. It is primarily characterized by a combination of partial albinism, hemophagocytic lymphohistiocytosis (HLH) or other immunodeficiency. However, neurological involvement at onset in GS2 and treatment has rarely been described.We describe a 3-year-old boy with GS2 in an Asian Chinese family. He presented with progressive neurological abnormalities following unremitting fever at onset. He developed HLH during the clinical course. A novel homozygous mutation (c.1 A > G) in RAB27A gene was subsequently identified. He was then treated by HLH-1994 protocol combined with ruxolitinib and experienced a dramatic remission. He subsequently underwent a successful haploidentical hematopoietic stem cell transplantation and stayed at a good condition.We reported an atypical form of GS2 manifesting as severe central nervous system involvement at onset and subsequent HLH, which was successfully rescued in time. This case also highlights the need for early consideration of immunologic and genetic evaluation for HLH in unexplained neuroinflammation in the diagnostic work up.
36	Exploring the Intersection of Isolated-CNS Hemophagocytic Lymphohistiocytosis and Pediatric Chronic Lymphocytic Inflammation With Pontine Perivascular Enhancement Responsive to Steroids.	34056941	CLIPPERS (chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids) is an extremely rare neurologic inflammatory condition. Fewer than 10 pediatric cases have been described.Debate persists as to whether it is a distinct disease or a clinical, radiologic, and histologic phenotype evolving into another disorder. We propose that CLIPPERS may be a clinical manifestation of an underlying state of immune-dysregulation.We describe the case of the youngest known report of CLIPPERS, an 18-month-old infant from Melbourne, Australia. Reviewing the literature for all reported pediatric cases, we identified that robust investigation and whole exome sequencing was underutilized and proposed diagnostic criteria were frequently unmet. Particular focus should be paid to genes known to cause familial hemophagocytic lymphohistiocytosis (HLH), with the CLIPPERS phenotype manifesting as a form of isolated central nervous system (CNS)-HLH in some patients. Curative treatment options such as hematopoietic stem cell transplantation may be appropriate for some patients and should be considered early.
37	Effect of LHCGR Gene Polymorphism (rs2293275) on LH Supplementation Protocol Outcomes in Second IVF Cycles: A Retrospective Study.	34046009	Infertility is a major concern for couples wanting to have progeny. Despite recent advances in the field of IVF, success rates still need improvement. Understanding the patient's variability and addressing it with personalized interventions may improve the success rate of fertilization and live births. This study examined the impact of a personalized pharmacogenomic approach on LH supplementation on the pregnancy and live birth rate outcomes in comparison with the traditional approaches. 193 patients undergoing a second IVF cycle in Krishna IVF Clinic received LH supplementation either as per the conventional methods or based on N312S (rs2293275) LHCGR gene polymorphism. Results showed a significant increase in pregnancy rate (P-value: 0.049) and a trend showing improvement in live birth rates (P-value: 0.082) when r-hLH supplementation protocol was decided as per the genotypes A/A, A/G, and G/G of the N312S variant in the respective patients. This stimulation regimen helped in providing optimum levels of r-hLH supplementation to patients with impaired hormone-receptor interacting activity, to achieve higher success in pregnancy and live birth rates.
38	Case Report: Hemophagocytic Lymphohistiocytosis and Non-Tuberculous Mycobacteriosis Caused by a Novel GATA2 Variant.	34040617	Hemophagocytic lymphohistiocytosis (HLH) is a disorder of uncontrolled immune activation with distinct clinical features including fever, cytopenia, splenomegaly, and sepsis-like symptoms. In a young adolescent patient a novel germline GATA2 variant (NM_032638.5 (GATA2): c.177C>G, p.Tyr59Ter) was discovered and had resulted in non-tuberculous mycobacterial (NTM) infection and aggressive HLH. Strikingly, impaired degranulation of cytotoxic T-lymphocytes (CTL) and natural killer (NK)-cells was detected in CD107a-analyses. The affected patient was treated with HLA-matched unrelated alloHSCT, and subsequently all hematologic and infectious abnormalities including HLH and NTM resolved. This case supports early alloHSCT in GATA2 deficiencies as curative approach regardless of active NTM infection. Future studies on GATA2 c.177C>G, p.Tyr59*Ter might unravel its potential role in cytotoxic effector cell function and its contribution to HLH pathogenesis.
39	Optimising Follicular Development, Pituitary Suppression, Triggering and Luteal Phase Support During Assisted Reproductive Technology: A Delphi Consensus.	34040586	A Delphi consensus was conducted to evaluate global expert opinions on key aspects of assisted reproductive technology (ART) treatment.Ten experts plus the Scientific Coordinator discussed and amended statements plus supporting references proposed by the Scientific Coordinator. The statements were distributed via an online survey to 35 experts, who voted on their level of agreement or disagreement with each statement. Consensus was reached if the proportion of participants agreeing or disagreeing with a statement was >66%.Eighteen statements were developed. All statements reached consensus and the most relevant are summarised here. (1) Follicular development and stimulation with gonadotropins (n = 9 statements): Recombinant human follicle stimulating hormone (r-hFSH) alone is sufficient for follicular development in normogonadotropic patients aged <35 years. Oocyte number and live birth rate are strongly correlated; there is a positive linear correlation with cumulative live birth rate. Different r-hFSH preparations have identical polypeptide chains but different glycosylation patterns, affecting the biospecific activity of r-hFSH. r-hFSH plus recombinant human LH (r-hFSH:r-hLH) demonstrates improved pregnancy rates and cost efficacy versus human menopausal gonadotropin (hMG) in patients with severe FSH and LH deficiency. (2) Pituitary suppression (n = 2 statements): Gonadotropin releasing hormone (GnRH) antagonists are associated with lower rates of any grade ovarian hyperstimulation syndrome (OHSS) and cycle cancellation versus GnRH agonists. (3) Final oocyte maturation triggering (n=4 statements): Human chorionic gonadotropin (hCG) represents the gold standard in fresh cycles. The efficacy of hCG triggering for frozen transfers in modified natural cycles is controversial compared with LH peak monitoring. Current evidence supports significantly higher pregnancy rates with hCG + GnRH agonist versus hCG alone, but further evidence is needed. GnRH agonist trigger, in GnRH antagonist protocol, is recommended for final oocyte maturation in women at risk of OHSS. (4) Luteal-phase support (n = 3 statements): Vaginal progesterone therapy represents the gold standard for luteal-phase support.This Delphi consensus provides a real-world clinical perspective on the specific approaches during the key steps of ART treatment from a diverse group of international experts. Additional guidance from clinicians on ART strategies could complement guidelines and policies, and may help to further improve treatment outcomes.
40	Quantitative analysis of the natural history of prolidase deficiency: description of 17 families and systematic review of published cases.	34040193	Prolidase deficiency is a rare inborn error of metabolism causing ulcers and other skin disorders, splenomegaly, developmental delay, and recurrent infections. Most of the literature is constituted of isolated case reports. We aim to provide a quantitative description of the natural history of the condition by describing 19 affected individuals and reviewing the literature.Nineteen patients were phenotyped per local institutional procedures. A systematic review following PRISMA criteria identified 132 articles describing 161 patients. Main outcome analyses were performed for manifestation frequency, diagnostic delay, overall survival, symptom-free survival, and ulcer-free survival.Our cohort presented a wide variability of severity. Autoimmune disorders were found in 6/19, including Crohn disease, systemic lupus erythematosus, and arthritis. Another immune finding was hemophagocytic lymphohistiocytosis (HLH). Half of published patients were symptomatic by age 4 and had a delayed diagnosis (mean delay 11.6 years). Ulcers were present initially in only 30% of cases, with a median age of onset at 12 years old.Prolidase deficiency has a broad range of manifestations. Symptoms at onset may be nonspecific, likely contributing to the diagnostic delay. Testing for this disorder should be considered in any child with unexplained autoimmunity, lower extremity ulcers, splenomegaly, or HLH.
41	Postpartum disseminated HSV-1 infection with hemophagocytic lymphohistiocytosis and fulminant neonatal herpes infection.	34037797	The present study describes a 19-year-old woman with systemic herpes simplex virus (HSV)-1 infection postpartum, and a fatal course of neonatal herpesvirus infection. The mother experienced an unusual disease course with hemophagocytic lymphohistiocytosis (HLH) and persistence of HSV-1 DNA for 15 weeks. Functional investigation of cells from the mother demonstrated significantly impaired induction of antiviral interferons and cytokines in response to viruses and various ligands in the context of normal activation of the transcription factors NF-κB and IRF3. Whole exome sequencing did not reveal any functionally validated genetic variants. We suggest that the functionally impaired antiviral responses, potentially caused by a mutation in CASP8 or other variants in non-coding regions of the genome, contributed to the unusually severe disease course in two generations with disseminated HSV-1 infection evolving into HLH in the mother, and a fatal neonatal HSV-1 infection.
42	Multiple-Organ Involvement in Familial Hemophagocytic Lymphohistiocytosis Type 2 Shown on FDG PET/CT.	34034321	FDG PET/CT in an 18-year-old man with known familial hemophagocytic lymphohistiocytosis (HLH) type 2 demonstrated abnormally in the brain, liver, and lymph nodes. Pathology showed no sign of lymphoproliferative disease. The patient underwent the chemotherapy of HLH. Six months after chemotherapy, a follow-up FDG PET/CT did not show any abnormal 18F-FDG activity, suggesting a complete response. The present case shows FDG PET/CT might not only be useful in staging the familial HLH but also able to evaluate the therapy response.
43	Therapy-related acute myeloid leukemia with KMT2A-SNX9 gene fusion associated with a hyperdiploid karyotype after hemophagocytic lymphohistiocytosis.	34034210	Therapy-related acute myeloid leukemia (t-AML) following treatment with topoisomerase-II inhibitors has been increasingly reported. These compounds (e.g. etoposide) promote DNA damage and are associated with KMT2A rearrangements. They are widely used as first-line treatment in hemophagocytic lymphohistiocytosis (HLH). Here we describe a newborn who developed t-AML after HLH treatment. We provide detailed clinical, cytogenetic, and molecular characteristics of this patient, including the identification of a novel gene fusion - KMT2A-SNX9 - in t-AML. Considering the dismal outcome of this case, we discuss the side-effects of etoposide administration during HLH treatment in infants.
44	A conserved klo-1-mpk-1 pathway regulates autophagy and modulates longevity in Caenorhabditis elegans.	34034091	There are six different longevity models in Caenorhabditis elegans. Previous studies have identified several convergence points, such as hlh-30, daf-16, and klf-3, required for lifespan extension in these longevity models. However, it is not clear whether there other such convergence points. In this study, based on analysis of transcriptome data, we found that the expression of klo-1/klotho was elevated in several longevity models. klo-1 was required for lifespan extension in the glp-1(e2141) and isp-1(qm150) mutants. klo-1 extended the lifespan of glp-1(e2141) and isp-1(qm150) worms by activating extracellular-signal-regulated kinase (ERK). In addition, klo-1 and mpk-1 (the homologous gene encoding ERK) regulated autophagy in glp-1(e2141) mutants, suggesting that klo-1 regulates lifespan by activating autophagy.
45	Polymorphisms and haplotypes of IL2RA, IL10, IFNG, IRF5, and CCR2 are associated with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children.	34031980	Cytokine storms are central to the development of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). Previous studies have shown that single-nucleotide polymorphisms (SNPs) of cytokine genes may be associated with the development of EBV-HLH in children. As such, we investigated the association between susceptibility to EBV-HLH in children and SNPs and haplotypes of genes encoding interleukin-2 receptor subunit alpha (IL2RA), interleukin-10 (IL10), interferon gamma (IFNG), interferon regulatory factor 5 (IRF5), and C-C chemokine receptor 2 (CCR2).Sixty-six children with EBV-HLH and 58 healthy EBV-seropositive controls were enrolled in this study. SNPs of IL2RA rs2104286, rs12722489, and rs11594656; IL10 rs1800896, rs1800871, and rs1800872; IFNG rs2430561, IRF5 rs2004640, and CCR2 rs1799864 were assayed and genotyped using the SNaPshot technique.Frequencies of the A allele of IL2RA rs2104286 and IL10 rs1800896, and C allele of IL-10 rs1800872 were significantly higher in the EBV-HLH group than in the control group. The AA genotype of IL2RA rs2104286 and IL10 rs1800896, and the CC genotype of IL10 rs1800872 might be associated with a significantly high risk of EBV-HLH. However, the frequencies of genotypes and alleles of IL2RA rs2104286, IL10 rs1800871, IFNG rs2430561, IRF5 rs2004640, and CCR2 rs1799864 were similar in both groups. Additionally, IL2RA AGT (rs2104286-rs12722489-rs11594656) and IL10 ACC (rs1800896-rs1800871-rs1800872) haplotypes were also associated with an increased risk of EBV-HLH.SNPs of IL2RA rs2104286, IL10 rs1800896 and rs1800872 and the haplotypes of IL2RA AGT and IL10 ACC were highly associated with susceptibility to EBV-HLH in children.
46	Anti-tumor effects of an ID antagonist with no observed acquired resistance.	34031428	ID proteins are helix-loop-helix (HLH) transcriptional regulators frequently overexpressed in cancer. ID proteins inhibit basic-HLH transcription factors often blocking differentiation and sustaining proliferation. A small-molecule, AGX51, targets ID proteins for degradation and impairs ocular neovascularization in mouse models. Here we show that AGX51 treatment of cancer cell lines impairs cell growth and viability that results from an increase in reactive oxygen species (ROS) production upon ID degradation. In mouse models, AGX51 treatment suppresses breast cancer colonization in the lung, regresses the growth of paclitaxel-resistant breast tumors when combined with paclitaxel and reduces tumor burden in sporadic colorectal neoplasia. Furthermore, in cells and mice, we fail to observe acquired resistance to AGX51 likely the result of the inability to mutate the binding pocket without loss of ID function and efficient degradation of the ID proteins. Thus, AGX51 is a first-in-class compound that antagonizes ID proteins, shows strong anti-tumor effects and may be further developed for the management of multiple cancers.
47	Case Report: Visceral Leishmaniasis and Hemophagocytic Lymphohistiocytosis: Three Clinical Cases, Three Different Pattern.	34029206	Visceral leishmaniasis (VL) is a neglected tropical disease with more than 30,000 cases annually reported worldwide. In Brazil, about 3,700 cases are annually reported. The VL clinical presentation is variable, from asymptomatic to severe cases with a high risk of death. We reported three cases of VL with clinical sign similarities but distinct development. All cases had bone marrow hemophagocytosis and hemophagocytic lymphohistiocytosis (HLH) criteria. HLH is a rare condition that may have secondary causes, including infectious and parasitic diseases, like VL. The delayed recognition of the secondary HLH (sHLH) association to VL may cause unfavorable outcomes and death.
48	Perspectives on a Way Forward to Implementation of Precision Medicine in Patients With Diabetic Kidney Disease; Results of a Stakeholder Consensus-Building Meeting.	34025424	Aim: This study aimed to identify from different stakeholders the benefits and obstacles of implementing precision medicine in diabetic kidney disease (DKD) and to build consensus about a way forward in order to treat, prevent, or even reverse this disease. Methods: As part of an ongoing effort of moving implementation of precision medicine in DKD forward, a two-day consensus-building meeting was organized with different stakeholders involved in drug development and patient care in DKD, including patients, patient representatives, pharmaceutical industry, regulatory agencies representatives, health technology assessors, healthcare professionals, basic scientists, and clinical academic researchers. The meeting consisted of plenary presentations and discussions, and small group break-out sessions. Discussion topics were based on a symposium, focus groups and literature search. Benefits, obstacles and potential solutions toward implementing precision medicine were discussed. Results from the break-out sessions were presented in plenary and formed the basis of a broad consensus discussion to reach final conclusions. Throughout the meeting, participants answered several statement and open-ended questions on their mobile device, using a real-time online survey tool. Answers to the statement questions were analyzed descriptively. Results of the open-ended survey questions, the break-out sessions and the consensus discussion were analyzed qualitatively. Results and conclusion: Seventy-one participants from 26 countries attended the consensus-building meeting in Amsterdam, April 2019. During the opening plenary on the first day, the participants agreed with the statement that precision medicine is the way forward in DKD (n = 57, median 90, IQR [75-100]). Lack of efficient tools for implementation in practice and generating robust data were identified as significant obstacles. The identified benefits, e.g., improvement of the benefit-risk ratio of treatment, offer substantive incentives to find solutions for the identified obstacles. Earlier and increased multi-stakeholder collaboration and specific training may provide solutions to alter clinical and regulatory guidelines that lie at the basis of both obstacles and solutions. At the end of the second day, the opinion of the participants toward precision medicine in DKD was somewhat more nuanced (n = 45, median 83, IQR [70-92]) and they concluded that precision medicine is an important way forward in improving the treatment of patients with DKD.
49	Enzyme replacement therapy and hematopoietic stem cell transplant: a new paradigm of treatment in Wolman disease.	34020687	Wolman disease is a rare, lysosomal storage disorder in which biallelic variants in the LIPA gene result in reduced or complete lack of lysosomal acid lipase. The accumulation of the substrates; cholesterol esters and triglycerides, significantly impacts cellular function. Untreated patients die within the first 12 months of life. Clinically, patients present severely malnourished, with diarrhoea and hepatosplenomegaly, many have an inflammatory phenotype, including with hemophagocytic lymphohistiocytosis (HLH). Hematopoietic stem cell transplant (HCT) had been historically the only treatment available but has a high procedure-related mortality because of disease progression and disease-associated morbidities. More recently, enzyme replacement therapy (ERT) with dietary substrate reduction (DSR) has significantly improved patient survival. However, ERT is life long, expensive and its utility is limited by anti-drug antibodies (ADA) and the need for central venous access.We describe five Wolman disease patients diagnosed in infancy that were treated at Royal Manchester Children's Hospital receiving ERT with DSR then HCT-multimodal therapy. In 3/5 an initial response to ERT was attenuated by ADA with associated clinical and laboratory features of deterioration. 1/5 developed anaphylaxis to ERT and the other patient died post HCT with ongoing HLH. All patients received allogeneic HCT. 4/5 patients are alive, and both disease phenotype and laboratory parameters are improved compared to when they were on ERT alone. The gastrointestinal symptoms are particularly improved after HCT, with reduced diarrhoea and vomiting. This allows gradual structured normalisation of diet with improved tolerance of dietary fat. Histologically there are reduced cholesterol clefts, fewer foamy macrophages and an improved villous structure. Disease biomarkers also show improvement with ERT, immunotherapy and HCT. Three patients have mixed chimerism after HCT, indicating a likely engraftment-defect in this condition.We describe combined ERT, DSR and HCT, multimodal treatment for Wolman disease. ERT and DSR stabilises the sick infant and reduces the formerly described prohibitively high, transplant-associated mortality in this condition. HCT abrogates the problems of ERT, namely attenuating ADA, the need for continuing venous access, and continuing high cost drug treatment. HCT also brings improved efficacy, particularly evident in improved gastrointestinal function and histology. Multimodal therapy should be considered a new paradigm of treatment for Wolman disease patients where there is an attenuated response to ERT, and for all patients where there is a well-matched transplant donor, in order to improve long term gut function, tolerance of a normal diet and quality of life.
50	Methods of Expression, Purification, and Preparation of the c-Myc b-HLH-LZ for Its Biophysical Characterization.	34019284	The b-HLH-LZ domain of c-Myc is a key target for the development of cancer therapies by blunting its binding to DNA with cell penetrant b-HLH-LZs and/or by stabilizing it into a state that cannot recognize Max to activate and amplify transcription of oncogenic genes. Although recent milestones have been reached with DNA binding blunting of c-Myc with the cell penetrant b-HLH-LZ Omomyc, the targeting of its b-HLH-LZ with small molecules, peptides, or proteins is lagging. As reviewed recently, the main problem relies in the intrinsically disordered nature of the b-HLH-LZ of c-Myc. This greatly complicates the classical approach of targeting a docking site with inhibitors. The solution state methods such as NMR are progressing towards the characterization of the ensembles of structures or states the b-HLH-LZ can adopt. However, the delicate balance that dictates the population of these dynamically interchanging states relies on its primary structure and the weak polar, electrostatic and hydrophobic interactions allowed. In this context, it is of the utmost importance to study the b-HLH-LZ of c-Myc in its WT background and avoid the use of tags such as His-tags. These tags could disrupt the balance of forces which could alter the conformational and physical transitions and states it can undergo and adopt. Here, we describe a robust protocol to express the WT b-HLH-LZ in E. coli and purify it, without the need of tags, to obtain the required quantities for solution state biophysical characterization such as NMR.
51	Secondary hemophagocytic lymphohystiocytosis in a Rubinstein Taybi syndrome patient.	34018455	Rubinstein-Taybi syndrome (RSTS) is an autosomal dominant disorder, caused by variants in CREBBP or EP300. Affected individuals present with distinctive craniofacial features, broad thumbs and/or halluces, intellectual disability and immunodeficiency. Here we report on one RSTS patient who experienced hemophagocytic lymphohystiocytosis (HLH) and disseminated herpes virus 1 ( HSV-1) disease. The clinical picture of RSTS is expanding to include autoinflammatory, autoimmune, and infectious complications. Prompt treatment of HLH and disseminated HSV-1 can lower the mortality rate of these life-threatening conditions.
52	Macrophage Activation Syndrome in a Case of Myasthenia Gravis with Concurrent Cytomegalovirus Infection.	34018170	Macrophage activation syndrome (MAS) or reactive hemophagocytic lymphohistiocytosis (HLH) refers to a set of clinical symptoms caused by the excessive activation and proliferation of macrophages. It was linked with autoimmune disease such as systemic-onset juvenile rheumatoid arthritis, systemic lupus erythematosus, rheumatoid arthritis, and dermatomyositis, etc. Herein we report a case of myasthenia gravis (MG) with concurrent cytomegalovirus (CMV) infection developed MAS.A 31-year-old female with history of MG for 2 years under stable control with azathioprine and prednisolone. She presented with persistent high fever for 2 weeks after an upper respiratory infection. Lab data revealed pancytopenia, elevated triglyceride, ferritin and C-reactive protein (CRP). A bone marrow aspiration confirmed hemophagocytosis. Investigation for occult infection revealed her plasma was positive for CMV IgG and IgM, and high for CMV viral load. She was then treated with 5 sessions of plasmapheresis and pulse steroid. Azathioprine was discontinued and replaced with cyclosporine. Gancylovir was given for her concurrent CMV infection. After 2 weeks of treatment, her fever gradually subsided, and her blood cell count, hepatobiliary enzymes, ferritin and CRP have returned to normal range. She was discharged in good recovery.MAS is a rare complication of systemic autoimmune disease with poor prognosis, which may be precipitated by concurrent infection. Early recognition of this syndrome and prompt immune modulation therapy is crucial for successful treatment.
53	Pediatric Hemophagocytic Lymphohistiocytosis - A Single Center Study of 118 Patients.	34016798	To describe the epidemiological features, outcomes and prognostic factors in diagnosis of pediatric hemophagocytic lymphohistiocytosis (HLH).Retrospective observational study. 118 children fulfilled the inclusion criteria for HLH between January, 2010 and December, 2019.Median age at diagnosis was 4 years (range13 days-15 years). Presenting features were fever (100%), hepatosplenomegaly (91%), neurological symptoms (23%), bicytopenia (76%), transaminitis (67.3%), increased full form (sIL-2r) (78%) and hemophagocytosis on bone marrow (75%). Median serum ferritin was 6504 ng/mL(44-297,000 ng/mL). Median follow up duration was 13.5 months (3 days to 102 months). Primary HLH was identified in 27 (23%) patients. Etiology of secondary HLH was infections 53 (45%), rheumatologic illnesses 21 (18%) and malignancies in 8 (6%) children. Treatment modalities were steroid only (25%), anti-infectious agent (58%), multi-agent chemotherapy (43%) and HSCT (40%); mortality among above treatment groups were 25%, 58%, 43% and 40% respectively. 15 patients (13%) had relapsed/refractory HLH who were treated with salvage chemotherapy and hematopoietic stem cell transplantation (HSCT). Mortality was significantly higher in patients with hyponatremia [OR (95% CI) 3.48 (1.35-8.99); P=0.008] hyperbilirubinemia [OR (95% CI) 2.0455 (0.88-4.75); P=0.002], coagulopathy [OR (95% CI) 2.92 (1.15-7.38); P=0.02] and sIL-2r ≥2400 U/mL [OR (95% CI) 9.05 (1.06-77.5); P=0.03).The overall mortality rate was 39 %; mortality within 30 days in 23%. Estimated overall survival (OS) and event free survival (EFS) at 3 years were 62% and 61%, respectively.Pediatric HLH is an aggressive disease with high mortality. Hyponatremia, hyperbilirubinemia, coagulopathy and increased sIL2 receptor level at diagnosis predicts poor outcome.
54	Gastric cancer complicated with hemophagocytic lymphohistiocytosis: case report and a brief review.	34012677	Hemophagocytic lymphohistiocytosis (HLH) comprises a group of severe immune function disorders that can lead to immune-mediated organ damage. There are two subtypes of HLH: primary and secondary. Secondary HLH is associated with infectious, oncologic, chemotherapeutic, and other underlying causes, and studies on HLH triggered by tumors have mainly focused on hematological malignancies. Secondary HLH in patients with solid tumors is rare. Here, we present two cases of gastric cancer complicated with HLH. The patient 1 was diagnosed as gastric cancer at stage I and got intractable fever after a distal subtotal gastrectomy without any evidence of infections or other complications. The patient 2 suffered from unresectable gastric adenocarcinoma and got fever, hemorrhagic rashes, and petechiae in mouth after six cycles of neoadjuvant chemotherapy. After detailed and comprehensive examinations, HLH was diagnosed in the two patients according to 2004 HLH diagnostic criteria, and the patients received treatment including immunosuppressive agents immediately. After therapy, the two patients showed partial remission, but both eventually died due to HLH relapse or progression of the primary tumor. The treatment regimen for HLH is intricate, and only a few relevant studies have focused on the treatment of cancer patients with HLH. The high mortality associated with this disease calls for more attention and additional research to improve the prognosis for these patients.
55	Analysis of the occurrence of hemophagocytic lymphohistiocytosis (HLH) features in patients with sepsis: a prospective study.	34006913	HLH syndrome may mimic sepsis but requires entirely different treatment. The aim of the study was to assess the occurrence of HLH features in patients with sepsis and the influence these exert on the patients' prognosis. The prospective study included 108 patients with suspected sepsis who were routinely evaluated according to HLH criteria. They were divided into group I (SOFA = 2, n = 57) and group II (SOFA ≥ 3, n = 51). Four patients were excluded from analysis: 1 with real HLH, 2 with Still's disease and 1 with lymphoma. The median (IQR) concentration of ferritin was 613.4 (850.3) ng/mL, however 6 patients revealed a remarkedly high ferritin concentration > 3000 ng/mL, including 2 with ferritin > 10,000 ng/mL. In total, 21 patients met ≥ 4/8 HLH criteria and were found to have sepsis with HLH-like syndrome (SHLS). Out of these, 19 responded to antimicrobials, 2 died due to infection. The sepsis patients presented with the following HLH criteria: fever (95.2%), hyperferritinemia (57.3%), splenomegaly (43.4%), reduced NK cell activity (35.2%), high sCD25 activity (27.4%) and rarely: hypertriglyceridemia (14.4%), duopenia (5.8%), hypofibrinogenemia (1.9%). Although group II patients had higher odds for SHLS presentation (OR 3.26, p = 0.026) and for death (OR 14.3, p = 0.013), SHLS occurrence had no impact on the risk of death (OR 0.77, p = 0.75). Sepsis patients can present with SHLS exclusively due to severe infection. Duopenia, hypertriglyceridemia, hypofibrinogenemia and high level of sCD25 are unusual in sepsis and might indicate real HLH syndrome. Hyperferritinemia, even as high as in real HLH syndrome, can occur in sepsis patients.
56	Reactions Related to CAR-T Cell Therapy.	33995389	The application of chimeric antigen receptor (CAR) T-cell therapy as a tumor immunotherapy has received great interest in recent years. This therapeutic approach has been used to treat hematological malignancies solid tumors. However, it is associated with adverse reactions such as, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), off-target effects, anaphylaxis, infections associated with CAR-T-cell infusion (CTI), tumor lysis syndrome (TLS), B-cell dysplasia, hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) and coagulation disorders. These adverse reactions can be life-threatening, and thus they should be identified early and treated effectively. In this paper, we review the adverse reactions associated with CAR-T cells, the mechanisms driving such adverse reactions, and strategies to subvert them. This review will provide important reference data to guide clinical application of CAR-T cell therapy.
57	Hemophagocytic lymphohistiocytosis associated with Epstein-Barr virus-positive diffuse large B-cell lymphoma, NOS of bone marrow-liver-spleen type: an autopsy case report.	33994430	Lymphoma-associated hemophagocytic lymphohistiocytosis (HLH) has a significantly poor prognosis among secondary HLH. We describe the rare case of a 74-year-old female with secondary HLH presenting with a rapidly fatal course. Post-mortem examination revealed Epstein-Barr virus (EBV) -positive diffuse large B-cell lymphoma (DLBCL). We were unable to make a definite antemortem diagnosis because the patient did not exhibit lymphadenopathy and bone marrow biopsy demonstrated hemophagocytosis without evidence of lymphoma. She died of multiple organ failure on the twelfth day of hospitalization despite a temporary response to steroids. Autopsy revealed diffuse lymphoma cell infiltration of the bone marrow, liver and spleen, suggesting "bone marrow-liver-spleen" (BLS)-type large B-cell lymphoma (LBCL). BLS-type LBCL is a rare and clinically aggressive lymphoma, usually associated with fever, cytopenia and HLH. The disease has a high mortality rate due to the delay in diagnosis and a highly aggressive clinical course. Further studies are required to improve our understanding of this rare extranodal DLBCL.
58	Prostate Cancer and Reactive Haemophagocytic Lymphohistiocytosis.	33987121	We report the case of a 76-year-old man presenting with reactive haemophagocytic lymphohistiocytosis (rHLH) in the setting of disseminated prostate cancer. This often fatal syndrome must be diagnosed early in order to maximize survival. Treatment should be initiated whenever the clinical diagnosis is suspected, even if the HLH-2004 criteria are not met. The HScore is a useful diagnostic tool for rHLH. In case of neurological symptoms, an extensive assessment must be performed. The goal of this case report is to raise awareness of this rare syndrome among oncologists.The association of prostate cancer and reactive haemophagocytic lymphohistiocytosis (rHLH) has rarely been described.This often fatal syndrome must be recognized early in order to start specific treatment and maximize survival.Specific treatment for rHLH must be accompanied by treatment of the triggering factors.
59	Lenalidomide-Associated Hemophagocytic Lymphohistiocytosis With Plasma Cell Phagocytosis.	33987058	Hemophagocytic lymphohistiocytosis (HLH) is a severe systemic inflammatory syndrome that is often fatal. In the adult population, it is believed to develop secondary to immune dysregulation due to rheumatologic, infectious, malignant, and recently, immunomodulatory drugs. It's co-occurrence with phagocytosis by non-macrophage cells has not been previously well defined. We present a case of lenalidomide-associated HLH with concurrent plasma cell hemophagocytosis in a patient with controlled multiple myeloma (MM).
60	Macrophage Activation in COVID-19 Patients in Intensive Care Unit.	33984075	We report six cases of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, admitted to intensive care unit (ICU), for whom bone marrow aspirate revealed hemophagocytosis. We compared their clinical presentation and laboratory findings to those that can be encountered during a hemophagocytic lymphohistiocytosis. These observations might evoke a macrophage activation mechanism different from the one encountered in the hemophagocytic lymphohistiocytosis (HLH).
61	NHR-49/PPAR-α and HLH-30/TFEB cooperate for C. elegans host defense via a flavin-containing monooxygenase.	33978570	The model organism Caenorhabditis elegans mounts transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, the regulation of which is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature, which was almost completely abrogated by deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Moreover, fmo-2/FMO5 induction required the nuclear hormone receptor, NHR-49/PPAR-α, which controlled host defense cell non-autonomously. These findings reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal FMOs as important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPAR-α during S. aureus infection, with implications for host defense and inflammation in higher organisms.
62	Clinical analysis and a novel risk predictive nomogram for 155 adult patients with hemophagocytic lymphohistiocytosis.	33977332	Recently, more and more attention has been paid on adult hemophagocytic lymphohistiocytosis (HLH), a disease with complicated symptoms and high mortality. In order to analyze the clinical characteristics and prediction risk factors of mortality, we designed a retrospective study with 1-year follow-up and included 155 patients admitted to Tongji Hospital diagnosed as HLH. One hundred seven patients formed the training cohort for nomogram development, and 48 patients formed the validation cohort to confirm the model's performance. All patients' clinical characteristics, laboratory results, medical records, and prognosis were analyzed. Among all the 107 patients in the training cohort, 46 were male and 61 were female, with the median age of 49.0 (IQR 31.0-63.0). The 1-year mortality rate was 43.9% (47/107) and 45.8% (22/48) in the training and validation cohort, respectively. And further multivariate logistic regression analysis in the training cohort showed that male (odds ratio 5.534, 95% CI 1.507-20.318, p = 0.010), altered mental status (11.876, 1.882-74.947, p = 0.008), serum ferritin ≥ 31,381 μg/L (8.273, 1.855-36.883, p = 0.006), and IL-6 ≥ 18.59 pg/mL (19.446, 1.527-247.642, p = 0.022) were independent risk factor of mortality. A nomogram included the four prediction factors mentioned above was also tabled to help clinicians evaluate the probability of poor outcome. Area under the receiver operating characteristic curve (AUROC) analysis, calibration curves, and decision curve analysis (DCA) certify the accuracy and the clinical usefulness of the nomogram. Our research reveals that male, altered mental status, serum ferritin ≥ 31,381 µg/L, and IL-6 ≥ 18.59 pg/mL are four independent predictors for poor prognosis. Doctors should pay more attention to patients with altered mental status, high serum ferritin, and IL-6 level, who have a higher risk of death.
63	Therapeutic administration of etoposide coincides with reduced systemic HMGB1 levels in macrophage activation syndrome.	33975537	Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. HMGB1 is a nuclear protein released extracellularly during proinflammatory lytic cell death or secreted by activated macrophages, NK cells, and additional cell types during infection or sterile injury. Extracellular HMGB1 orchestrates central events in inflammation as a prototype alarmin. TLR4 and the receptor for advanced glycation end products operate as key HMGB1 receptors to mediate inflammation.Standard ELISA and cytometric bead array-based methods were used to examine the kinetic pattern for systemic release of HMGB1, ferritin, IL-18, IFN-γ, and MCP-1 before and during treatment of four children with critical MAS. Three of the patients with severe underlying systemic rheumatic diseases were treated with biologics including tocilizumab or anakinra when MAS developed. All patients required intensive care therapy due to life-threatening illness. Add-on etoposide therapy was administered due to insufficient clinical response with standard treatment. Etoposide promotes apoptotic rather than proinflammatory lytic cell death, conceivably ameliorating subsequent systemic inflammation.This therapeutic intervention brought disease control coinciding with a decline of the increased systemic HMGB1, IFN-γ, IL-18, and ferritin levels whereas MCP-1 levels evolved independently.Systemic HMGB1 levels in MAS have not been reported before. Our results suggest that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of HMGB1 antagonists. They also advocate therapeutic etoposide administration in severe MAS and provide a possible biological explanation for its mode of action.
64	An autopsy series of an oft-missed ante-mortem diagnosis: hemophagocytic lymphohistiocytosis.	33968822	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal syndrome resulting from a hyperactivated immune system. Diverse patient profiles and clinical presentations often result in misdiagnosis. This article describes the varied clinical presentations and autopsy findings in three patients with this entity. The etiopathogenesis of HLH, its disparate and confounding clinical features, the diagnostic criteria, and management principles are also briefly reviewed.
65	Graft-versus-host disease after liver transplantation is associated with bone marrow failure, hemophagocytosis, and DNMT3A mutations.	33961341	Graft-versus-host disease after liver transplantation (LT-GVHD) is rare, frequently fatal, and associated with bone marrow failure (BMF), cytopenias, and hyperferritinemia. Given hyperferritinemia and cytopenias are present in hemophagocytic lymphohistiocytosis (HLH), and somatic mutations in hematopoietic cells are associated with hyperinflammatory responses (clonal hematopoiesis of indeterminate potential, CHIP), we identified the frequency of hemophagocytosis and CHIP mutations in LT-GVHD. We reviewed bone marrow aspirates and biopsies, quantified blood/marrow chimerism, and performed next-generation sequencing (NGS) with a targeted panel of genes relevant to myeloid malignancies, CHIP, and BMF. Twelve marrows were reviewed from 9 LT-GVHD patients. Ten aspirates were evaluable for hemophagocytosis; 7 had adequate DNA for NGS. NGS was also performed on marrow from an LT cohort (n=6) without GVHD. Nine of 10 aspirates in LT-GVHD patients showed increased hemophagocytosis. Five (71%) of 7 with LT-GVHD had DNMT3A mutations; only 1 of 6 in the non-GVHD LT cohort demonstrated DNMT3A mutation (p=0.04). Only 1 LT-GVHD patient survived. BMF with HLH features was associated with poor hematopoietic recovery, and DNMT3A mutations were over-represented, in LT-GVHD patients. Identification of HLH features may guide prognosis and therapeutics. Further studies are needed to clarify the origin and impact of CHIP mutations on the hyperinflammatory state.
66	Epstein-Barr virus induced haemophagocytic lymphohistiocytosis.	33952568	Haemophagocytic lymphohistiocytosis (HLH) is a rare condition of uncontrolled immune activation as a result of an inherited genetic defect or in response to malignancy, autoimmune disease, rheumatological disease, AIDS infection or post-transplant immunosuppression. Described here is the case of a 19-year-old Caucasian man who presented with complaints of worsening fever, new-onset jaundice and lethargy after failing treatment for suspected infectious mononucleosis. Physical examination was significant for fever and splenomegaly while laboratory results revealed transaminitis, cytopaenia, indirect hyperbilirubinaemia and elevated ferritin, raising the likelihood of both autoimmune haemolytic anaemia and HLH. He tested positive for Epstein-Barr virus (EBV), and bone marrow biopsy revealed hypercellular marrow with haemophagocytosis and no evidence of malignancy. High dose steroids were initiated with significant improvement in haemoglobin, resulting in a final diagnosis of HLH secondary to acute EBV infection. The patient was discharged on continued high-dose prednisone with planned taper and consideration of outpatient rituximab therapy for 4 weeks. High clinical suspicion and prompt evaluation were critical to early treatment and decreased morbidity.
67	Secondary hemophagocytic lymphohistiocytosis: a case report.	33944851	Hemophagocytic lymphohistiocytosis (HLH) is a rare hematologic syndrome presenting either as an inherited life-threatening inflammatory disorder in children or as a secondary disease in adults. Inherited HLH involves inborn defects in lymphocytes and includes autosomal recessive and X-linked disorders characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. Secondary or acquired HLH occurs in the settings of infections, systemic connective tissue disease and lymphoid malignancies, possibly due to underlying genetic predisposition to develop HLH. The mechanisms leading to secondary HLH have yet to be fully determined and the disease remains frequently undiagnosed and thereby untreated. Herewith we report the case of an 83-year old Caucasian male who referred to our Division of Internal Medicine and Nephrology due to marked asthenia associated with fever, mental confusion, drowsiness and hyporexia, who was ultimately diagnosed with HLH secondary to anaplastic B cell lymphoma. This case report illustrates the difficulties in the diagnostic workup of HLH, mainly related to early identification of the underlying disease and rapid instauration of appropriate therapy.
68	Hemophagocytic lymphohistiocytosis presented with fever of unknown origin: A case study and literature review.	33936693	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening clinical syndrome, which may present with FUO. The possible diagnosis of HLH must be considered in the differential diagnosis when a patient presents with FUO.
69	Current status of the diagnosis and treatment of hemophagocytic lymphohistiocytosis in adults.	33935031	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of defective apoptosis, a disruption of the regulatory pathway that terminates immune and inflammatory responses. Fever, cytopenia, splenomegaly, and/or hemophagocytosis are typical findings of this syndrome. HLH can be induced by genetic disorders (familial) or secondary causes. Familial HLH is rare, while secondary causes in adults include infection, autoimmunity, and malignancy. HLH in adults tends to be confused with or misdiagnosed as sepsis, mainly due to similar clinical manifestations and laboratory findings, which make it difficult to diagnose HLH rapidly and adopt immunosuppressive agents and/or chemotherapy adequately. Treatment of pediatric HLH using HLH-2004 or multi-agent chemotherapy can be applied in adult patients, although the dose and type of drug need to be adjusted. It is highly recommended that allogenic hematopoietic stem cell transplantation should be used in patients who become reactivated or are refractory to the initial treatment as soon as possible to improve survival. Future clinical trials are warranted to determine more suitable treatments for adult patients with HLH.
70	Hyperinflammation: On the pathogenesis and treatment of macrophage activation syndrome.	33934408	Macrophage activation syndrome (MAS) is a subtype of hemophagocytic lymphohistiocytosis (HLH) diseases. The underlying mechanism of these life-threatening disorders is impaired granule-mediated cytotoxicity exerted by natural killer (NK) cells and T lymphocytes. This function is meant for elimination of virus-infected cells, malignant cells and to prevent exaggerated immune responses. The normal outcome after an attack by NK or cytotoxic T cells is apoptosis of the target cell. This prevents cytotoxic inflammatory responses in adjacent tissues which occur after lytic cell death. Extensive cell lysis can even produce a cytokine storm, as evidenced in MAS. Programmed proinflammatory lytic cell death, pyroptosis, caused by activated inflammasomes is central in the pathogenesis of MAS. Pyroptosis mediates IL-18 cytokine release, which robustly stimulates NK and T cells to produce IFN-γ, the key macrophage-activating signal which initiates a burst of inflammatory cytokines and chemokines. Lytic cell death also mediates a discharge of the prototype alarmin high mobility group box protein 1 (HMGB1), a proinflammatory molecule present in all cells and that mediates the pathogenesis of MAS as outlined here. Therapeutic options to control causal factors operating in the pathogenesis of MAS are also discussed.
71	Difficult Balance Between EBV Treatment and Posttransplant Immunosuppression: A Successful Transplant in a Child With Recurrent Epstein-Barr Virus-Induced Hemophagocytic Lymphohistiocytosis.	33933286	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening clinical syndrome. HLH can be classified into 2 major forms: primary and secondary. Viral infections are frequently implicated in the onset of active HLH episodes. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for primary HLH and refractory/relapsed HLH after proper chemoimmunotherapy, although following immunosuppressive therapy may lead to infectious complications, including viral infections.We report a case of a 6-year-old boy with Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis. The patient underwent an allo-HSCT from a 10/10 HLA-matched unrelated donor. Because he received myeloablative and immunosuppressive treatment, another EBV reactivation occurred, as well as cytomegalovirus (CMV) reactivation. After antiviral therapy, on day +27, elimination of EBV and CMV was achieved. Repeated chimerism tests evaluated decreasing donor chimerism; graft-versus-host disease prophylaxis was reduced from day +32 and eventually withdrawn. Later on, the patient developed acute graft-versus-host disease (skin rush, gastrointestinal dysfunction). Immunosuppressive agents (methylprednisolone, cyclosporine) were applied once again, which led to an increase of CMV viremia and polyomavirus (BK virus) primary infection.Virus infection can induct a severe disorder, such as HLH, and recur after its treatment. We believe our case represents dynamic changes in immunologic reaction to viral infection, which depend on modifications in treatment after allo-HSCT. These observations underscore the importance and difficulty of balancing immunosuppressive therapy and infection control.
72	Ferulic Acid Supplementation Increases Lifespan and Stress Resistance via Insulin/IGF-1 Signaling Pathway in C. elegans.	33924155	Ferulic acid (FA) is a naturally-occurring well-known potent antioxidant and free radical scavenger. FA supplementation is an effective strategy to delay aging, but the underlying mechanism remains unknown. In the present study, we examined the effects of FA on lifespan extension and its mechanism of FA in Caenorhabditis elegans (C. elegans). Results suggested that FA increased the lifespan of C. elegans, rather than altering the growth of E. coli OP50. Meanwhile, FA promoted the healthspan of C. elegans by improving locomotion and reducing fat accumulation and polyQ aggregation. FA increased the resistance to heat and oxidative stress through reducing ROS. The upregulating of the expression of the hlh-30, skn-1, and hsf-1 were involved in the FA-mediated lifespan extension. Furthermore, FA treatment had no impact on the lifespan of daf-2, hlh-30, skn-1, and hsf-1 mutants, confirming that insulin/IGF-1 signaling pathway and multiple longevity mechanisms were associated with the longevity mechanism of FA. We further found that mitochondrial signaling pathway was modulation involved in FA-mediated lifespan extension. With the results from RNA-seq results and mutants lifespan assay. These findings contribute to our knowledge of the lifespan extension and underlying mechanism of action of FA in C. elegans.
73	HLH or sepsis: the truth is in the T cells.	33914076	NaN
74	Mortal Interaction Between Hemophagocytic Syndrome and Newly Developed Heart Failure.	3390976533909766	Hemophagocytic syndrome (HPS) ia s devastating hyperinflammatory syndrome. Heart failure (HF) with preserved ejection fraction (HFpEF) status is closely correlated with increased inflammation, both systemic and intramyocardial.This study sought to determine mortality predictors and reliable follow-up parameters in HPS that developed HFpEF during the clinical course.Thirty-nine patients, diagnosed as HPS, according to HLH 2004 diagnostic criteria, with an HScore of ≥169 and proven bone marrow aspiration or biopsy, were recruited retrospectively. Both traditional, serum C-reactive protein, albumin and ferritin levels with lymphocyte, and platelet counts, as well as non-traditional risk factors, neutrophil-to-lymphocyte count (NLR), monocyte-to-lymphocyte count (MLR), mean platelet volume (MPV), and N-Terminal pro-brain natriuretic peptide (NTproBNP), were investigated retrospectively. The relationship between time-changed laboratory values both among themselves and with mortality. The overall significance level was set at 5%.This study showed that temporal change of cardiothoracic ratio (CTR), serum NTproBNP, ferritin, CRP, and albumin levels were detected as mortality predictors (p<0.05, for all) in the univariate analysis. Lymphocyte and platelet counts with NLR and MPV values were also significant (p<0.05). The relationship between NT-proBNP and increased systemic inflammatory markers proved to be significant. In addition to traditional risk factors, serum ferritin levels, NLR, MLR, and MPV levels also proved to be significantly correlated with each other.Accompanied by reliable follow-up parameters, rapid diagnosis and aggressive anti-inflammatory treatment with tight volume control can be life-saving in HPS patients who suffer from HFpEF. Close monitoring of inflammation may predict the outcome of patients suffering from HFpEF.A síndrome hemofagocítica (SHF) é uma síndrome hiperinflamatória debilitante. O status da insuficiência cardíaca (IC) com fração de ejeção preservada (ICFEP) está intimamente relacionado ao aumento da inflamação sistêmica e intramiocárdica.este estudo pretende determinar os preditores de mortalidade e os parâmetros de monitoramento confiáveis nos casos de SHF que desenvolveram a ICFEP durante seu curso clínico.Trinta e nove pacientes, diagnosticados com SHF de acordo com os critérios diagnósticos do estudo HLH 2004 com Hscore ≥169, e com aspiração ou biópsia de medula óssea comprovada, foram recrutados retrospectivamente. Foram investigados retrospectivamente os fatores de risco tradicionais, como proteína C reativa sérica, níveis de albumina e ferritina com contagens de linfócitos e plaquetas, e fatores não tradicionais, como relação neutrófilolinfócito (NLR), relação linfócito-monócito (MLR), volume plaquetário médio (MPV) e pró-peptídeo natriurético cerebral N-terminal (NTproBNP). Analisou-se a relação entre os valores laboratoriais alterados ao longo do tempo entre si e com a mortalidade. O nível de significância geral foi de 5%.Foi demonstrado que a alteração temporal dos níveis de índice cardiotorácico (ICT), NTproBNP sérico, ferritina, PCR e albumina foram detectados como sendo preditores de mortalidade (p<0,05, para todos) em análise univariada. As contagens de linfócitos e plaquetas com valores de NLR e MPV também foram significativos (p<0,05). A relação entre NT-proBNP e o aumento dos marcadores inflamatórios sistêmicos também foi considerada significativa. Além de fatores de risco tradicionais, os níveis de ferritina sérica, e os níveis de NLR, MLR e MPV foram considerados significativamente correlacionados entre si.Acompanhado de parâmetros de monitoramento confiáveis, o diagnóstico rápido e o tratamento antiinflamatório agressivo com controle rígido de volume podem salvar vidas de pacientes com SHF que sofrem de complicações por ICFEP. O monitoramento rígido da inflamação pode prever o resultado do paciente que sofre de ICFEP.
75	Hemophagocytic lymphohistiocytosis, a new cause of death during 'post-acute COVID-19 syndrome?' A case report.	33897909	'Post-acute coronavirus disease 2019 (COVID-19) syndrome' is a new term that was coined to describe a constellation of persistent symptoms and new complications following recovery from COVID-19 initial illness. Identifying post-COVID-19 complications is crucial for early diagnosis and subsequent initiation of therapies to ensure more favorable outcomes. Severe COVID-19-related hemophagocytic lymphohistiocytosis (HLH) has been previously described during the acute phase of the infection. It is characterized by hyperinflammation, multi-organ involvement, and hemophagocytosis. We report the case of a 69-year-old woman who presented, two weeks after recovery from COVID-19, with hyperosmolar state and high inflammatory markers. Bone marrow aspirate, flow cytometry, extensive viral panel and total body scan were performed. Review of bone marrow aspirate smear showed hemophagocytosis. Flow cytometry, viral panel, and total body scan were normal. The diagnosis of post COVID-19-related HLH was made. Unfortunately, the patient died despite adequate treatment. To our knowledge, this is the first case establishing a high probability of COVID-19-related HLH given its occurrence two weeks following a documented biological and clinical recovery. Awareness and early recognition of HLH features following COVID-19 recovery should prompt timely initiation of therapy.
76	Comment on: Gudu T, Stober C, Cope AP et al. Baricitinib set to join the Covid-19 therapeutic arsenal? Rheumatology (Oxford). 2021;60:1585-1587.	33895812	NaN
77	Transgenic mice overexpressing the LH receptor in the female reproductive system spontaneously develop endometrial tumour masses.	33893331	The receptor for the luteinizing hormone (LH-R) is aberrantly over expressed in cancers of the reproductive system. To uncover whether LH-R over expression has a causative role in cancer, we generated a transgenic (TG) mouse which overexpresses the human LH-R (hLH-R) in the female reproductive tract, under the control of the oviduct-specific glycoprotein (OGP) mouse promoter (mogp-1). The transgene was highly expressed in the uterus, ovary and liver, but only in the uterus morphological and molecular alterations (increased proliferation and trans-differentiation in the endometrial layer) were detected. A transcriptomic analysis on the uteri of young TG mice showed an up regulation of genes involved in cell cycle control and a down regulation of genes related to the immune system and the metabolism of xenobiotics. Aged TG females developed tumor masses in the uteri, which resembled an Endometrial Cancer (EC). Microarray and immunohistochemistry data indicated the deregulation of signaling pathways which are known to be altered in human ECs. The analysis of a cohort of 126 human ECs showed that LH-R overexpression is associated with early-stage tumors. Overall, our data led support to conclude that LH-R overexpression may directly contribute to trigger the neoplastic transformation of the endometrium.
78	Clinical Management of Relapsed/Refractory Hemophagocytic Lymphohistiocytosis in Adult Patients: A Review of Current Strategies and Emerging Therapies.	33888986	Haemophagocytic lymphohistiocytosis (HLH) is a severe disorder with high mortality. The aim of this review is to update clinical management of relapsed/refractory HLH in adults, with a focus on current and new therapies.We searched relevant articles in Embase and PUBMED with the MESH term "hemophagocytic lymphohistiocytosis; refractory; relapsing; adult."One hundred eight papers were found; of these, 22 were retained for this review. The treatment of HLH in adult is based on the HLH-94 regimen. The response rate is lower than in pediatric patients, and 20-30% are refractory to this therapy. DEP regimen and allogenic hematopoietic stem cell transplantation (HSCT) are associated with complete response and partial response in 27% and 49.2%, respectively. However, many patients fail to achieve a stable condition before HSCT, and mortality is higher in them. New drugs have been developed, such as emapalumab, ruxolitinib, and alemtuzumab, and they may be used as bridges to the curative HSCT. They are relatively well tolerated and have few or mild side effects. With these agents, the rate of partial response ranges from 14.2% to 100%, while the rate of complete response is highly variable according to study and medication used. The number of patients who achieved HSCT ranged from 44.8% to 77%, with a survival rate of 55.9% to 100%. However, the populations in these studies are mainly composed of mixed-age patients (pediatric and adult patients), and studies including only adult patients are scarce.Relapsed or refractory HLH in adult patients is associated with poor outcome, and consolidation with HSCT may be required in some cases. Mortality related to HSCT is mainly due to active HLH disease before HSCT and post HSCT complications. New drugs, such as empalumab, ruxolitinib, and alemtuzumab are interesting since these agents may be used as bridges to HSCT with increases in the numbers of patients proceeding to HSCT and survival rate.
79	Treatment of Severe Hypertriglyceridemia With Insulin Infusions in Severe COVID-19: A Case Series.	33882724	Rapid onset of severe hypertriglyceridemia was quickly recognized in critical COVID-19 patients. Associated causes have been due to secondary hemophagocytic lymphohystiocytosis (HLH) syndrome, medication-induced, or acute liver failure. Statins, omega-3 polyunsaturated acids, niacin, and fibrates are common oral lipid lowering therapy options in patients at risk for hypertriglyceridemia. The severity of hypertriglyceridemia in COVID-19 patients with triglyceride values reaching greater than 1,000 mg/dL put them at a heightened risk of pancreatitis and therefore an essential need to acutely lower their levels. We present a case series of 5 patients who achieved rapid triglyceride lowering through continuous insulin infusion therapy.A retrospective chart review of 48 critical COVID-19 patients who were admitted from March 22 to April 15, 2020 was conducted. Inclusion criteria consisted of mechanical ventilation and continuous insulin infusion to treat severe hypertriglyceridemia resulting with 5 eligible patients in this case report.In addition to standard oral lipid lowering therapies, continuous insulin infusion successfully treated severe hypertriglyceridemia in critically ill COVID-19 patients. None of the patients experienced pancreatitis or hypoglycemia necessitating cessation of insulin. Further studies are needed to show the optimum dose and duration of insulin infusion as monotherapy and in combination with oral therapies.
80	Primary varicella-zoster virus infection of the immunocompromised associated with acute pancreatitis and hemophagocytic lymphohistiocytosis: A case report.	33879665	Primary varicella-zoster virus (VZV) infection may be associated with hemophagocytic lymphohistiocytosis (HLH), as well as with acute pancreatitis. However, there is few data concerning the evolution and the optimal treatment of these rare associations.A 57-year-old immunocompromised woman, who was treated for chronic lymphocytic leukemia 3 years prior to admission, was hospitalized with abdominal pain revealing severe acute pancreatitis. The day after admission, a pruritic rash appeared on her face, trunk, and limbs, sparing the palmoplantar regions. At the same time, fever, thrombocytopenia (27 × 109/L), major hyperferritinemia (11,063 μg/mL), hypertriglyceridemia (2.56 mmol/L) and elevated lactate dehydrogenase levels (1441 IU/L) suggested HLH.The diagnosis of chickenpox (varicella) was established. Primary VZV infection was then confirmed: cutaneous and plasma VZV polymerase chain reactions were positives, VZV serology was negative for IgG.Treatment with aciclovir was started intravenously after the onset of the rash, for a total of 10 days. A 48-h surveillance in intensive care was carried out.Acute pancreatitis and biological abnormalities evolved favorably under aciclovir. Platelet count was normalized 6 days after admission to hospital.A favorable outcome of primary VZV infection associated with severe acute pancreatitis and probable HLH in an immunocompromised patient is possible with aciclovir alone.
81	Ehrlichia-induced hemophagocytic lymphohistiocytosis after autologous stem cell transplant.	33877729	Hemophagocytic lymphohistiocytosis (HLH) causes multiorgan failure due to the release of multiple cytokines mediating widespread inflammation. We present a patient with multiple myeloma on maintenance chemotherapy with the anti-CD38 monoclonal antibody daratumumab after autologous stem cell transplant (ASCT) who developed fatal HLH secondary to Ehrlichiosis.
82	Multisystem inflammation and susceptibility to viral infections in human ZNFX1 deficiency.	33872655	Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known.We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease.Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids.Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes.ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease.
83	Adult onset type 2 familial hemophagocytic lymphohistiocytosis with PRF1 c.65delC/c.163C>T compound heterozygous mutations: A case report.	33869605	Familial hemophagocytic lymphohistiocytosis (FHL) is a primary immunodefici-ency disease caused by gene defects. The onset of FHL in adolescents and adults may lead clinicians to ignore or even misdiagnose the disease. To the best of our knowledge, this is the first report to detail the clinical features of type 2 FHL (FHL2) with compound heterozygous perforin (PRF1) defects involving the c.163C>T mutation, in addition to correlation analysis and a literature review.We report a case of a 27-year-old male patient with FHL2, who was admitted with a persistent fever and pancytopenia. Through next-generation sequencing technology of hemophagocytic lymphohistiocytosis (HLH)-related genes, we found compound heterozygous mutations of PRF1: c.65delC (p.Pro22Argfs*29) (frameshift mutation, paternal) and c.163C>T (p.Arg55Cys) (missense mutation, maternal). Although he did not receive hematopoietic stem cell transplantation, the patient achieved complete remission after receiving HLH-2004 treatment protocol. To date, the patient has stopped taking drugs for 15 mo, is in a stable condition, and is under follow-up observation.The delayed onset of FHL2 may be related to the PRF1 mutation type, pathogenic variation pattern, triggering factors, and the temperature sensitivity of some PRF1 mutations. For individual, the detailed reason for the delay in the onset of FHL warrants further investigation.
84	Performance of HScore in Reactive Hemophagocytic Lymphohistiocytosis.	33867732	Hemophagocytic lymphohistiocytosis (HLH) is a life threatening systemic hyperinflammatory disorder that leads to multiple organ damage. The most widely used diagnostic guidelines are the HLH 2004 guidelines proposed by the Histiocyte society. Recently, Fardet et al. has proposed a Scoring system, "HScore" for diagnosis of reactive HLH. To test the performance of "HScore" in diagnosis of reactive HLH and compare the credibility of the same with the existing gold standard 2004 guidelines in a cohort of Indian patients. This was a descriptive study of cases referred to the department of Pathology for bone marrow examination with suspicion or diagnosis of HLH from Jan 2015 to June 2017. The clinical records of these patients were analysed and diagnosed as positive and negative for HLH using the HLH 2004 guidelines and HScore, the scoring system developed by Fardet et al. Fifty cases fulfilled the inclusion criteria. The variables fever, hepato and /or splenomegaly, high triglyceride levels and marrow hemophagocytosis are highly significant to predict an individuals risk of HLH. We propose a cut-off of ≥ 166 for the diagnosis of HLH by using HScore. If 4 of the 7 criteria are fulfilled by HLH 2004 guidelines, there is an 80% probability of having HLH by HScore. The HScore is less restrictive in confirming a diagnosis of HLH compared to the HLH 2004 guidelines. HScore is a simple, and cost effective tool for diagnosis of reactive HLH. The higher the HScore, more is the probability of HLH.
85	Germline variants in UNC13D and AP3B1 are enriched in COVID-19 patients experiencing severe cytokine storms.	33867526	Critically ill coronavirus disease 2019 (COVID-19) is characterized by severe cytokine storms, a hyperinflammatory condition intimately related to the development of fatal outcomes. Why some individuals seem particularly vulnerable to severe cytokine storms is still unknown. Primary immunodeficiency (PID)-related genes are inherited factors that dysregulate host inflammatory responses to infection, especially hemophagocytic lymphohistiocytosis (HLH)-related genes, established as contributors to the development of excessive cytokine storms. We analyzed the association between PID gene variants with severe cytokine storms in COVID-19. We conducted whole-exome sequencing in 233 hospitalized COVID-19 patients and identified four PID gene (UNC13D, AP3B1, RNF168, DHX58) variants were significantly enriched in COVID-19 patients experiencing severe cytokine storms. The total percentage of COVID-19 patients with variants in UNC13D or AP3B1, two typical HLH genes, was dramatically higher in high-level cytokine group than in low-level group (33.3 vs. 5.7%, P < 0.001). Germline variants in UNC13D and AP3B1 were associated with the development of severe cytokine storms, fatal outcomes in COVID-19. These findings advance the understanding of individual susceptibility to severe cytokine storms and help optimize the current management of COVID-19.
86	Investigation of MRI-based radiomics model in differentiation between sinonasal primary lymphomas and squamous cell carcinomas.	33860416	To develop and validate an MRI-based radiomics model in differentiation between sinonasal primary lymphomas and squamous cell carcinomas (SCCs).One-hundred-and-fifty-four patients were enrolled (74 individuals with SCCs and 80 with lymphomas). After feature analysis and feature selection with variance threshold and least absolute shrinkage and selection operator (LASSO) methods, an MRI-based radiomics model with the support vector machine (SVM) classifier was constructed in differentiation between lymphomas and SCCs. Areas under the receiver operating characteristic curves (AUCs) of the MRI-based radiomics model were compared with those of radiologists using Delong test.Five features (T1 original shape Compactness2, T1 wavelet-HHH first-order Total Energy, T2 wavelet-HLH GLCM Informational Measure of Correlation1, T1 wavelet-LHL GLCM Inverse Variance and T1 square GLRLM Long Run Low Gray Level Emphasis) were finally selected in the radiomics model. The AUC values in differentiation between lymphomas and SCCs were 0.94 for the training dataset and 0.85 for the validation dataset, respectively. For all the patient datasets, the AUC values of radiomics model, readers 1, 2 and 3 were 0.92, 0.76, 0.77 and 0.80, respectively. For the validation datasets, no significant difference was found between the AUCs of the radiomics model and those of the three radiologist (P = 0.459, 0.469, 0.738 for radiologist 1, 2 and 3, respectively).An MRI-based radiomics model can help to differentiate sinonasal lymphomas from SCCs with high accuracy.
87	RHOG: Rac1-ing up another HLH gene.	33856445	NaN
88	The occurrence and treatment of hemophagocytic lymphohistiocytosis caused by multiple factors: a case report and literature review.	33849134	Hemophagocytic lymphohistiocytosis (HLH) is a high-fatality disease caused by hereditary or acquired immune dysfunction, and is characterized by pathological inflammatory response. Primary HLH (pHLH) has hereditary genetic defects, and secondary HLH (sHLH) is caused by a variety of underlying diseases. Here, we report the case of a patient with aggressive natural killer cell leukemia and HLH-related gene defects who achieved long-term survival after treatment. A 20-year-old man presented to our hospital with symptoms of fever and fatigue. Investigations revealed splenomegaly, cytopenia, hyperferritinemia, hypofibrinogenemia, elevated levels of soluble CD25 (sCD25), and hemophagocytosis in bone marrow. Bone marrow flow cytometry showed 23.4% abnormal natural killer cells, the cells were CD2, CD7, CD16, CD94, NKG2A positive, met the diagnosis of aggressive NK-Cell leukemia. Investigation of the patient's pedigree revealed that mutations of pHLH-related genes (LYST and UNC13D) were inherited from his father and mother, but neither of the parents had the disease. The patient received hematopoietic stem-cell transplantation (HSCT), after which he achieved complete remission. As of 2020-10-19, the patient's survival has exceeded 3 years, and he has returned to his normal life. A variety of factors contribute to the onset of HLH, and this case gives greater insight into the etiology of HLH. Allogeneic HSCT is a key treatment for HLH patients with underlying genetic mutations.
89	Improved transplant outcomes with myeloablative conditioning for hemophagocytic lymphohistiocytosis in HLA-matched and mismatched donors: a national multicenter retrospective study.	33846559	We report the results of national retrospective study of 45 children with hemophagocytic lymphohistiocytosis (HLH) who underwent allogeneic hematopoietic stem-cell transplantation (HSCT) in Israel between the years 2000-2018. Donors were either HLA-matched (n = 26), partially mismatched (n = 7), haploidentical (n = 8), or cord-blood (n = 4). Myeloablative conditioning (MAC) was used in 20 procedures, and reduced-intensity conditioning (RIC) in 25. Forty-two patients engrafted, two had primary graft failure (one successfully retransplanted), one died prior to engraftment, and two developed secondary graft failure. Of the eight patients who had mixed donor chimerism at day 30 (5-95%), five achieved stable mixed or full donor chimerism. The 5-year probabilities of overall survival and event-free survival (EFS) were 86% and 82%, respectively. Five-year EFS was lower for patients receiving RIC compared to MAC (72% vs. 100%, p = 0.018) and following alternative-donor transplant (68% vs. 92% for HLA-matched donors, p = 0.034), mostly due to increased transplant-related mortality (TRM). Thus, both HLA-matched and alternative donor transplant procedures may benefit form a myeloablative conditioning regimen.
90	Central nervous system involvement in adult-onset relapsing hemophagocytic lymphohistiocytosis responsive to maintenance treatment with anakinra.	33845282	A 43 year-old male presented with a relapsing and progressive systemic inflammatory disorder with central nervous system (CNS) involvement. After a two years follow up, he was diagnosed with hemophagocytic lymphohistiocytosis (HLH), based on clinical, laboratory and radiological findings. Treatment was started with anakinra, a recombinant humanised interleukin-1 (IL-1) receptor antagonist. Clinical response was good. Laboratory and radiological findings showed no disease activity throughout a five years follow-up period. Several immunosuppressive agents have been used in HLH without any good outcomes. This is the first case report of HLH with CNS involvement responsive to chronic treatment with anakinra.
91	Challenges in improving global outcomes for hemophagocytic lymphohistiocytosis.	33843440	NaN
92	The Efficacy of Etoposide-Based Therapy in Adult Secondary Hemophagocytic Lymphohistiocytosis.	33827086	Data supporting the use of etoposide-based therapy in hemophagocytic lymphohistiocytosis (HLH) arise largely from pediatric studies. There is a lack of comparable data among adult patients with secondary HLH. We conducted a retrospective study to assess the impact of etoposide-based therapy on outcomes in adult secondary HLH. The primary outcome was overall survival. The log-rank test was used to compare Kaplan-Meier distributions of time-to-event outcomes. Multivariable Cox proportional hazards modeling was used to estimate adjusted hazard ratios (HRs) with 95% confidence intervals (CIs). Ninety adults with secondary HLH seen between January 1, 2009, and January 6, 2020, were included. Forty-two patients (47%) received etoposide-based therapy, while 48 (53%) received treatment only for their inciting proinflammatory condition. Thirty-three patients in the etoposide group (72%) and 32 in the no-etoposide group (67%) died during follow-up. Median survival in the etoposide and no-etoposide groups was 1.04 and 1.39 months, respectively. There was no significant difference in survival between the etoposide and no-etoposide groups (log-rank p = 0.4146). On multivariable analysis, there was no association between treatment with etoposide and survival (HR for death with etoposide = 1.067, 95% CI: 0.633-1.799, p = 0.8084). Use of etoposide-based therapy was not associated with improvement in outcomes in this large cohort of adult secondary HLH patients.
93	Rituximab-containing immuno-chemotherapy regimens are effective for the elimination of EBV for EBV-HLH with only and mainly B lymphocytes of EBV infection.	33826999	Patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) have a poor prognosis. This study investigated the efficacy of rituximab-containing immuno-chemotherapy regimens for EBV-HLH. In this study, 15 patients were treated with rituximab-containing regimens. The treatment efficacy and adverse events were evaluated. In 10 patients, EBV DNA became negative after the first course of treatment. The lymphocyte types infected by EBV in the 10 patients were only infected with B cells and mainly infected with B cells. In the other 5 patients, the EBV DNA of peripheral blood mononuclear cells (PBMC) before and after treatment with the regimens had no statistical difference (P = 0.111). In addition, in these 5 patients, EBV mainly infected T and NK cells. Among the 5 patients without a significant decline in EBV DNA of PBMC, 2 patients received allogeneic hematopoietic stem cell transplantation and turned negative for EBV DNA. This study suggests that rituximab-containing regimens are effective therapy for EBV-HLH with only and mainly B lymphocytes infected by EBV, especially for eliminating EBV.
94	Death of unknown cause? Post-mortem diagnosis of fulminant course of an EBV-associated secondary hemophagocytic lymphohistiocytosis.	33824685	HLH is a life-threatening disease, which is characterized by a dysregulated immune response with uncontrolled T cell and macrophage activation. The often fulminant course of the disease needs a fast diagnostic work-up to initiate as soon as possible the appropriate therapy. We present herein the case of a 71-year-old patient with rapidly progressive hyperinflammatory syndrome, which post mortem resulted in the diagnosis of EBV-associated HLH. With this case report, we intend to highlight the relevance of the HScore in the diagnosis of HLH, to create a greater awareness for EBV as a trigger of HLH, and to demonstrate the importance of treating EBV-associated HLH as early as possible.
95	The Presence of Secondary Evans Syndrome in AIDS Patients with Talaromyces marneffei Infection.	33824596	Talaromyces marneffei (T.M) is a common opportunistic fungus in human immunodeficiency virus (HIV) infection individual. Secondary Evans syndrome in AIDS patients with Talaromyces marneffei infection has not been reported before. Here, we described cases related to this comorbidity.AIDS patients diagnosed with Talaromyces marneffei infection from 2016 to 2020 at Xixi Hospital of Hangzhou were included in this retrospective study.Total 76 AIDS patients with T.M infection were enrolled. The most common symptoms were fever and cough (70/76; 55/76, respectively). 53/76 (69.74%) patients got positive results of direct antiglobulin test. 14/76 AIDS-T.M patients combined with secondary Hemophagocytic lymphohistiocytosis (HLH). Five patients were diagnosed with AIDS-T.M associated Evans syndrome. There were severe inflammatory reaction, liver dysfunction, coagulation dysfunction and immunodeficiency status in AIDS-T.M patients with secondary Evans syndrome. All patients received antifungal therapy and three patients received corticosteroids for Evans syndrome treatment. One patient died due to sepsis.AIDS-T.M patients with secondary Evans syndrome is extremely rare and we need to be alert to the occurrence of secondary Evans syndrome in AIDS-T.M patients. Clinicians should timely start effective antifungal treatments with suspicious T.M infection in AIDS patients.
96	Do COVID-19 Infections Result in a Different Form of Secondary Hemophagocytic Lymphohistiocytosis.	33803997	The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in significant morbidity and mortality across the world, with no current effective treatments available. Recent studies suggest the possibility of a cytokine storm associated with severe COVID-19, similar to the biochemical profile seen in hemophagocytic lymphohistiocytosis (HLH), raising the question of possible benefits that could be derived from targeted immunosuppression in severe COVID-19 patients. We reviewed the literature regarding the diagnosis and features of HLH, particularly secondary HLH, and aimed to identify gaps in the literature to truly clarify the existence of a COVID-19 associated HLH. Diagnostic criteria such as HScore or HLH-2004 may have suboptimal performance in identifying COVID-19 HLH-like presentations, and criteria such as soluble CD163, NK cell activity, or other novel biomarkers may be more useful in identifying this entity.
97	Ruxolitinib treatment permits lower cumulative glucocorticoid dosing in children with secondary hemophagocytic lymphohistiocytosis.	33794928	This study aimed to analyze the effects of ruxolitinib on children with secondary hemophagocytic lymphohistiocytosis (HLH).Eleven pediatric patients diagnosed with HLH and treated with ruxolitinib (ruxolitinib group: group R) between November 2017 and August 2018 were retrospectively analyzed. Eleven age-matched pediatric patients with HLH undergoing conventional treatment (control group: group C) during the same period were also analyzed.In group R, three patients who did not respond to methylprednisolone (MP) pulse and intravenous immunoglobulin (IVIG) therapies were treated with Ruxolitinib and their temperature decreased to normal levels. Four patients had normal temperature after conventional treatment (dexamethasone and etoposide, with or without cyclosporine A), but they had severe organ involvement, including obvious yellowing of the skin, increased liver enzyme levels and neuropsychiatric symptoms, and they were all ameliorated with ruxolitinib treatment. Four patients were relieved with ruxolitinib therapy alone. In group C, the body temperatures of eleven patients decreased to normal levels after conventional treatment. The body temperature of group R patients decreased to normal levels more rapidly than that of group C patients. The glucocorticoid dosage in group R was significantly lower than that in group C. Both groups were followed-up for 2-2.5 years. No obvious adverse drug reactions to ruxolitinib were observed during treatment and follow-up.Ruxolitinib might be an effective drug in controlling body temperature and reducing inflammation indicators. It might be a potential replacement for glucocorticoid therapy for HLH treatment in children, thereby reducing or avoiding glucocorticoid-related adverse reactions.
98	Secondary haemophagocytic lymphohistiocytosis (HLH) triggered by Salmonella typhi.	33790033	NaN
99	Case of haemophagocytic lymphohistiocytosis following Epstein-Barr virus infection.	33789863	Haemophagocytic lymphohistiocytosis (HLH) is a rare diagnosis that carries a high degree of mortality. We present this case of a previously healthy 22-year-old woman, who was admitted acutely ill to the hospital. One week prior, she had been seen by her primary care physician for fatigue and malaise. At that time, she was noted to have anterior and posterior cervical lymphadenopathy. She was referred to the emergency room and was diagnosed with acute Epstein-Barr virus (EBV) mononucleosis based on her clinical symptoms and positive heterophile antibody test. She was discharged after an uneventful 48-hour stay on the wards. She represented 7 days after discharge with cough, fatigue, nausea, vomiting, epigastric abdominal pain, diarrhoea, weight loss and subjective fevers. She had also reported haematemesis, epistaxis and melaena. Vital signs included temperature 36.9°C, blood pressure 90/50 mm Hg, heart rate 130 beats per minute and respiratory rate 32 breaths per minute. Physical examination was notable for an acutely ill appearing woman with scleral icterus, hepatosplenomegaly and palpable cervical and axillary lymphadenopathy. Complete blood count showed pancytopaenia with haemoglobin 59 g/L (normal 120-160 g/L), white blood cell count 2.7×109/L (normal 4-10.5×109/L) and platelet count 50×109/L (normal 150-450×109/L). The white blood cell count differential included 58% neutrophils (normal 38%-77%) with immature neutrophils in band form elevated at 45% (normal <14%), 16% lymphocytes (normal 20%-48%), 7% monocytes (normal <12%) and no eosinophils (normal <6%). Blood smear revealed anisocytosis, poikilocytosis and hypochromia. Coagulation panel showed elevated levels of d-dimer level at 1.39 µg/mL (normal <0.45 µg/mL), prolonged prothrombin time at 34.4 s (normal 11-15 s), prolonged activated partial thromboplastin time of 55.6 s (normal 25-34 s), prolonged international normalised ratio at 3.31 (normal <1.1) and low fibrinogen 60 mg/dL (normal >200 mg/dL). Lipid panel showed cholesterol at 114 mg/dL (normal 125-200 mg/dL), triglycerides 207 mg/dL (normal 30-150 mg/dL), high-density lipoprotein cholesterol 10 mg/dL (normal 40-60 mg/dL) and low-density lipoprotein cholesterol 63 mg/dL (normal <100 mg/dL). Other lab abnormalities included elevated ferritin of 6513 ng/mL (normal 10-150 ng/mL) and elevated lactate dehydrogenase of 1071 unit/L (normal 95-240 unit/L). Soluble interleukin-2 receptor alpha level was elevated at 60 727 units/mL (normal 223-710 units/mL). Fluorodeoxyglucose-positron emission tomography (FDG-PET) scan showed abnormal tracer localisation within the paratracheal, hilar, pelvic, abdominal and subcarinal lymph nodes, along with FDG-PET positive hepatosplenomegaly. A bone marrow biopsy showed hypercellular marrow (95% cellularity) with trilineage haematopoiesis, haemophagocytic cells, polytypic plasmacytosis and T-cell lymphocytosis, along with positive latent membrane protein-1 immunohistochemical staining for EBV. EBV quantitative DNA PCR showed >1 million copies. These findings were consistent with a diagnosis of HLH secondary to EBV infection. Despite intense therapy with the HLH-94 protocol, the patient expired from her illness after a prolonged hospital course.
100	Hemophagocytic lymphohistiocytosis induced by Toxoplasma gondii infection diagnosed by a bone marrow biopsy and DNA next-generation sequencing in an allogeneic hematopoietic stem cell transplant recipient.	33783930	In the United States, toxoplasmosis following allogeneic hematopoietic stem transplant (allo-HCT) is very rare with a rate only between 0.5% and 2%. The reported rates of hemophagocytic lymphohistiocytosis (HLH) following allo-HCT range between 0.3% and 17%. Secondary HLH due to toxoplasmosis infection is extremely rare. Herein, we report a case of secondary HLH due to toxoplasmosis following allo-HCT. The diagnosis was reached by a bone marrow biopsy and confirmed by DNA next generation sequencing and immunohistochemical (IHC) staining. The IHC staining included CD1a, a stain previously known to react with cells infected by Leishmania, here we show CD1a staining of macrophages infected with Toxoplasma gondii. Our report highlights the utility of bone marrow biopsy in diagnosing parasitic infection underlying HLH in post-transplant settings. The pre-transplant evaluation of patients from low endemic countries, is a great opportunity to obtain a travel history to determine the risks and the preventative measures against opportunistic infections including toxoplasmosis.
101	Small Molecule Rescue of ATXN3 Toxicity in C. elegans via TFEB/HLH-30.	33782863	Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a polyglutamine expansion disease arising from a trinucleotide CAG repeat expansion in exon 10 of the gene ATXN3. There are no effective pharmacological treatments for MJD, thus the identification of new pathogenic mechanisms, and the development of novel therapeutics is urgently needed. In this study, we performed a comprehensive, blind drug screen of 3942 compounds (many FDA approved) and identified small molecules that rescued the motor-deficient phenotype in transgenic ATXN3 Caenorhabditis elegans strain. Out of this screen, five lead compounds restoring motility, protecting against neurodegeneration, and increasing the lifespan in ATXN3-CAG89 mutant worms were identified. These compounds were alfacalcidol, chenodiol, cyclophosphamide, fenbufen, and sulfaphenazole. We then investigated how these molecules might exert their neuroprotective properties. We found that three of these compounds, chenodiol, fenbufen, and sulfaphenazole, act as modulators for TFEB/HLH-30, a key transcriptional regulator of the autophagy process, and require this gene for their neuroprotective activities. These genetic-chemical approaches, using genetic C. elegans models for MJD and the screening, are promising tools to understand the mechanisms and pathways causing neurodegeneration, leading to MJD. Positively acting compounds may be promising candidates for investigation in mammalian models of MJD and preclinical applications in the treatment of this disease.
102	A Minimal Parameter Set Facilitating Early Decision-making in the Diagnosis of Hemophagocytic Lymphohistiocytosis.	33779897	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune dysregulation syndrome characterized by uncontrolled immune cell activation. Timely diagnosis is important, since early treatment can improve survival rates. However, completing all assessments needed to reach ≥5 positive criteria out of the 8 HLH-2004 criteria can be time consuming and may delay timely initiation of treatment. Hence, we applied a data-driven approach to identify a minimal parameter set for early decision-making towards the initiation of HLH-specific treatment. We retrospectively evaluated 165 patients from five Dutch tertiary hospitals with suspected HLH. Sixteen pHLH (median age 0.5 years) and 70 sHLH patients (median age 8.7 years) were identified using the HLH-2004 criteria. Clustering analysis and multi-receiver operator characteristics were used to identify parameters distinctive of HLH. The presence of either increased ferritin, cytopenia in ≥2 lineages, or splenomegaly distinguished HLH from non-HLH cases with a negative predictive value of 100%. A minimal parameter set consisting of 2 major criteria (phagocytosis and splenomegaly) and 3 minor criteria (cytopenia, increased ferritin, and increased triglycerides/low fibrinogen) predicted HLH with 95% (88-99) sensitivity and 94% (86-98) specificity. This finding was replicated in an independent retrospective validation cohort of 109 US patients (n = 109). By dividing a subset of the HLH-2004 criteria into major and minor criteria, this strategy uses the evaluation of less than 5 criteria to quickly identify patients with HLH. When confirmed in a prospective setting, this approach could be of value for timely diagnosis and treatment of HLH.
103	Host-commensal interaction promotes health and lifespan in Caenorhabditis elegans through the activation of HLH-30/TFEB-mediated autophagy.	33770762	Gut homeostasis is maintained by the close interaction between commensal intestinal microbiota and the host, affecting the most complex physiological processes, such as aging. Some commensal bacteria with the potential to promote healthy aging arise as attractive candidates for the development of pro-longevity probiotics. Here, we showed that heat-inactivated human commensal Lactobacillus fermentum BGHV110 (BGHV110) extends the lifespan of Caenorhabditis elegans and improves age-related physiological features, including locomotor function and lipid metabolism. Mechanistically, we found that BGHV110 promotes HLH-30/TFEB-dependent autophagy to delay aging, as longevity assurance was completely abolished in the mutant lacking HLH-30, a major autophagy regulator in C. elegans. Moreover, we observed that BGHV110 partially decreased the content of lipid droplets in an HLH-30-dependent manner and, at the same time, slightly increased mitochondrial activity. In summary, this study demonstrates that specific factors from commensal bacteria can be used to exploit HLH-30/TFEB-mediated autophagy in order to promote longevity and fitness of the host.
104	Lysinuric protein intolerance mimicking N-acetylglutamate synthase deficiency in a nine-year-old boy.	33763330	We report a 9-year-old boy with lysinuric protein intolerance (LPI). He had developmental delay, short stature, failure to thrive, high-protein food aversion, hypothyroidism, growth hormone deficiency, features of hemophagocytic lymphohistiocytosis (HLH), decreased bone mineral density and multiple thoracic spine compression fractures on X-ray. LPI was suspected, but urine amino acid profile and normal orotic acid did not suggest biochemical diagnosis of LPI. Targeted next generation sequencing panel for HLH (including SLC7A7) was organized. Due to elevated glutamine in plasma amino acid analysis, a metabolic consultation was initiated and his asymptomatic post-prandial ammonia was 295 μmol/L. We then suspected n-acetylglutamate synthase or carbamoyl-phosphate synthase I deficiency due to marked hyperammonemia, elevated glutamine level, normal orotic acid, and normalization of ammonia at 2 h of carglumic acid (200 mg/kg/d). His targeted next generation sequencing panel for HLH revealed homozygous pathogenic variant in SLC7A7 ((NM_001126106.2): c.726G>A (p.Trp242*)) and confirmed the diagnosis of LPI. We emphasize the importance of genetic investigations in the diagnosis of LPI.
105	Hemophagocytic lymphohistiocytosis in COVID-19: Case reports of a stepwise approach.	33761694	The immunologic syndrome induced by severe acute coronavirus disease 2019 (COVID-19) is yet not fully understood. Typical patterns of clinical and laboratory features match secondary hemophagocytic lymphohistiocytosis (sHLH). However, the optimal approach to COVID-19 patients testing positive for sHLH is still unclear.Three patients with COVID-19 are reviewed. All showed hyperinflammation and cytokine storm, necessitating intensive care treatment including mechanical ventilation.Secondary hemophagocytic lymphohistiocytosis due to severe COVID-19; diagnosed via HScore.A treatment regimen of methylprednisolone, pentaglobin, and anakinra was developed and administered.One patient survived the ICU stay. Two other patients, in whom sHLH was diagnosed too late, deceased.A routine screening of COVID-19 patients for secondary HLH by using the HScore is feasible; especially those patients deteriorating clinically with no sufficient response to shock management might be at particular high risk. A stepwise therapeutic approach comprising corticosteroids, immunoglobulins and anakinra, accompanied by immunoadsorption, may dampen cytokine storm effects, and potentially reduce mortality.
106	Diagnostic Challenges in Pediatric Hemophagocytic Lymphohistiocytosis.	33761058	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune dysregulation that encompasses a broad range of underlying genetic diseases and infectious triggers. Monogenic conditions, autoimmune diseases, and infections can all drive the phenotype of HLH and associated immune hyperactivation with hypercytokinemia. A diagnosis of HLH usually requires a combination of clinical and laboratory findings; there is no single sensitive and specific diagnostic test, which often leads to "diagnostic dilemmas" and delays in treatment initiation. Ferritin levels, one of the most commonly used screening tests, were collected across a large tertiary care pediatric hospital to identify the positive predictive value for HLH. Herein, we present several cases that illustrate the clinical challenges of confirming an HLH diagnosis. Additionally, we report on the utility of establishing a formal multi-disciplinary group to aid the prompt diagnosis and treatment of patients presenting with HLH-like pathophysiologies.
107	Scrub typhus with secondary hemophagocytic lymphohistiocytosis in a 3-month-old child from a tertiary care hospital of Odisha.	33753698	Scrub typhus is one of the re-emerging infectious diseases in India, whereas hemophagocytic lymphohistiocytosis (HLH) results from an uncontrolled and ineffective hyperinflammatory response to a variety of triggers. HLH is categorized into primary and secondary type with infection being one of the leading causes of secondary HLH. Here, we report a case of 3-month-old girl diagnosed with scrub typhus associated with secondary HLH as both the age of presentation and the association are rarely reported in literature.
108	Adjustments to pharmacologic therapies for hemophagocytic lymphohistiocytosis while on extracorporeal support.	33751818	Hemophagocytic lymphohistiocytosis (HLH) is an immune dysregulatory syndrome characterized by severe inflammation and end-organ damage. Due to significant organ dysfunction, patients often require extracorporeal membrane oxygenation (ECMO) and continuous renal replacement therapy (CRRT). In this report, we describe consideration for adjusting treatment in the context of extracorporeal organ support. We describe agents commonly used and dosing adjustments made in light of extracorporeal organ support. We report six cases that illustrate the feasibility of initiating standard HLH therapies in patients requiring these modalities.
109	The Spectrum of Clinical, Immunological, and Molecular Findings in Familial Hemophagocytic Lymphohistiocytosis: Experience From India.	33746956	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of immune dysregulation characterized by hyperactivation of the immune system, excessive cytokine secretion and severe systemic inflammation. HLH is classified as familial (FHL) when associated with mutations in PRF1, UNC13D, STX11, and STXBP2 genes. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. This study is a retrospective analysis of 101 molecularly characterized FHL patients over the last 10 years from 20 different referral centers in India. FHL2 and FHL3 together accounted for 84% of cases of FHL in our cohort. Patients belonging to different FHL subtypes were indistinguishable based on clinical and biochemical parameters. However, flow cytometry-based assays viz. perforin expression and degranulation assay were found to be specific and sensitive in diagnosis and classification of FHL patients. Molecular characterization of respective genes revealed 76 different disease-causing mutations including 39 (51%) novel mutations in PRF1, UNC13D, STX11, and STXBP2 genes. Overall, survival was poor (28%) irrespective of the age of onset or the type of mutation in our cohort. Altogether, this article sheds light on the current scenario of FHL in India. Our data reveal a wide genetic heterogeneity of FHL in the Indian population and confirms the poor prognosis of FHL. This study also emphasizes that though mutational analysis is important for diagnostic confirmation of FHL, flow cytometry based assays help significantly in rapid diagnosis and functional validation of novel variants identified.
110	Hemophagocytic Lymphohistiocytosis in the Emergency Department: Recognizing and Evaluating a Hidden Threat.	33745765	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hematologic disorder resulting from an ineffective and pathologic activation of the immune response system that may mimic common emergency department presentations, including sepsis, acute liver failure, disseminated intravascular coagulation, and flu-like illnesses such as coronavirus disease 2019 (COVID-19).This narrative review provides a summary of the disease and recommendations for the recognition and diagnostic evaluation of HLH with a focus on the emergency clinician.Though the condition is rare, mortality rates are high, ranging from 20% to 80% and increasing with delays in treatment. Importantly, HLH has been recognized as a severe variation of the cytokine storm associated with COVID-19. Common features include a history of infection or malignancy, fever, splenomegaly or hepatomegaly, hyperferritinemia, cytopenias, coagulopathies, abnormal liver enzymes, and hypertriglyceridemia. Using specific features of the history, physical examination, laboratory studies, and tools such as the HScore, HLH-2004/2009, and hyperferritinemia thresholds, the emergency clinician can risk-stratify patients and admit for definitive testing. Once diagnosed, disease specific treatment can be initiated.This review describes the relevant pathophysiology, common presentation findings, and a framework for risk stratification in the emergency department.
111	Peripheral immune profile of children with Talaromyces marneffei infections: a retrospective analysis of 21 cases.	33743629	Talaromyces marneffei (T. marneffei) is an opportunistic pathogen that infects immunodeficient children. The aim of the study is to determine the clinical features and peripheral immune state of Talaromyces marneffei (T. marneffei) infections in children for early detection and diagnosis.We retrospectively reviewed 21 pediatric patients who were diagnosed with T. marneffei infections and were followed up in the Guangzhou Women and Children's Medical Center from January 2010 to January 2020. For each patient, we collected and analyzed clinical characteristics, peripheral immunological results, genetic tests, complications and prognosis.Common clinical features of the patients included fever (20/21, 95.24%), cough (17/21, 80.95%) and hepatomegaly (17/21, 80.95%). Severe complications included septic shock (12/21, 57.14%), hemophagocytic lymphohistiocytosis (HLH) (11/21, 52.38%), acute respiratory distress syndrome (ARDS) (10/21, 47.62%), multiple organ dysfunction syndrome (MODS) (9/21, 42.86%), and disseminated intravascular coagulation (DIC) (7/21, 33.33%). Eleven children (11/21, 52.38%) eventually died of T. marneffei infections. All patients were HIV negative. Seven cases revealed reduced antibody levels, especially IgG. Higher levels of IgE were detected in 9 cases with an obvious increase in two patients. Ten patients showed decreased complement C3 levels, some of whom had low C4 levels. Three patients displayed decreased absolute T lymphocyte counts, including the CD 4+ and CD 8+ subsets. A reduction in NK cells was present in most patients. No patient had positive nitro blue tetrazolium (NBT) test results. Nine patients were screened for common genetic mutations. Of the cases, one case had no disease-specific gene mutation. Four children had confirmed hyperimmunoglobulin M syndrome (HIGM) with CD40LG variation, one case had severe combined immunodeficiency (SCID), and one case had hyper-IgE syndrome (HIES). One patient was identified as having a heterozygous mutation in STAT3 gene; however, he showed no typical clinical manifestations of HIES at his age. One patient had a mutated COPA gene with uncertain pathogenic potential. Another patient was diagnosed with HIES that depended on her clinical features and the National Institutes of Health (NIH) scoring system.T. marneffei infections in HIV-negative children induced severe systemic complications and poor prognosis. Children with T. marneffei infections commonly exhibited abnormal immunoglobulin levels in peripheral blood, particularly decreased IgG or increased IgE levels, further suggesting possible underlying PIDs in these patients.
112	Effect of different conditioning regimens on survival and engraftment for children with hemophagocytic lymphohistiocytosis undergoing allogeneic hematopoeitic stem cell transplantation: A single institution experience.	33740322	Hemophagocytic lymphohistiocytosis (HLH), a rare hyperinflammatory immuneregulatory disorder, is a challenge in hematopoietic stem cell transplantation (HSCT) because of the high rate of mixed chimerism, relapse, and graft failure (GF) unless intensive myeloablative regimens are used. However, historically conventional myeloablative regimens (conv MA) are associated with high toxicity and mortality.We retrospectively compared transplant outcomes between three preparative regimens of varying intensities: Conv MA (n = 15), reduced-intensity conditioning (RIC, n = 12), and a treosulfan-based reduced-toxicity conditioning (RTC, n = 9).Patients in the RIC cohort had a higher incidence of mixed donor chimerism and five patients (42%) developed secondary GF (P = .002) compared to the other two regimens. There was a higher incidence of veno-occlusive disease and intensive care unit (ICU) admissions in the Conv MA cohort. With the RTC regimen, there was a similar 2-year overall survival (89, 73, and 83%; P = .87), but improved compound EFS (lack of relapse, GF, second transplant or additional donor cell infusions, or death; 89, 73, and 42%, P = .041) in RTC, Conv MA, and RIC regimen, respectively.The intensity of the preparative regimen has a significant impact on outcome of HSCT for HLH. The newly described treosulfan-based RTC provides for a stable graft with a reasonable toxicity profile.
113	Acute cerebral atrophy in autoimmune encephalitis complicated by haemophagocytic lymphohistiocytosis.	33731389	Autoimmune encephalitis is a disease characterised by neural-specific antibodies. This case report presents a 20-year-old young man with a recent history of suspected viral encephalitis who presented with recurrent fevers and episodes of confusion. He was found to have anti-N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid 1 receptor (AMPAR1) positive autoantibodies and was diagnosed with autoimmune encephalitis. He subsequently developed global cerebral atrophy and was found to meet diagnostic criteria for haemophagocytic lymphohistiocytosis (HLH). This patient's presentation was consistent with existing literature showing that autoimmune encephalitis may develop after an initial viral meningoencephalitis. However, concurrent anti-NMDAR and anti-AMPAR1 positive autoimmune encephalitis has not been reported in literature to date, and this case report represents one instance of its presentation. We speculate that multiple antibodies against neural surface antigens may increase the risk for systemic immune activation leading to HLH and acute cerebral atrophy.
114	Ruxolitinib in conjunction with the HLH-94 protocol for Epstein-Barr virus-related hemophagocytic lymphohistiocytosis in the intensive care unit: A case report.	33726009	The HLH-94 protocol is a standard induction treatment for hemophagocytic lymphohistiocytosis. However, about 30% of patients may not respond. Ruxolitinib has been clinically proven to be an effective treatment for hemophagocytic lymphohistiocytosis (HLH).A previously healthy 14-year-old girl presented to the local hospital with a 4-day history of persistent fever and sore throat.Clinical and laboratory tests revealed fever >38.5°C, hepatosplenomegaly, pancytopenia, hypertriglyceridemia, hypofibrinogenemia, hyperferritinemia, and an elevated interleukin-2 receptor level.This patient was treated with ruxolitinib and the HLH-94 protocol.The patient's clinical and some laboratory indices improved. Unfortunately, vital signs such as respiratory function and consciousness did not improve.This case report highlights the effect of using ruxolitinib in conjunction with the HLH-94 protocol. However, safety evaluation of this regimen was not performed because critically ill patient died too fast.
115	HLH-30-dependent rewiring of metabolism during starvation in C. elegans.	33724708	One of the most fundamental challenges for all living organisms is to sense and respond to alternating nutritional conditions in order to adapt their metabolism and physiology to promote survival and achieve balanced growth. Here, we applied metabolomics and lipidomics to examine temporal regulation of metabolism during starvation in wild-type Caenorhabditis elegans and in animals lacking the transcription factor HLH-30. Our findings show for the first time that starvation alters the abundance of hundreds of metabolites and lipid species in a temporal- and HLH-30-dependent manner. We demonstrate that premature death of hlh-30 animals under starvation can be prevented by supplementation of exogenous fatty acids, and that HLH-30 is required for complete oxidation of long-chain fatty acids. We further show that RNAi-mediated knockdown of the gene encoding carnitine palmitoyl transferase I (cpt-1) only impairs survival of wild-type animals and not of hlh-30 animals. Strikingly, we also find that compromised generation of peroxisomes by prx-5 knockdown renders hlh-30 animals hypersensitive to starvation, which cannot be rescued by supplementation of exogenous fatty acids. Collectively, our observations show that mitochondrial functions are compromised in hlh-30 animals and that hlh-30 animals rewire their metabolism to largely depend on functional peroxisomes during starvation, underlining the importance of metabolic plasticity to maintain survival.
116	Characteristics of primary Epstein-Barr virus infection disease spectrum and its reactivation in children, in Suzhou, China.	33719067	Epstein-Barr virus (EBV) is a globally prevalent herpesvirus associated with multiple diseases. This study aimed to determine the characteristics of primary EBV infection disease spectrum and reactivation in children, in Suzhou, China. All children admitted to the Children's Hospital of Soochow University between May 2018 and September 2020 with suspected EBV-associated disease and subjected to the indirect immunofluorescence assay for EBV-specific antibodies and plasma EBV-DNA assays were included. Of the 3567 children, 2782 (78.0%) tested positive for EBV. The positive rates of viral capsid antigen (VCA)-IgM, VCA-IgG, early antigen (EA)-IgG, nuclear antigen (EBNA)-IgG, and plasma EBV-DNA were 12.1%, 74.6%, 37.9%, 35.6%, and 31.1%, respectively. The lowest VCA-IgG and EBNA-IgG seropositivity rates occurred at ages between 8 and 36 months, then increased gradually in the older age groups. The EBV-IgM seropositivity rate was the highest in those aged 36 to <72 months. Primary EBV infection was more common in children aged 36 to <72 months. In past infections, reactivation mainly occurred in 8 to <36 months. The most common disease caused by primary EBV infection was infectious mononucleosis (56.1%), followed by a respiratory infection (17.0%). Respiratory infection (30.0%), EBV infection (29.2%), and hemophagocytic lymphohistiocytosis (HLH) (15.8%) were the commonest diseases caused by EBV reactivation. EBV reactivation was a risk factor for HLH (adjusted odds ratio, 16.4; 95% confidence interval, 7.9-34.0). Among reactivated patients, the viral load of HLH was higher than that of EBV infection and respiratory infection (p < .01). This is a retrospective large sample study that explored the characteristics of primary EBV infection disease spectrum and reactivation in children.
117	Single-Cell RNA Sequencing and Quantitative Proteomics Analysis Elucidate Marker Genes and Molecular Mechanisms in Hypoplastic Left Heart Patients With Heart Failure.	33718359	To probe markers and molecular mechanisms of the hypoplastic left heart (HLH) by single-cell RNA sequencing (scRNA-seq) and quantitative proteomics analysis.Following data preprocessing, scRNA-seq data of pluripotent stem cell (iPSC)-derived cardiomyocytes from one HLH patient and one control were analyzed by the Seurat package in R. Cell clusters were characterized, which was followed by a pseudotime analysis. Markers in the pseudotime analysis were utilized for functional enrichment analysis. Quantitative proteomics analysis was based on peripheral blood samples from HLH patients without heart failure (HLH-NHF), HLH patients with heart failure (HLH-HF), and healthy controls. Hub genes were identified by the intersection of pseudotime markers and differentially expressed proteins (DE-proteins), which were validated in the GSE77798 dataset, RT-qPCR, and western blot.Cardiomyocytes derived from iPSCs were clustered into mesenchymal stem cells, myocardium, and fibroblast cells. Pseudotime analysis revealed their differentiation trajectory. Markers in the three pseudotime clusters were significantly associated with distinct biological processes and pathways. Finally, three hub genes (MMP2, B2M, and COL5A1) were identified, which were highly expressed in the left (LV) and right (RV) ventricles of HLH patients compared with controls. Furthermore, higher expression levels were detected in HLH patients with or without HF than in controls.Our findings elucidate marker genes and molecular mechanisms of HLH, deepening the understanding of the pathogenesis of HLH.
118	Cardiovascular Drug Therapy during Interstage After Hybrid Approach: A Single-Center Experience in 51 Newborns with Hypoplastic Left Heart.	33713024	Newborns with hypoplastic left heart (HLH) are usually palliated with the Norwood procedure or a hybrid stage I procedure. Hybrid is our preferred approach. Given the critical relationship between stage I, interstage, and comprehensive stage II or advanced biventricular repair, we hypothesized that appropriate drug treatment is a significant therapeutic cornerstone, especially for the management of the high-risk interstage.We report a single-center observational study addressing the cardiovascular effects of, in particular, oral β-blockers and the additional use of angiotensin-converting enzyme (ACE) and mineralocorticoid inhibitors.In total, 51 newborns-30 with HLH syndrome (HLHS) and 21 with HLH complex (HLHC)-with a median bodyweight of 3.0 kg (range 1.9-4.4; nine with bodyweight ≤ 2500 g) underwent an uneventful "Giessen hybrid approach" using a newly approved duct stent. All patients were discharged home with a single, double or triple therapy consisting of ß-blockers, ACE and mineralocorticoid inhibitors; 90% of the patients received bisoprolol, 10% received propranolol, 72% received lisinopril, and 78% received spironolactone. Resting heart rate decreased from 138 bpm (range 112-172; n = 51) at admission to 123 bpm (range 99-139; n = 51) at discharge and 110 bpm before stage II/biventricular repair/heart transplantation (range 90-140; n = 37) accompanied by favorable bodyweight gain. No side effects were evident.In view of drug risk/benefit profiles, as well as the variable morphology and hemodynamics, the highly selective β1-adrenoceptor blocker bisoprolol is our preferred drug for treatment of HLHS/HLHC in the interstage. We avoid using ACE inhibitor monotherapy and exclude potential risks for coronary and cerebral perfusion pressure beforehand.
119	The Treatment Effect of Protamine on Severe Coagulopathy in Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis: Case Reports and Literature Review.	33707840	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening disease, which is characterized by severe systemic inflammation with cytokine storm as well as histologic evidence of hemophagocytosis. Besides, coagulopathy and hemorrhages are two common severe complications in HLH patients. Recent literatures indicate that Epstein-Barr virus (EBV) infection is one of the important triggers for the disease. In the study, we present three cases of EBV related HLH (EBV-HLH) with coagulopathy in patients with distinct backgrounds. Case 1 is a 45-year-old female diagnosed with EBV associated NK/T cell lymphoproliferative disorder (EBV-T/NK-LPD) and EBV-HLH. Case 2 is a 17-year-old male with a diagnosis of EBV-T-LPD and EBV-HLH. Case 3 is a 51-year-old male and also diagnosed with EBV-T-LPD and EBV-HLH. All cases were given with treatment with HLH-94 protocol, and the symptoms of the three patients improved. Furthermore, during the treatment, protamine, which has not been reported in the literature previously, was given to the three cases with EBV-HLH, and our results showed that after treatment with protamine, the coagulopathy and bleedings in these patients were improved rapidly. Unfortunately, the three patients relapsed soon and died despite intensive treatment. However, these cases suggest that protamine may serve as a potential treatment option for coagulation associated with EBV-HLH. Besides, the study helps us improve the understanding of the EBV-HLH related coagulation disorders, and provide a potential strategy for future treatment of the disease.
120	Haemophagocytic lymphohistiocytosis secondary to brucellosis in a young child.	33692068	Brucellosis is a common zoonotic disease worldwide. It has protean clinical manifestation and sometimes may has a life-threatening complication. A 4-year-old boy presented with a history of fever, myalgia and appetite loss for 3 weeks. On examination, he had hepatosplenomegaly. The initial working diagnosis was an infection, autoimmune disease and malignancy. Investigations showed positive Brucella serology, and he was started on rifampicin and cotrimoxazole. He was further investigated because of persistent fever, which revealed evidence of haemophagocytic lymphohistiocytosis (HLH). He continued treatment for brucellosis, except rifampicin which was replaced with doxycyclin due to a worsening liver function. The child showed complete clinical and biochemical improvement after 6 weeks of therapy. HLH is a life-threatening condition and should be suspected in children with brucellosis, who did not respond to appropriate antibiotics treatment. Secondary HLH does not always require specific therapy; it may improve with adequate treatment of the underlying condition.
121	Ectopic expression of the atypical HLH FaPRE1 gene determines changes in cell size and morphology.	33691964	PACLOBUTRAZOL RESISTANCE (PRE) genes code atypical HLH transcriptional regulators characterized by the absence of a DNA-binding domain but present an HLH dimerization domain. In vegetative tissues, the function of these HLH proteins has been related with cell elongation processes. In strawberry, three FaPRE genes are expressed, two of them (FaPRE2 and FaPRE3) in vegetative tissues while FaPRE1 is fruit receptacle-specific. Ubiquitous FaPRE1 accumulation produced elongated flower receptacles and plants due to the elongation of the main aerial vegetative organs, with the exception of leaves. Histological analysis clearly demonstrated that the observed phenotype was due to significant changes in the parenchymal cell's morphology. In addition, transcriptomic studies of the transgenic elongated flower receptacles allowed to identify a small group of differentially expressed genes that encode cell wall-modifying enzymes. Together, the data seem to indicate that, in the strawberry plant vegetative organs, FaPRE proteins could modulate the expression of genes related with the determination of the size and shape of the parenchymal cells.
122	Emapalumab for adult and pediatric patients with hemophagocytic lymphohistiocytosis.	33686916	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory syndrome. Standard treatment is based on immunosuppressive, cytotoxic drugs and hematopoietic stem cell transplantation (HSCT) in primary HLH. Interferon-gamma (IFN-γ) plays a key pathogenic role. Emapalumab, a monoclonal antibody directed against IFN-γ, is the first target therapy approved for primary HLH with refractory, recurrent or progressive disease or intolerance to conventional therapy.We reviewed the pharmacological characteristics, safety, efficacy and clinical uses of emapalumab. We summarized the results of current standard treatment based on chemo-immunosuppressive protocols and outlined the alternative options available.Emapalumab is an effective treatment for HLH with a good safety profile. Its efficacy was demonstrated in a phase II/III study on primary HLH pediatric patients with refractory, relapsing HLH or intolerance to first-line treatment. The use of emapalumab allowed most patients to proceed to HSCT, with a high estimated probability of survival 12 months after transplantation. The outcomes in patients who underwent transplantation compare favorably with those reported previously with either myeloablative or reduced-intensity conditioning regimens. The potential role of emapalumab in the treatment of secondary HLH and as a prevention of graft failure after HSCT deserves to be further assessed.
123	A network-informed analysis of SARS-CoV-2 and hemophagocytic lymphohistiocytosis genes' interactions points to Neutrophil extracellular traps as mediators of thrombosis in COVID-19.	33684134	Abnormal coagulation and an increased risk of thrombosis are features of severe COVID-19, with parallels proposed with hemophagocytic lymphohistiocytosis (HLH), a life-threating condition associated with hyperinflammation. The presence of HLH was described in severely ill patients during the H1N1 influenza epidemic, presenting with pulmonary vascular thrombosis. We tested the hypothesis that genes causing primary HLH regulate pathways linking pulmonary thromboembolism to the presence of SARS-CoV-2 using novel network-informed computational algorithms. This approach led to the identification of Neutrophils Extracellular Traps (NETs) as plausible mediators of vascular thrombosis in severe COVID-19 in children and adults. Taken together, the network-informed analysis led us to propose the following model: the release of NETs in response to inflammatory signals acting in concert with SARS-CoV-2 damage the endothelium and direct platelet-activation promoting abnormal coagulation leading to serious complications of COVID-19. The underlying hypothesis is that genetic and/or environmental conditions that favor the release of NETs may predispose individuals to thrombotic complications of COVID-19 due to an increase risk of abnormal coagulation. This would be a common pathogenic mechanism in conditions including autoimmune/infectious diseases, hematologic and metabolic disorders.
124	Diagnosis of GATA2 Deficiency in a Young Woman with Hemophagocytic Lymphohistiocytosis Triggered by Acute Systemic Cytomegalovirus Infection.	33684095	BACKGROUND Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease characterized by an intense immunologic response that results in multiorgan dysfunction. It typically manifests as a result of a familial genetic immunodeficiency disorder or secondary to a trigger such as an infection, malignancy, or autoimmune disease. The major factors involved in the development of the disease are an individual's genetic propensity to develop HLH, such as rare associated mutations, or inflammatory processes that trigger the immune system to go haywire. CASE REPORT Before the COVID-19 pandemic, a 22-year-old woman with a history of congenital absence of the right kidney, right-sided hearing loss, and leukopenia presented with a 3-week history of generalized malaise, fever, chest pain, cough, and shortness of breath. She developed an acute systemic cytomegalovirus infection further complicated by HLH. Based on her history and clinical course, an underlying primary immunodeficiency was suspected. An immunodeficiency gene panel revealed a monoallelic mutation in GATA2, a gene that encodes zinc-transcription factors responsible for the regulation of hematopoiesis. CONCLUSIONS GATA2 deficiency encompasses a large variety of mutations in the GATA2 gene and leads to disorders associated with hematologic and immunologic manifestations of monocytopenia and B-, and natural killer-cell deficiency. Over time, affected individuals are at high risk of developing life-threatening infections and serious hematologic complications, such as myelodysplastic syndromes and/or leukemias. We aimed to illustrate the importance of identifying an underlying genetic disorder associated with secondary HLH to help guide acute and long-term management.
125	The conserved regulator of autophagy and innate immunity hlh-30/TFEB mediates tolerance of enterohemorrhagic Escherichia coli in Caenorhabditis elegans.	33683370	Infection with antibiotic-resistant bacteria is an emerging life-threatening issue worldwide. Enterohemorrhagic Escherichia coli O157: H7 (EHEC) causes hemorrhagic colitis and hemolytic uremic syndrome via contaminated food. Treatment of EHEC infection with antibiotics is contraindicated because of the risk of worsening the syndrome through the secreted toxins. Identifying the host factors involved in bacterial infection provides information about how to combat this pathogen. In our previous study, we showed that EHEC colonizes in the intestine of Caenorhabditis elegans. However, the host factors involved in EHEC colonization remain elusive. Thus, in this study, we aimed to identify the host factors involved in EHEC colonization. We conducted forward genetic screens to isolate mutants that enhanced EHEC colonization and named this phenotype enhanced intestinal colonization (Inc). Intriguingly, four mutants with the Inc phenotype showed significantly increased EHEC-resistant survival, which contrasts with our current knowledge. Genetic mapping and whole-genome sequencing (WGS) revealed that these mutants have loss-of-function mutations in unc-89. Furthermore, we showed that the tolerance of unc-89(wf132) to EHEC relied on HLH-30/TFEB activation. These findings suggest that hlh-30 plays a key role in pathogen tolerance in C. elegans.
126	Super refractory status in a case of Febrile Infection-Related Epilepsy Syndrome due to hemophagocytic lymphocytic histiocytosis.	33681644	A 14-year-old boy presented with a prodromal respiratory infection followed by super refractory status epilepticus. A diagnosis of Febrile Infection-Related Epilepsy Syndrome (FIRES) was made. Initial MRI study and CSF analysis were normal. He required multiple anticonvulsants owing to the refractory nature of the seizures. The course of the illness was rather stormy, laced with various medical problems viz. hepatic dysfunction, sepsis, hemodynamic, and hematological abnormalities which posed several challenges in the management. Hemophagocytic lymphocytic histiocytosis (HLH) was identified as the etiology of the illness and was treated but without success. The case report highlights the several immunomodulatory strategies that were employed to treat the disease, despite which the outcome was unfavorable.
127	Lemierre's Syndrome with Severe Thrombocytopenia without Disseminated Intravascular Coagulation: A Case Report and Literature Review.	33681457	Lemierre's syndrome (LS) is an uncommon clinical entity characterized by a primary oropharyngeal infection with subsequent septic thrombophlebitis. Diagnosis is made with clinical or radiographic evidence of internal jugular vein (IJV) thrombosis, along with metastatic focus such as lungs or joints. Life threatening thrombocytopenia in Lemierre's syndrome in the absence of disseminated intravascular coagulation (DIC) is rarely reported. We present a case of a 41-year-old woman with Lemierre's syndrome caused by beta-hemolytic group C streptococci and fusobacterium species manifested as worsening pharyngitis, IJV thrombosis, and complicated by severe thrombocytopenia.
128	Secondary Hemophagocytic Lymphohistiocytosis in a Young Hispanic Adult.	33680624	Hemophagocytic lymphohistiocytosis (HLH) is a disease caused by a severe immune system reaction that involves an overwhelming inflammatory response with overproduction of cytokines and hemophagocytosis. HLH is classified as primary HLH or familial HLH (PHLH or FHLH) and secondary HLH. PHLH is due to mutations in several genes that regulate immune cells, while secondary HLH is triggered by a severe illness (viral infections or malignancies) that induce an excessive immune response that is difficult to control. We present a case of a young Hispanic adult female with a medical history of diabetes mellitus type 1 and hepatitis E that was diagnosed with HLH secondary to lymphoma caused by Epstein Barr virus infection. The patient was started on broad-spectrum antibiotics and steroid therapy; however, the patient succumbed to the disease. HLH is associated with high mortality, mainly because it is not a very common entity and patients usually present critically ill and deteriorate very fast. Immunosuppression and treatment of the underlying disorder is the target of the treatment of HLH, however, the prognosis remains poor.
129	Thrombotic microangiopathy led to acute kidney injury in an infant with hemophagocytic lymphohistiocytosis: A case report.	33680118	Hemophagocytic lymphohistiocytosis (HLH) is a rare but fatal clinical syndrome frequently complicated by acute kidney injury (AKI) and acute tubular necrosis. Renal thrombotic microangiopathy (TMA) is a specific pathological feature of childhood HLH and few cases have been reported among infants. The present study presents a rare case of HLH with TMA in an infant. A 15-month-old infant with a week-long history of fever was admitted to hospital. The infant presented with AKI and subsequently a reduction in platelet and hemoglobin levels. TMA was diagnosed by kidney biopsy and the clinical, laboratory and bone marrow biopsy findings met the criteria of HLH. Due to a progressive increase in serum creatinine levels, hemodialysis was initiated on the second day following admission. Dexamethasone was administered to treat both the fever and HLH. The patient's body temperature returned to a normal range and platelet and hemoglobin levels were stable after 14 days of admission. Renal function stabilized on day 21. The results of genetic testing did not identify any disease-related variations. Childhood HLH is a severe condition and mortality can be reduced by early diagnosis and correct treatment. For patients with HLH and AKI, the possible role of TMA should be considered. Renal biopsy can help to identify the cause of AKI and can be performed when the patient's condition is stable.
130	Life-Threatening Influenza, Hemophagocytic Lymphohistiocytosis and Probable Vaccine-Strain Varicella in a Novel Case of Homozygous STAT2 Deficiency.	33679716	STAT2 is a transcription factor that plays an essential role in antiviral immunity by mediating the activity of type I and III interferons (IFN-I and IFN-III). It also has a recently established function in the negative regulation of IFN-I signaling. Homozygous STAT2 deficiency is an ultra-rare inborn error of immunity which provides unique insight into the pathologic consequence of STAT2 dysfunction. We report here a novel genetic cause of homozygous STAT2 deficiency with several notable clinical features. The proband presented aged 12 months with hemophagocytic lymphohistiocytosis (HLH) closely followed by clinical varicella, both occurring within three weeks of measles, mumps, and rubella (MMR) and varicella vaccinations. There was a history of life-threatening influenza A virus (IAV) disease 2 months previously. Genetic investigation uncovered homozygosity for a novel nonsense variant in STAT2 (c. 1999C>T, p. Arg667Ter) that abrogated STAT2 protein expression. Compatible with STAT2 deficiency, dermal fibroblasts from the child demonstrated a defect of interferon-stimulated gene expression and a failure to mount an antiviral state in response to treatment with IFN-I, a phenotype that was rescued by lentiviral complementation by wild type STAT2. This case significantly expands the phenotypic spectrum of STAT2 deficiency. The occurrence of life-threatening influenza, which has not previously been reported in this condition, adds STAT2 to the list of monogenetic causes of this phenotype and underscores the critical importance of IFN-I and IFN-III to influenza immunity. The development of probable vaccine-strain varicella is also a novel occurrence in STAT2 deficiency, implying a role for IFN-I/III immunity in control of attenuated varicella zoster virus in vivo and reinforcing the susceptibility to pathologic effects of live-attenuated viral vaccines in disorders of IFN-I immunity. Finally, the occurrence of HLH in this case reinforces emerging links to hyperinflammation in patients with STAT2 deficiency and other related defects of IFN-I signaling-highlighting an important avenue for further scientific enquiry.
131	Granulomatous Tubercular Hepatitis Presenting as Secondary Hemophagocytic Lymphohistiocytosis: A Case Report and Systematic Review of the Literature.	33679052	Hemophagocytic lymphohistiocytosis is a life-threatening disorder characterized by persistent pathologic activation of cytotoxic T lymphocytes, natural killer cells, and macrophages. We present details of a young patient who presented with high-grade fever, jaundice, and breathlessness. On investigations, he had hepatitis, anemia, neutropenia, and coagulopathy. He also had hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. Bone marrow aspiration revealed histiocytosis, and transjugular liver biopsy revealed necrotizing granulomas positive for Mycobacterium tuberculosis on acid-fast bacilli staining. He was successfully managed with a combination of immunosuppressants and antitubercular therapy. Tuberculosis associated hemophagocytosis syndrome is rare and should be considered in patients with unexplained hemophagocytosis syndrome, especially in tuberculosis-endemic regions. Prompt recognition and treatment with antitubercular treatment and immunosuppressants are associated with good outcomes.
132	Secondary Hemophagocytic Lymphohistiocytosis and Autoimmune Cytopenias: Case Description and Review of the Literature.	33672504	Hemophagocytic lymphohistocytosis (HLH) is a rare hyperinflammatory condition which may be primary or secondary to many diseases, including hematologic malignancies. Due to its life-threatening evolution, a timely diagnosis is paramount but challenging, since it relies on non-specific clinical and laboratory criteria. The latter are often altered in other diseases, including autoimmune cytopenias (AIC), which in turn can be secondary to infections, systemic autoimmune or lymphoproliferative disorders. In the present article, we describe two patients presenting at the emergency department with acute AICs subsequently diagnosed as HLH with underlying diffuse large B cell lymphoma. We discuss the diagnostic challenges in the differential diagnosis of acute cytopenias in the internal medicine setting, providing a literature review of secondary HLH and AIC.
133	Hemophagocytic lymphohistiocytosis associated with ocrelizumab treatment in a patient with multiple sclerosis.	33666121	Hemophagocytic lymphohistiocytosis (HLH) is a rarely recognized hyperinflammatory condition of high death risk.The objective was to describe a case of HLH in a patient with multiple sclerosis (MS) treated with ocrelizumab.Clinical observation, laboratory testing, and use of HLH-2004 criteria for HLH diagnosis.A 32-year-old Caucasian female developed HLH during ocrelizumab treatment. She met six of the eight HLH criteria including fever, splenomegaly, cytopenia, hypertriglyceridemia and hypofibrinogenemia, high serum ferritin level, and low natural killer (NK) cells.HLH should be considered in the differential diagnosis in MS patients displaying a fever and malaise syndrome following administration of ocrelizumab.
134	Use of the JAK Inhibitor Ruxolitinib in the Treatment of Hemophagocytic Lymphohistiocytosis.	33664745	Hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome driven by overactive T cells and macrophages that abundantly secrete numerous pro-inflammatory cytokines, including interferon (IFN)-gamma, interleukin (IL)-1-beta, IL-2, IL-6, IL-10, IL-18, and tumor necrosis factor (TNF). The release of these and other cytokines underlies many of the clinical and pathologic manifestations of HLH, which if left untreated, can lead to multi-organ failure and death. The advent of etoposide-based regimens, such as the Histiocyte Society HLH-94 and HLH-2004 protocols, has substantially decreased the mortality associated with HLH. Nevertheless, the 5-year survival remains low at ~60%. To improve upon these results, studies have focused on the use of novel cytokine-directed therapies to dampen inflammation in HLH. Among the agents being tested is ruxolitinib, a potent inhibitor of the Janus Kinase (JAK) and Signal Transducer and Activation of Transcription (STAT) pathway, which functions downstream of many HLH-associated cytokines. Here, we review the basic biology of HLH, including the role of cytokines in disease pathogenesis, and discuss the use of ruxolitinib in the treatment of HLH.
135	Secondary Leukemia in a Patient With EBV-HLH Carrying Heterozygous STXBP2 Variant.	33661178	Hemophagocytic lymphohistiocytosis (HLH) is a symptom with severe systemic hyperinflammation. A 26-month-old male presented with Epstein-Barr virus associated HLH with a heterozygous variant of syntaxin-binding protein-2 (STXBP2). Complete remission was achieved with the HLH-2004 protocol, but the disease soon relapsed. Four weeks after re-installing HLH-2004 protocol, HLH was resolved. The cumulative dosage of etoposide was 2100 mg/m2. He developed acute promyelocytic leukemia 17 months later. The patient underwent standard chemotherapy and since remained in complete remission. In conclusion, a regular screening of malignance might be in necessity for the patients harboring gene variants associated with familial HLH.
136	Diagnostic and therapeutic caveats in Griscelli syndrome.	33660295	Griscelli syndrome (GS) is a rare autosomal recessive disease with characteristic pigment distribution, and there are currently 3 types according to the underlying genetic defect and clinical features. We present the case of a girl born from consanguineous parents who presented with predominant neurologic symptoms, silvery hair and granulomatous skin lesions. Cerebral magnetic resonance revealed diffuse white matter lesions, and central nervous system (CNS) lymphocytic infiltration was suspected. The patient underwent haematopoietic stem cell transplantation with graft failure and autologous reconstitution. She developed elevated liver enzyme with a cholestatic pattern. Multiple liver biopsies revealed centrilobular cholestasis and unspecific portal inflammation that improved with immunomodulatory treatment. She was revealed to have an impaired cytotoxicity in NK cells and a decreased expression of RAB27A. However, no variants were found in the gene. All types of GS present with pigment dilution and irregular pigment clumps that can be seen through light microscopy in hair and skin biopsy. Dermic granulomas and immunodeficiency with infectious and HLH predisposition have been described in GS type 2 (GS2). Neurologic alterations might be seen in GS type 1 (GS1) and GS type 2 (GS2), due to different mechanisms. GS1 presents with neurologic impairment secondary to myosin Va role in neuronal development and synapsis. Meanwhile, GS2 can present with neurologic impairment secondary to SNC HLH. Clinical features and cytotoxicity might aid in differentiating GS1 and GS2, especially since treatment differs.
137	Hemophagocytic Lymphohistiocytosis Gene Mutations in Adult Patients Presenting With CLIPPERS-Like Syndrome.	33658321	To determine whether adult cases of Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS) may be related to familial hemophagocytic lymphohistiocytosis (HLH) causes, we have screened patients with adult-onset CLIPPERS for mutations in primary HLH-associated genes.In our cohort of 36 patients fulfilling the criteria for probable or definite CLIPPERS according to the CLIPPERS-2017 criteria, we conducted a first study on 12 patients who consented to genetic testing. In these 12 patients, systemic HLH criteria were searched, and genetic analysis of 8 genes involved in primary HLH was performed.Four definite and 8 probable CLIPPERS were enrolled (n = 12). Mutations involved in HLH were identified in 2 definite and 2 probable CLIPPERS (4/12). Three of them had biallelic PRF1 mutations with reduced perforin expression in natural killer cells. The remaining patient had biallelic UNC13D mutations with cytotoxic lymphocyte impaired degranulation. None of the mutated patients reached the criteria for systemic HLH. During follow-up, 3 of them displayed atypical findings for CLIPPERS, including emergence of systemic non-Hodgkin lymphoma (1/3) and confluent gadolinium-enhancing lesions on brain MRI (3/3).In our patients presenting with adult-onset CLIPPERS, one-third have HLH gene mutations. This genetic treatable condition should be searched in patients with CLIPPERS, especially in those presenting with atypical findings.
138	Haemophagocytic lymphohistiocytosis secondary to intrauterine cytomegalovirus infection.	33656138	Congenital cytomegalovirus infection causes lethal diseases with neurological, visual, auditory and systemic injuries, including the hemophagocytic syndrome. Hemophagocytic lymphohistiocytosis (HLH) can be caused by primary hereditary immunological defects, as well as several infectious triggering factors, such as viruses, bacteria and fungus, among them the cytomegalovirus (CMV). Here we present the case report of a male newborn male, delivered by cesarean at term (gestation age of 39 weeks), weighing 3,250 g, with suffusion skin lesions spread throughout the body, anemia, generalized edema, hepatosplenomegaly, thrombocytopenia associated with grunts and difficulty breathing, treated with ganciclovir after receiving the diagnosis of congenital CMV infection. After a few days of hospitalization, the patient presented with high fever, persistent hepatosplenomegaly and pancytopenia, in addition to elevated ferritin and triglycerides, receiving the diagnosis of HLH treated with immunosuppressive therapy, corticosteroids and intravenous human immunoglobulin. The present case report highlights the importance for health professionals to carry out the investigation of congenital diseases, especially in developing countries, as well as their complications, such as HLH.
139	Hemophagocytic lymphohistiocytosis and myelodysplastic syndrome: a case report and review of the literature.	33648567	Hemophagocytic lymphohistiocytosis (HLH) is characterized by hyperinflammation and life-threatening cytopenias. Survival is poor, and management is pivotal on rapid identification of the disease. HLH is associated with hematologic malignancies, however correlation with myelodysplastic syndromes (MDS) is exceedingly unusual. Although minimizing overwhelming hyperinflammation by treating hemophagocytosis are central for HLH outcome, there is urgent necessity to identify potential initiating mechanisms that could assist in therapy design.Here, we describe an elderly African American patient who developed rapid onset of cytopenias and coagulopathy associated with hepatic and bone marrow hemophagocytosis. We analyze four additional similar cases to isolate clinical, laboratory and cytogenetic findings expected in patients exhibiting concurrent HLH and MDS. HLH linked with MDS retains common HLH features associated with systemic hyperinflammation such as fever, hypotension, hepatosplenomegaly, hyperferritinemia, coagulopathy and rapidly evolving cytopenias. Typical MDS chromosomic abnormality such as trisomy 8 was frequently observed in our studied cases.Our case describes difficulties while managing HLH in MDS patients. Diagnosis should be based on identifying HLH appropriate criteria and if possible karyotypic abnormalities normally observed in MDS.
140	Acute Hepatitis-A Virus Infection as a Rare Cause of Hemophagocytic Lymphohistiocytosis.	33645198	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder of the mononuclear phagocytic system, characterised by histiocyte and lymphocyte activation. It can be classified as primary and secondary HLH. Primary HLH usually presents in childhood, and is associated with gene mutations. Secondary HLH usually presents in adulthood, and is due to an underlying infection, autoimmune disease or malignancy. We describe a case of HLH secondary to acute hepatitis-A virus infection, which was characterised by persistent fever, pancytopenia, splenomegaly, hyperferritinemia, and hemophagocytosis observed in the bone marrow. Key Words: Hemophagocytic lymphohistiocytosis, Hepatitis-A, Mononuclear phagocytes.
141	In vitro effects of the endocrine disruptor p,p'DDT on human choriogonadotropin/luteinizing hormone receptor signalling.	33638691	Dichlorodiphenyltrichloroethane (p,p'DDT) is an endocrine-disrupting chemical (EDC). Several studies showed an association between p,p'DDT exposure and reprotoxic effects. We showed that p,p'DDT was a positive allosteric modulator of human follitropin receptor (FSHR). In contrast, we demonstrated that p,p'DDT decreased the cyclic AMP (cAMP) production induced by human choriogonadotropin (hCG). This study evaluated further the effects of p,p'DDT on Gs-, β-arrestin 2- and steroidogenesis pathways induced by hCG or luteinizing hormone (LH). We used Chinese hamster ovary cells line stably expressing hCG/LHR. The effects of 10-100 µM p,p'DDT on cAMP production and on β-arrestin 2 recruitment were measured using bioluminescence and time-resolved resonance energy transfer technology. The impact of 100 µM of p,p'DDT on steroid secretion was analysed in murine Leydig tumor cell line (mLTC-1). In cAMP assays, 100 µM p,p'DDT increased the EC50 by more than 300% and reduced the maximum response of the hCG/LHR to hCG and hLH by 30%. This inhibitory effect was also found in human granulosa cells line and in mLTC-1 cells. Likewise, 100 µM p,p'DDT decreased the hCG- and hLH-promoted β-arrestin 2 recruitment down to 14.2 and 26.6%, respectively. Moreover, 100 µM p,p'DDT decreased by 30 and 47% the progesterone secretion induced by hCG or hLH, respectively, without affecting testosterone secretion. This negative effect of p,p'DDT was independent of cytotoxicity. p,p'DDT acted as a negative allosteric modulator of the hCG/LHR signalling. This emphasizes the importance of analyzing all receptor-downstream pathways to fully understand the deleterious effects of EDC on human health.
142	[Bile acid diarrhea, stepchild of chronic diarrhea - prevalence, diagnosis and treatment. Update 2021].	33634438	Bile acid diarrhea is one of the most frequently undiagnosed causes of chronic diarrhea. A variety of different pathophysiologic causes can underlie chronic diarrhea. Even after exclusion of the more frequent causes, up to 5 % of the population remains affected by unexplained chronic diarrhea. In up to 50 % within this cohort, bile acid diarrhea is the underlying cause.The various pathophysiologies leading to bile acid diarrhea are well characterized. In this way, bile acid diarrhea can be divided into primary, secondary and tertiary subtypes. Common to all causes is the increased amount of bile acids in the colon and in the faeces and the resulting secretory-osmotic diarrhea, in more severe forms in combination with steatorrhea. The diagnosis of bile acid diarrhea follows a clear algorithm which, in addition to the search for the cause and possibly a therapeutic trial, recognizes the 75SeHCAT test as the reference method for the detection of an increased loss of bile acids. In view of the chronic nature of the symptoms and the need for permanent, lifelong therapy, the use of a one-time, reliable diagnostic test is justified, though the test is currently only available at a few centers. In addition to the treatment of identifiable underlying diseases, the current treatment includes the use of drugs that bind bile acids, with additional nutritional recommendations and vitamin substitutions.The present review article summarizes the pathophysiology and importance of bile acid diarrhea and discusses the current approach towards diagnosis and treatment.Die chologene Diarrhö ist eine der häufigsten nicht diagnostizierten Ursachen der chronischen Diarrhö. Zahlreiche verschiedene Pathophysiologien können einer chronischen Diarrhö zugrunde liegen. Auch nach Ausschlussdiagnostik der häufigeren Ursachen verbleiben bis zu 5 % der Bevölkerung von einer ungeklärten chronischen Diarrhö betroffen. In diesem Kollektiv findet sich in bis zu 50 % als Ursache eine chologene Diarrhö.Die verschiedenen Pathophysiologien, die zu einer chologenen Diarrhö führen, sind gut charakterisiert. Danach lässt sich die chologene Diarrhö in einen primären, einen sekundären und einen tertiären Subtyp unterteilen. Allen Ursachen gemein sind die erhöhte Menge an Gallensäuren im Kolon und im Fäzes und die dadurch bedingte sekretorisch-osmotische Diarrhö, bei schwereren Formen in Kombination mit einer Steatorrhö. Die Diagnostik der chologenen Diarrhö folgt einem klaren Algorithmus, der neben der Ursachensuche und dem diagnostischen Therapieversuch den 75SeHCAT-Test als Referenzverfahren für den Nachweis eines Gallensäurenverlusts angibt. Aufgrund der Chronizität der Beschwerden und der Notwendigkeit einer dauerhaften, lebenslangen Therapie scheint eine einmalige sichere Diagnosestellung prinzipiell sinnvoll; der Test ist allerdings derzeit nur an wenigen Zentren verfügbar. Die Therapie umfasst neben der Behandlung identifizierbarer Grundkrankheiten den Einsatz von Gallensäuren bindenden Arzneimitteln, Ernährungsempfehlungen und Vitaminsubstitutionen.Der vorliegende Übersichtsartikel fasst Pathophysiologie und Stellenwert der chologenen Diarrhö zusammen und diskutiert die aktuelle Diagnostik und Therapie.
143	Macrophage Activation Syndrome Upon Initiation of Adalimumab in a Patient With Longstanding Rheumatoid Arthritis.	33633871	Macrophage activation syndrome (MAS) is a subset of hemophagocytic lymphohistiocytosis (HLH) described in patients with rheumatological disorders. Some triggers of MAS and HLH include infection, malignancy, rheumatological disease, HIV, and rarely medications such as immunosuppressants. In recent medical literature, biologic agents are increasingly recognized as a potential trigger, but the mechanism behind this remains poorly understood. We describe the case of a patient who developed MAS after initiating adalimumab and propose a potential pathophysiological link between biologics and this syndrome.
144	Applying an Age-specific Definition to Better Characterize Etiologies and Outcomes in Neonatal Acute Liver Failure.	33633086	Neonatal acute liver failure (ALF) is a rare disease with high mortality for which no standard age-specific definition exists. To advance the understanding of neonatal ALF, we characterize the etiology, presenting features, treatment, and outcomes in infants within 1 month of life.We performed a single-center 11-year retrospective chart review of neonates ≤30 days of life with ALF as defined by an INR of ≥2.0. Comparisons were made by etiology and survival with native liver (SNL). Estimated survival was performed using the Kaplan-Meier method.Forty-three patients met inclusion criteria for neonatal ALF. Etiologies included viral infection (23%), gestational alloimmune liver disease with neonatal hemochromatosis (GALD-NH) (21%), cardiac-associated ischemia (16%), other ischemia (14%), genetic etiologies (9%), Trisomy 21-associated myelodysplasia (TAM) (7%), hemophagocytic lymphohistiocytosis (HLH) (2%), and not identified (7%). Infants with viral etiologies had the highest alanine aminotransferase (ALT) at presentation (1179 IU/L, interquartile range [IQR] 683-1585 IU/L) in contrast to low levels in GALD-NH (23 IU/L, IQR 18-64 IU/L). Across all etiologies, only 33% were alive at 1 year. Overall median survival was 74 days; 17 days for viral infection and 74 days for GALD-NH. Among laboratory values at presentation, alpha-fetoprotein (AFP) was significantly higher in patients that survived with their native liver (P = 0.04).Overall, outcome for neonatal ALF is poor. Although initial laboratory values can differentiate viral infection or GALD-NH, further studies are needed to identify laboratory parameters that predict SNL by etiology to ultimately improve patient outcomes.
145	A "ligand-targeting" peptide-drug conjugate: Targeted intracellular drug delivery by VEGF-binding helix-loop-helix peptides via receptor-mediated endocytosis.	33630870	As a new alternative to antibody-drug conjugates, we generated "ligand-targeting" peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity (KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy.
146	Hemophagocytic Lymphohistiocytosis Secondary to Prostatic Adenocarcinoma.	33628667	Hemophagocytic lymphohistiocytosis (HLH) is caused by excessive immune activation. It can be primary in the setting of genetic defects or secondary in the setting of infection, inflammation, and malignancy. Here we present the fourth reported case of secondary HLH in association with prostatic adenocarcinoma and the diagnostic challenges of this rare, life-threatening condition. This is a 78-year-old male who presented to the hospital with fever and generalized weakness for three days and found to have splenomegaly, pancytopenia, markedly elevated transaminases, and ferritin. Bone marrow biopsy revealed hemophagocytes. He underwent an extensive evaluation to identify the etiology. Para-aortic lymph node biopsy was consistent with prostatic adenocarcinoma. The diagnosis of HLH needs a high index of suspicion because the presentation is nonspecific. HLH is a rapidly progressive and potentially fatal condition underscoring the need for a prompt evaluation if this condition is suspected.
147	Busulfan-based reduced toxicity conditioning regimen used in hematopoietic stem cell transplantation in HLH patients results in sustained donor chimerism.	33625293	NaN
148	Cytomorphologic Features Found in Cerebrospinal Fluid Specimens of Hemophagocytic Lymphohistiocytosis Patients.	33624013	Central nervous system involvement is present in 70% of patients with hemophagocytic lymphohistiocytosis (CNS-HLH). CNS-HLH is defined by neurologic deficits, neuroimaging abnormalities, or positive cerebrospinal fluid (CSF) findings. The CSF cytomorphologic spectrum of CNS-HLH, however, has not been well investigated.A retrospective review was performed on 64 CSF specimens from pediatric and adult patients with HLH. Ten patients had clinicoradiologic evidence of CNS involvement.We identified five CSF cytomorphologic patterns: (1) hemophagocytosis, (2) vacuolated macrophages without evidence of hemophagocytosis, (3) monocytes and/or nonvacuolated macrophages, (4) acellular specimens, and (5) bloody specimens. Patterns 1 and 2 were common in CNS-HLH and rare in patients without CNS involvement. The CSF cytomorphologic patterns did not correlate well with WBC counts or protein concentration.Our study offers a comprehensive view of the cytomorphologic features seen in CSF specimens from patients with HLH.
149	Study of the diagnostic criteria for hemophagocytic lymphohistiocytosis in neonatal and pediatric patients with severe sepsis or septic shock.	33622175	Septic shock is a major public health concern. However, the clinical and laboratory criteria for sepsis overlap with those for hemophagocytic lymphohistiocytosis (HLH), and their differentiation can be challenging. The aim of this study was to compare HLH criteria among patients diagnosed with neonatal sepsis and childhood sepsis and to study the outcomes in patients fulfilling the diagnostic criteria for HLH. A cross-sectional study included 50 neonates and children with severe sepsis and/or septic shock. Clinical and laboratory data and HLH diagnostic criteria were studied in relation to patients outcome. Of all patients, 18% fulfilled three of the eight HLH diagnostic criteria, 2% fulfilled four criteria, and 4% fulfilled five criteria. All patients who fulfilled three or more of the criteria died. Mortality was higher in the presence of more positive HLH criteria and in pediatric age groups. However, the distributions of the HLH criteria were comparable for pediatric and neonatal patients with severe sepsis/septic shock, and their mortality rates were not significantly different when based on the criteria.
150	Cytokine release syndrome in COVID-19: a major mechanism of morbidity and mortality.	33616462	The coronavirus disease 2019 (COVID-19) triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) erupted in Hubei Province of China in December 2019 and has become a pandemic. Severe COVID-19 patients who suffer from acute respiratory distress syndrome (ARDS) and multi-organ dysfunction have high mortality. Several studies have shown that this is closely related to the cytokine release syndrome (CRS), often loosely referred to as cytokine storm. IL-6 is one of the key factors and its level is positively correlated with the severity of the disease. The molecular mechanisms for CRS in COVID-19 are related to the effects of the S-protein and N-protein of the virus and its ability to trigger NF-κB activation by disabling the inhibitory component IκB. This leads to activation of immune cells and the secretion of proinflammatory cytokines such as IL-6 and TNF-α. Other mechanisms related to IL-6 include its interaction with GM-CSF and interferon responses. The pivotal role of IL-6 makes it a target for therapeutic agents and studies on tocilizumab are already ongoing. Other possible targets of treating CRS in COVID-19 include IL-1β and TNF-α. Recently, reports of a CRS like illness called multisystem inflammatory syndrome in children (MIS-C) in children have surfaced, with a variable presentation which in some cases resembles Kawasaki disease. It is likely that the immunological derangement and cytokine release occurring in COVID-19 cases is variable, or on a spectrum, that can potentially be governed by genetic factors. Currently, there are no approved biological modulators for the treatment of COVID-19, but the urgency of the pandemic has led to numerous clinical trials worldwide. Ultimately, there is great promise that an anti-inflammatory modulator targeting a cytokine storm effect may prove to be very beneficial in reducing morbidity and mortality in COVID-19 patients.
151	Increasing ferritin predicts early death in adult hemophagocytic lymphohistiocytosis.	33595184	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of pathologic immune activation. Most studies on adult HLH have evaluated prognostic factors for overall survival; factors predicting early mortality have not been sufficiently investigated.This was a collaborative study between Henry Ford Hospital and Barnes-Jewish Hospital. We identified all adult HLH patients with at least 2 ferritin levels within 30 days from admission.One-hundred twenty-four patients were identified. There were 77 males and 47 females; the median age at diagnosis was 48 years. Multivariate analysis showed that age (OR = 11.41; 95% CI:2.71-48.04; P = .001), hepatomegaly (OR = 15.68; 95% CI:3.24-75.96; P = .001), hyponatremia (OR = 5.94; 95% CI:1.76-20.1; P = .004), hypoalbuminemia (OR = 7.47; 95% CI:2.08-26.85; P = .002), and increasing ferritin levels (OR = 19.46; 95% CI:4.69-80.71; P < .001) were significant predictors of 30-day mortality. Patients with declining ferritin by more than 35% from the ferritin peak were more likely to survive the first 30 days of admission (OR = 4.33; 95% CI:1.04-18.1; P = .033). By risk stratifying our cohort, we identified changes in ferritin levels to be the most significant prognostic factor of 30-day mortality among other risk factors. Further investigating the prognostic utility of ferritin showed that increasing ferritin during the 1st week of admission (data available for 44 patients) was the only significant predictor of 30-day mortality.To the best of our knowledge, this is the first study reporting changes in ferritin to be a predictor for early death in adult HLH. Changes in ferritin might be a useful indicator of adult HLH disease activity and early prognosis.
152	Efficacy of moderately dosed etoposide in macrophage activation syndrome - hemophagocytic lymphohistiocytosis (MAS-HLH).	33589555	Macrophage activation syndrome (MAS) constitutes one subtype of the hyperinflammatory syndrome hemophagocytic lymphohistiocytosis (HLH), and the term MAS-HLH was recently proposed for HLH with underlying autoimmune/autoinflammatory conditions. The mortality of MAS-HLH has been estimated to 5-10%. Here we report our experiences with moderately dosed etoposide in severe MAS-HLH, administered with the objective to effectively reduce severe hyperinflammatory activity with limited side effects.In addition to conventional anti-inflammatory treatment, moderately dosed etoposide was administered to seven children affected by rapidly progressing MAS-HLH with central nervous system (n=5) and/or pulmonary (n=5) involvement. Three had underlying systemic onset juvenile idiopathic arthritis (sJIA), two atypical sJIA (no arthritis at diagnosis), and two systemic lupus erythematosus. We performed lymphocyte cytotoxicity analyses in all seven and genetic analyses in six.All children promptly responded to moderately dosed etoposide (50-100 mg/m2 once weekly), added to conventional MAS-HLH treatment which was considered insufficient. The mean accumulated etoposide dose was 671 mg/m2 (range 300-1,050 mg/m2), as compared to 1,500 mg/m2 recommended the first 8 weeks of the HLH-94/HLH-2004 protocols. One child developed neutropenic fever and another neutropenic sepsis (neutrophils 0.3x109/L at therapy onset). Five/seven children had low percentages (<5%) circulating NK-cells prior to or in association with diagnosis; NK-cell activity was pathologically low in two/five children studied. Disease-causing variants in HLH-associated genes were not found. All children were alive at latest follow-up (2-9 years after onset); neurological symptoms had normalized in four/five affected children.Moderately dosed etoposide may be beneficial in severe and/or refractory MAS-HLH.
153	Non-EBV infection-associated hemophagocytic lymphohistiocytosis: a distinct subgroup where pathogen-directed therapy is essential and favorable outcomes are expected.	33586592	EBV is the most prevalent cause of infection-associated hemophagocytic lymphohistiocytosis (IAHLH), non-EBV IAHLH is observed clinically but less documented. We conducted a retrospective research enrolled 36 cases from 1/1/2015 to 31/12/2019. Intriguingly, 92% cases were immunocompetent individuals prior to the onset of HLH. Clinically, the cardinal features were prolonged high fever, splenomegaly and hemophagocytosis. Bicytopenia occurred in most patients, besides, liver dysfunction was characterized by increased transaminase, bilirubin, alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (γ-GGT) and lactate dehydrogenase (LDH). Immunomodulatory agents should be added to control the overwhelming inflammatory storm without delay. Once a certain pathogen was identified as the causative factor of HLH, cytotoxic agents were withdrawn, specific pathogen-directed treatment was initiated. Further, glucocorticoids were tapered off when a stable state of HLH was achieved. After treatment, about 70% patients were fully recovered without relapse. All in all, non-EBV IAHLH is a special group of HLH with admirable outcome.
154	[Hemophagocytic lymphohistiocytosis in critically ill patients].	33580314	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammation syndrome. In adults, secondary HLH is mostly observed. HLH is often triggered by infections, malignancies or autoimmune disorders. However, HLH cases in association with immunotherapies have been described recently. HLH in critically ill patients is often difficult to differentiate from sepsis. Both conditions can also be present at the same time. Early diagnosis and timely initiation of an adequate immunosuppressive therapy are essential for the further course and the prognosis of HLH. Therefore, HLH should represent a differential diagnosis in critically ill patients with persistent fever and additional symptoms (e.g. enlarged spleen, neurologic symptoms) or laboratory parameters (e.g. hyperferritinemia, cytopenia, increased transaminases) compatible with HLH. The diagnosis of HLH is made using the HLH-2004 criteria. The probability of the presence of HLH can be calculated using the HScore. High-dose corticosteroids represent the cornerstone of HLH treatment. Etoposide, immunoglobulins, anakinra or other drugs are added depending on the trigger. The course of HLH is influenced by the time of treatment initiation, the underlying trigger and the response to treatment. Generally, the prognosis of critically ill HLH patients is poor.Bei der hämophagozytischen Lymphohistiozytose (HLH) handelt es sich um ein Hyperinflammationssyndrom bedingt durch aberrant aktivierte Makrophagen und T‑Zellen. Beim Erwachsenen ist in erster Linie die erworbene Form anzutreffen. Häufige Auslöser sind Infektionen, Malignome und Autoimmunerkrankungen. Zuletzt wurden zudem zunehmend Fälle berichtet, in denen das Auftreten im Zusammenhang mit stattgehabten Immuntherapien zu sehen war. Auf der Intensivstation ist die HLH aufgrund des ähnlichen klinischen Erscheinungsbilds oft schwer von der Sepsis abzugrenzen. Zum Teil liegen beide zeitgleich vor. Die frühzeitige Diagnosestellung und Einleitung einer adäquaten immunsuppressiven Therapie ist für den weiteren Verlauf und die Prognose der HLH essenziell. Deshalb muss bei kritisch kranken Patienten mit persistierendem Fieber und entsprechenden Symptomen (z. B. Splenomegalie, neurologische Auffälligkeiten) oder Laborveränderungen (z. B. erhöhter Ferritinwert, Zytopenie von 2 oder 3 Zellreihen, erhöhte Transaminasen) das Vorliegen einer HLH in Betracht gezogen werden. Die Diagnose wird mithilfe der HLH-2004-Kriterien gestellt. Mit dem HScore kann die Wahrscheinlichkeit des Vorliegens einer HLH berechnet werden. Hochdosierte Kortikosteroide stellen den Grundpfeiler der HLH-Therapie dar. Je nach Auslöser werden Etoposid, Immunglobuline, Anakinra oder weitere Medikamente ergänzt. Der Verlauf hängt neben einem frühzeitigen Behandlungsbeginn vom Auslöser sowie dem Ansprechen auf die Therapie ab. Insgesamt ist die Prognose der HLH trotz maximaler intensivmedizinischer Behandlung ungünstig und sie ist mit einer hohen Letalität assoziiert.
155	Central Nervous System Hemophagocytic Lymphohistiocytosis (CNS-HLH) from Leptomeningeal Anaplastic Large Cell Lymphoma : Mild Clinical Neurologic Syndrome with Extensive Multifocal White Matter Disease.	33576835	NaN
156	Systemic and Nodular Hyperinflammation in a Patient with Refractory Familial Hemophagocytic Lymphohistiocytosis 2.	33570715	Familial hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome resulting from defective cytotoxicity. A previously healthy 3-month-old female presented with fever, irritability, abdominal distention, and tachypnea. She ultimately met all eight HLH-2004 diagnostic criteria, accompanied by elevated CXCL9. Initial empiric anti-inflammatory treatment included anakinra and IVIg, which stabilized ferritin and cytopenias. She had molecular and genetic confirmation of perforin deficiency and was started on dexamethasone and etoposide per HLH-94. She clinically improved, though CXCL9 and sIL-2Ra remained elevated. She was readmitted at week 8 for relapsed HLH without clear trigger and HLH-94 induction therapy was reinitiated. Her systemic HLH symptoms failed to respond and she soon developed symptomatic CNS HLH. She was incidentally found to have multifocal lung and kidney nodules, which were sterile and consisted largely of histiocytes and activated, oligoclonal CD8 T cells. The patient had a laboratory response to salvage therapy with alemtuzumab and emapalumab, but progressive neurologic decline led to withdrawal of care. This report highlights HLH foci manifest as pulmonary/renal nodules, demonstrates the utility of monitoring an array of HLH biomarkers, and suggests possible benefit of earlier salvage therapy.
157	Macrophage activation syndrome in a newborn: report of a case associated with neonatal lupus erythematosus and a summary of the literature.	33568193	Macrophage activation syndrome (MAS) is a life-threatening hyperinflammatory syndrome and is caused by a severely dysregulated immune response. It has rarely been associated with neonatal lupus.We present a female neonate with MAS born to a mother who had cutaneous lupus erythematosus with circulating anti-nuclear antibodies (ANA), anti-SSA, anti-SSB and anti-extractable nuclear antigen (anti-ENA) antibodies. Because of neonatal lupus (NLE) with a total atrioventricular block, epicardial pacemaker implantation was required on the sixth day of life. Following surgery she developed non-remitting fever and disseminated erythematous skin lesions. A diagnosis of MAS was made based on these symptoms, with hyperferritinemia, elevated transaminases, hypertriglyceridemia, and a skin biopsy that showed hemophagocytosis. Our patient was treated with steroids for 3 months with good effect. No relapse has occurred.MAS is a rare complication of neonatal lupus that may be difficult to diagnose, but needs to be treated promptly. In this article, pathogenesis and overlap of MAS and hemophagocytic lymphohistiocytosis (HLH) has been described. Diagnosis of MAS can be difficult. Different diagnostic criteria are used in both diagnosing MAS and HLH. Validated criteria for diagnosis of MAS in other disease than systemic onset JIA have not been validated yet. In NLE, diagnosing MAS is even more difficult, since skin lesions are already common in NLE. We show the potential additional value of skin biopsy in diagnosing MAS.
158	Wolman's disease presenting with secondary hemophagocytic lymphohistiocytosis: a case report from Saudi Arabia and literature review.	33568092	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal syndrome that is characterized by strong activation of the immune system from hyperinflammatory cytokines. Symptoms of HLH patients include fever, hepatosplenomegaly, cytopenia, and hyperferritinemia. Inherited HLH is classified as primary, whereas secondary HLH (sHLH) occurs when acquired from non-inherited reasons that include severe infection, immune deficiency syndrome, autoimmune disorder, neoplasm, and metabolic disorder. Wolman's disease (WD) is a rare and fatal infantile metabolic disorder caused by lysosomal acid lipase deficiency, that exhibits similar clinical signs and symptoms as HLH. This paper reports the case of an infant diagnosed with WD and who presented with sHLH.A 4-month-old infant presenting with hepatosplenomegaly, failure to thrive, and other abnormalities. WD diagnosis was confirmed by the presence of the LIPA gene homozygous deletion c.(428 + 1_967-1)_(*1_?)del. The infant also met the HLH-2004 diagnostic criteria.Metabolic disorder such as WD should be investigated in infants fulfilling the HLH criteria to diagnose the underlying condition. More studies are needed to understand the link between WD and sHLH and to identify appropriate therapies.
159	Therapeutic Drug Monitoring and Pharmacokinetic Analysis of Cyclosporine in a Pediatric Patient with Hemophagocytic Lymphohistiocytosis Complicated by Diabetes Insipidus: A Grand Round.	33560100	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease. Initial therapy is based on etoposide, dexamethasone, and cyclosporine (CSA). The pharmacokinetics (PKs) of CSA and other drugs are sometimes altered in patients with HLH complicated by diabetes insipidus (DI) but the precise mechanisms remain unknown.In this study, the authors present a case of a 4-year-old boy with HLH complicated by DI. CSA concentrations were determined by enzyme multiplied immunoassay technique; noncompartmental PK analysis of the plasma concentration-time data was performed using PKSolver; and linear regression analysis was performed to determine linearity of relationship between urine output and C0 levels of CSA.Although C0 values of CSA were lower than the target levels, the patient was successfully treated and a good clinical outcome was achieved. Linear regression analysis showed a strong negative correlation between urine output and the serum trough concentration (C0) of CSA, pharmacokinetic analysis showed the main PK parameters of CSA as follows: C0, 50.2 mcg/L; peak concentration (Cmax), 723.4 mcg/L; area under the curve0-24, 7478.2 mcg·h/L; clearance, 0.77 L/h/kg, elimination half-life, 5.3 hours, and volume of distribution, 6.0 L/kg.To the best of the authors' knowledge, this is the first report of the CSA PK profile in a patient with HLH complicated by DI. The authors suppose that a large fluid output and input leads to extensive CSA distribution. These results suggest that the monitoring of the Cmax and area under the curve of CSA might be more clinically and pharmacokinetically significant than that of C0 in patients with HLH complicated by DI. This case highlights the importance of therapeutic drug monitoring and demonstrates PK parameters of CSA in a pediatric patient with HLH complicated by DI.
160	Ruxolitinib-combined doxorubicin-etoposide-methylprednisolone regimen as a salvage therapy for refractory/relapsed haemophagocytic lymphohistiocytosis: a single-arm, multicentre, phase 2 trial.	33559893	We performed a multicentre, non-randomised trial (NCT03533790) to investigate the efficacy of ruxolitinib combined with the doxorubicin-etoposide-methylprednisolone (Ru-DEP) regimen as a salvage therapy for refractory/relapsed haemophagocytic lymphohistiocytosis (HLH). All patients failing to achieve a complete or partial response 2 weeks after initial HLH-94/HLH-04 regimen or relapsed after remission were enrolled in the study between June 2018 and June 2019. The efficacy was evaluated 2 weeks after initiating Ru-DEP salvage therapy. Fifty-four eligible patients with refractory/relapsed (R/R) HLH were enrolled. One case could not be evaluated for efficacy. Excluding 12 patients who had previously received the DEP regimen, the overall response rate was 32 of 41 (78·0%) patients, with eight of 41 (19·5%) achieving complete response and 24 of 41 (58·5%) attaining a partial response. Of the R/R HLH patients who had previously received the DEP regimen, 7 of 12 (58·3%) achieved a partial response. Ferritin and soluble CD25 concentrations were significantly lower (P < 0·05), while the platelet count increased significantly (P = 0·034), and triglycerides decreased significantly (P = 0·002) compared with those before treatment. The Ru-DEP regimen may be a safe and effective salvage therapy, remaining effective in refractory/relapsed HLH following DEP treatment, especially in macrophage activation syndrome. In addition, the regimen can be considered for patients with contraindications to glucocorticoid, especially those with gastrointestinal bleeding.
161	Management of hemophagocytic lymphohistiocytosis (HLH) associated with chimeric antigen receptor T-cell (CAR-T) therapy using anti-cytokine therapy: an illustrative case and review of the literature.	33559517	NaN
162	Inferences of glia-mediated control in Caenorhabditis elegans.	33559247	Astrocytes modulate synaptic transmission; yet, it remains unclear how glia influence complex behaviors. Here, we explore the effects of Caenorhabditis elegans astrocyte-like cephalic glia (CEPglia ) and the glia-specific bHLH transcription factor HLH-17 on mating behavior and the defecation motor program (DMP). In C. elegans, male mating has been explicitly described through the male tail circuit and is characterized by coordination of multiple independent behaviors to ensure that copulation is achieved. Furthermore, the sex-specific male mating circuitry shares similar components with the DMP, which is complex and rhythmic, and requires a fixed sequence of behaviors to be activated periodically. We found that loss of CEPglia reduced persistence in executing mating behaviors and hindered copulation, while males that lacked HLH-17 demonstrated repetitive prodding behavior that increased the time spent in mating but did not hinder copulation. During the DMP, we found that posterior body wall contractions (pBocs) and enteric muscle contractions (EMCs) were differentially affected by loss of HLH-17 or CEPglia in males and hermaphrodites. pBocs and EMCs required HLH-17 activity in both sexes, whereas loss of CEPglia alone did not affect DMP in males. Our data suggest that CEPglia mediate complex behaviors by signaling to the GABAergic DVB neuron, and that HLH-17 activity influences those discrete steps within those behaviors. Collectively, these data provide evidence of glia as a link in cooperative regulation of complex and rhythmic behavior that, in C. elegans links circuitry in the head and the tail.
163	Biophysical Properties and Cytotoxicity of Feruloylated Helix Lucorum Hemocyanin.	33558913	For the first time Helix lucorum hemocyanin (HlH) has been feruloylated. Two HlH conjugates with 40- and 120- ferulic acid residues were prepared, denoted as FA-HlH-1 and FA-HlH-2. Expectedly, the feruloylation of HlH induced a rearrangement of the protein molecule, a decrease in the ?-helical structure at the expense of ß-structures was observed. Besides, the FA-HlH conjugates were more prone to aggregation, which is probably due to the stabilization of the partially unfolded protein molecules by non-covalent bonding. Interestingly, the thermal stability of HlH was not affected by the modification. The native and feruloylated HlH were not toxic to normal fibroblasts (BJ cells). We observed a decrease in cell viability of breast cancer MCF-7 cells to about 66% after a 48h exposure to 70 µg/well of FA-HlH-2.
164	[Therapeutic Effect of PE+CRRT Combined with Chemotherapy on Children with Severe EBV-HLH and Non-EBV-HLH].	33554831	To investigate the difference in the therapeutic effect of plasma exchange and continuous renal replacement therapy (PE+CRRT) combined with chemotherapy in the treatment of children with severe Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and non-EBV-HLH.The clinical data of 21 cases of all children with severe HLH treated by PE+CRRT combined with chemotherapy from January 2017 to January 2020 were collected and retrospectively analyzed. According to the presence of EBV infection, the children were divided into EBV+ group and EBV- group. The differences of the observation indexes between the children in the two groups and the improvement of the observation indexes of each group before and after treatment were compared.Among the 21 children, 14 were divided into the EBV+ group and 7 were divided into the EBV- group. There was no difference in age, sex and the number of organ damage between the children in the two groups (P>0.05). Duration of PE+CRRT was longer in the EBV+ group as compared with the EBV- group (P<0.05). Before treatment, the ANC in the EBV+ group was lower than that in the EBV- group (P<0.05), and there was no significant difference in the other observation indexes between the two groups (P>0.05). After treatment, Hb, Fib, APTT, SF, ALT, AST, LDH, Alb, CHE, TBil and TBA of the children in the EBV+ group were significantly improved as compared with those before treatment (P<0.05), but ANC, PLT, TG showed not improve (P>0.05); Fib, APTT, SF, LDH, Alb, and CHE in the EBV- group were significantly as improved compared with those before treatment (P<0.05), while the ANC, PLT, Hb, TG, ALT, AST, TBil, and TBA were not improved (P>0.05). After treatment, the differences of Fib and SF in the children between the EBV+ group and the EBV- group were statistically significant (P<0.05), and there was no significant difference in the other observation indexes of the children between the two groups (P>0.05). Compared with the children before treatment, EBV-DNA in the EBV+ group were decreased significantly in 2-4 weeks after treatment (P<0.05). After PE+CRRT combined with chemotherapy, the overall survival rate of the children with severe HLH was 66.7%, and there was no significant difference in overall survival rate between EBV+ group and EBV- group (P>0.05).PE+CRRT combined with chemotherapy can reduce serum ferritin quickly, then improve organ function, and increase the overall survival rate of severe HLH, and it is a good effect on children with severe EBV-HLH and non-EBV-HLH.PE+CRRT联合化学免疫疗法治疗儿童重型EBV-HLH和非EBV-HLH的疗效观察.探讨血浆置换与连续性肾脏替代疗法(PE+CRRT)联合化学免疫疗法在儿童重型EB病毒相关噬血细胞综合征（EBV-HLH）和非EBV-HLH中疗效的差异.收集并回顾性分析2017年1月至2020年1月所有接受PE+CRRT联合化学免疫疗法的21例重型HLH患儿的临床资料，根据是否存在EBV感染，将患儿分为EBV+组和EBV-组，比较两组间各观察指标的差异及各组治疗前后各观察指标的改善情况.21例患儿中，EBV+组14例，EBV-组7例，两组年龄、性别及器官损害数无差异（P>0.05）。与EBV-组相比，EBV+组行PE+CRRT的持续时间更长（P<0.05)。治疗前，EBV+组ANC较EBV- 组明显降低（P<0.05），其余观察指标两组间比较差异无统计学意义（P>0.05）。治疗后，EBV+组Hb、Fib、APTT、SF、ALT、AST、LDH、Alb、CHE、TBil及TBA较治疗前均可见明显改善（P<0.05），ANC、PLT、TG与治疗前相比未见改善（P>0.05）；EBV- 组Fib、APTT、SF、LDH、Alb、CHE较治疗前明显改善（P<0.05），ANC、PLT、Hb、TG、ALT、AST、TBil、TBA与治疗前相比未见改善（P>0.05）。治疗后，EBV+组和EBV-组间Fib、SF差异具有统计学意义（P<0.05），两组间其余观察指标差异无统计学意义（P>0.05）。与治疗前相比，治疗后2-4周，EBV+组EBV-DNA显著降低(P<0.05)。接受PE+CRRT联合化学免疫疗法后，重型HLH患儿总生存率为66.7%，EBV+组和EBV- 组患儿总生存率差异无统计学意义（P>0.05）.PE+CRRT联合化学免疫疗法可快速降低血清铁蛋白、改善器官功能，提高重型HLH总生存率，且对儿童重型EBV-HLH和非EBV-HLH均有较好的疗效，是治疗HLH的有效方法.
165	Inhibitor of Differentiation 4 (ID4) represses mammary myoepithelial differentiation via inhibition of HEB.	33554073	Inhibitor of differentiation (ID) proteins dimerize with basic HLH (bHLH) transcription factors, repressing transcription of lineage-specification genes across diverse cellular lineages. ID4 is a key regulator of mammary stem cells; however, the mechanism by which it achieves this is unclear. Here, we show that ID4 has a cell autonomous role in preventing myoepithelial differentiation of basal cells in mammary organoids and in vivo. ID4 positively regulates proliferative genes and negatively regulates genes involved in myoepithelial function. Mass spectrometry reveals that ID4 interacts with the bHLH protein HEB, which binds to E-box motifs in regulatory elements of basal developmental genes involved in extracellular matrix and the contractile cytoskeleton. We conclude that high ID4 expression in mammary basal stem cells antagonizes HEB transcriptional activity, preventing myoepithelial differentiation and allowing for appropriate tissue morphogenesis. Downregulation of ID4 during pregnancy modulates gene regulated by HEB, promoting specialization of basal cells into myoepithelial cells.
166	Hemophagocytic Lymphohistiocytosis Secondary to Disseminated Histoplasmosis in Rheumatologic Disease.	33552603	Hemophagocytic lymphohistiocytosis (HLH) was originally described in pediatric patients presenting with fever, hepatosplenomegaly, and blood cell abnormalities. Later, HLH was recognized to occur in adults, often associated with hematologic malignancies or serious infections.Patients presenting with HLH are critically ill, and rapid diagnosis is key. In adults, the search for the trigger must begin promptly as time to diagnosis effects survival. The underlying trigger in our patients was Histoplasma capsulatum infection, which is rare in the southwestern United States. Prompt diagnosis led to recovery in one patient, while the other did not survive.
167	Elevated serum gasdermin D N-terminal implicates monocyte and macrophage pyroptosis in adult-onset Still's disease.	33550379	Elevation of serum IL-18 in adult-onset Still's disease (AOSD) and systemic JIA (sJIA) suggests the role of the inflammasome in these diseases. Gasdermin D is a pore-forming protein playing central roles in inflammasome-mediated inflammation, but its role in rheumatic disease is unknown. We aimed to elucidate the auto-inflammatory mechanisms in AOSD and sJIA.Patients with AOSD, sJIA, hemophagocytic lymphohistiocytosis (HLH) and Behçet's disease followed at Yokohama City University (YCU), or US National Institutes of Health (NIH) were included in the study. Disease activity was evaluated by the modified Pouchot score. Ferritin and N-terminal gasdermin D levels in serum and culture supernatant were measured by ELISA. Primary monocytes (Mo) were stimulated with GM-CSF or M-CSF and differentiated into M1 macrophages (Mφ) or M2Mφ, respectively. The number of Mo/Mφ and their viability were monitored over time.Patients with active AOSD and sJIA had increased levels of serum gasdermin D N-terminal, which correlated with serum ferritin and IL-18 levels. Mo-derived Mφ from active AOSD patients showed reduced cell viability and increased cell death. The number of cultured Mφ cells on day nine was negatively correlated with the serum ferritin and gasdermin D levels. Higher ferritin and gasdermin D levels were observed in the M1Mφ culture supernatant of active AOSD patients. Gasdermin D inhibitors reduced the pyroptosis-mediated ferritin release in Mo.Elevation of serum gasdermin D N-terminal provides evidence for inflammasome activation triggering gasdermin D-mediated Mo and Mφ pyroptosis in AOSD and possibly sJIA.
168	Scrub Typhus-Associated Hemophagocytic Lymphohistiocytosis: Not a Rare Entity in Pediatric Age Group.	33547467	Our goal was to study the demographic, clinical and laboratory profile and outcome of scrub typhus-associated hemophagocytic lymphohistiocytosis (HLH) in the pediatric age group.We conducted a prospective observational study in a tertiary care teaching hospital over a period of 1 year. Children in the age group of 1 month to 12 years with IgM ELISA positive for scrub typhus were included in the study. HLH was diagnosed using HLH-2004 criteria. Demographic, clinical and laboratory profile, treatment and outcome of HLH patients were noted and also compared with non-HLH scrub typhus patients.Among 58 children with scrub typhus infection, 18 had HLH. The mean age of patients with HLH was 35.3 ± 44.8 months and 61% were male. Anemia, thrombocytopenia and hyperferritinemia were seen in all the patients. Hypertriglyceridemia, hypofibrinogenemia and coagulopathy were noted in 78%, 56% and 44%, respectively. All the patients were treated with intravenous doxycycline for an average duration of 9.5 days. Intravenous immunoglobulin and methylprednisolone were given in 33% and 22% cases, respectively. Complications like acute respiratory distress syndrome (ARDS) (p = 0.001) and MODS (p = 0.004) were significantly high in the HLH group. Younger age (<3 years), fever > 7 days, presence of convulsion, ARDS and MODS were the clinical predictors of scrub typhus-associated HLH.HLH in scrub typhus infected children is being increasingly recognized. Younger age, prolonged fever, presence of convulsion, ARDS and MODS should alert clinicians of the risk of HLH. Treating the primary cause usually cures the disease and immunomodulator therapy need not be routinely administered.
169	Association of Long-Term Trajectories of Neighborhood Socioeconomic Status With Weight Change in Older Adults.	33544146	Studying long-term changes in neighborhood socioeconomic status (SES) may help to better understand the associations between neighborhood exposure and weight outcomes and provide evidence supporting neighborhood interventions. Little previous research has been done to examine associations between neighborhood SES and weight loss, a risk factor associated with poor health outcomes in the older population.To determine whether improvements in neighborhood SES are associated with reduced likelihoods of excessive weight gain and excessive weight loss and whether declines are associated with increased likelihoods of these weight outcomes.This cohort study was conducted using data from the National Institutes of Health-AARP (formerly known as the American Association of Retired Persons) Diet and Health study (1995-2006). The analysis included a cohort of 126 179 adults (aged 50-71 years) whose neighborhoods at baseline (1995-1996) were the same as at follow-up (2004-2006). All analyses were performed from December 2018 through December 2020.Living in a neighborhood that experienced 1 of 8 neighborhood SES trajectories defined based on a national neighborhood SES index created using data from the US Census and American Community Survey. The 8 trajectory groups, in which high, or H, indicated rankings at or above the sample median of a specific year and low, or L, indicated rankings below the median, were HHH (ie, high in 1990 to high in 2000 to high in 2010), or stable high; HLL, or early decline; HHL, or late decline; HLH, or transient decline; LLL, or stable low; LHH, or early improvement; LLH, or late improvement; and LHL, or transient improvement.Excessive weight gain and loss were defined as gaining or losing 10% or more of baseline weight.Among 126 179 adults, 76 225 (60.4%) were men and the mean (SD) age was 62.1 (5.3) years. Improvements in neighborhood SES were associated with lower likelihoods of excessive weight gain and weight loss over follow-up, while declines in neighborhood SES were associated with higher likelihoods of excessive weight gain and weight loss. Compared with the stable low group, the risk was significantly reduced for excessive weight gain in the early improvement group (odds ratio [OR], 0.87; 95% CI, 0.79-0.95) and for excessive weight loss in the late improvement group (OR, 0.89; 95% CI, 0.80-1.00). Compared with the stable high group, the risk of excessive weight gain was significantly increased for the early decline group (OR, 1.19; 95% CI, 1.08-1.31) and late decline group (OR, 1.13; 95% CI, 1.04-1.24) and for excessive weight loss in the early decline group (OR, 1.15; 95% CI, 1.02-1.28). The increases in likelihood were greater when the improvement or decline in neighborhood SES occurred early in the study period (ie, 1990-2000) and was substantiated throughout the follow-up (ie, the early decline and early improvement groups). Overall, we found a linear association between changes in neighborhood SES and weight outcomes, in which every 5 percentile decline in neighborhood SES was associated with a 1.2% to 2.4% increase in the risk of excessive weight gain or loss (excessive weight gain: OR, 1.01; 95% CI, 1.00-1.02 for women; OR, 1.02; 95% CI, 1.01-1.03 for men; excessive weight loss: OR, 1.02; 95% CI, 1.01-1.03 for women; OR, 1.02; 95% CI, 1.01-1.03 for men; P for- trend < .0001).These findings suggest that changing neighborhood environment was associated with changes in weight status in older adults.
170	Secretion of von Willebrand Factor and Suppression of ADAMTS-13 Activity by Markedly High Concentration of Ferritin.	33539188	Hyperferritinemia is associated with poor outcomes in critically ill patients with sepsis, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndromes (MAS) and coronavirus disease 19 (COVID-19). Autopsies of hyperferritinemic patients that succumbed to either sepsis, HLH, MAS or COVID-19 have revealed disseminated microvascular thromboses with von Willebrand factor (VWF)-, platelets-, and/or fibrin-rich microthrombi. It is unknown whether high plasma ferritin concentration actively promotes microvascular thrombosis, or merely serves as a prognostic biomarker in these patients. Here, we show that secretion of VWF from human umbilical vein endothelial cells (HUVEC) is significantly enhanced by 100,000 ng/ml of recombinant ferritin heavy chain protein (FHC). Ferritin fraction that was isolated by size exclusion chromatography from the plasma of critically ill HLH patients promoted VWF secretion from HUVEC, compared to similar fraction from non-critically ill control plasma. Furthermore, recombinant FHC moderately suppressed the activity of VWF cleaving metalloprotease ADAMTS-13. These observations suggest that a state of marked hyperferritinemia could promote thrombosis and organ injury by inducing endothelial VWF secretion and reducing the ADAMTS-13 activity.
171	Predictive Ki-67 Proliferation Index of Cervical Squamous Cell Carcinoma Based on IVIM-DWI Combined with Texture Features.	33531882	This study aims to determine whether IVIM-DWI combined with texture features based on preoperative IVIM-DWI could be used to predict the Ki-67 PI, which is a widely used cell proliferation biomarker in CSCC.A total of 70 patients were included. Among these patients, 16 patients were divided into the Ki-67 PI <50% group and 54 patients were divided into the Ki-67 PI ≥50% group based on the retrospective surgical evaluation. All patients were examined using a 3.0T MRI unit with one standard protocol, including an IVIM-DWI sequence with 10 b values (0-1,500 sec/mm2). The maximum level of CSCC with a b value of 800 sec/mm2 was selected. The parameters (diffusion coefficient (D), microvascular volume fraction (f), and pseudodiffusion coefficient (D ∗ )) were calculated with the ADW 4.6 workstation, and the texture features based on IVIM-DWI were measured using GE AK quantitative texture analysis software. The texture features included the first order, GLCM, GLSZM, GLRLM, and wavelet transform features. The differences in IVIM-DWI parameters and texture features between the two groups were compared, and the ROC curve was performed for parameters with group differences, and in combination.The D value in the Ki-67 PI ≥50% group was lower than that in the Ki-67 PI <50% group (P < 0.05). A total of 1,050 texture features were obtained using AK software. Through univariate logistic regression, mPMR feature selection, and multivariate logistic regression, three texture features were obtained: wavelet_HHL_GLRLM_ LRHGLE, lbp_3D_k_ firstorder_IR, and wavelet_HLH_GLCM_IMC1. The AUC of the prediction model based on the three texture features was 0.816, and the combined D value and three texture features was 0.834.Texture analysis on IVIM-DWI and its parameters was helpful for predicting Ki-67 PI and may provide a noninvasive method to investigate important imaging biomarkers for CSCC.
172	The conserved autoimmune-disease risk gene TMEM39A regulates lysosome dynamics.	33531362	TMEM39A encodes an evolutionarily conserved transmembrane protein and carries single-nucleotide polymorphisms associated with increased risk of major human autoimmune diseases, including multiple sclerosis. The exact cellular function of TMEM39A remains not well understood. Here, we report that TMEM-39, the sole Caenorhabditis elegans (C. elegans) ortholog of TMEM39A, regulates lysosome distribution and accumulation. Elimination of tmem-39 leads to lysosome tubularization and reduced lysosome mobility, as well as accumulation of the lysosome-associated membrane protein LMP-1. In mammalian cells, loss of TMEM39A leads to redistribution of lysosomes from the perinuclear region to cell periphery. Mechanistically, TMEM39A interacts with the dynein intermediate light chain DYNC1I2 to maintain proper lysosome distribution. Deficiency of tmem-39 or the DYNC1I2 homolog in C. elegans impairs mTOR signaling and activates the downstream TFEB-like transcription factor HLH-30. We propose evolutionarily conserved roles of TMEM39 family proteins in regulating lysosome distribution and lysosome-associated signaling, dysfunction of which in humans may underlie aspects of autoimmune diseases.
173	Ruxolitinib as adjunctive therapy for secondary hemophagocytic lymphohistiocytosis: A case series.	33523540	Hemophagocytic lymphohistiocytosis (HLH) is a cytokine storm syndrome associated with mortality rates of up to 88%. Standard therapy with high-dose glucocorticoids and etoposide used in adults is extrapolated from pediatric trials, with significant toxicity in older patients and those with poor performance status. The JAK1/2 inhibitor ruxolitinib has recently gained attention as a treatment option for HLH due to its broad cytokine-modulating abilities and safety profile. Herein we report our center's experience using ruxolitinib in the treatment of adult-onset secondary HLH.We report four patients with profound secondary HLH provoked by diverse triggers, including invasive pulmonary aspergillosis on background systemic lupus erythematosus, disseminated tuberculosis, and T-cell lymphoma treated with ruxolitinib as monotherapy or combination therapy in upfront and salvage settings.All four patients had rapid, sustained improvement in clinical status, inflammatory markers, and hematological cell counts followed by durable remission. Three patients developed manageable infectious complications postruxolitinib.This series demonstrates the effective use of JAK inhibition with ruxolitinib to control pathological immune activation in critically ill patients with secondary HLH and otherwise limited therapeutic options. JAK inhibition is also an area of urgent investigation for the treatment of cytokine storm associated with COVID-19.
174	COVID-19-associated cytokine storm syndrome and diagnostic principles: an old and new Issue.	33522893	SARS-CoV-2 has claimed 2,137,908 lives in more than a year. Some COVID-19 patients experience sudden and rapid deterioration with the onset of fatal cytokine storm syndrome (CSS), which have increased interest in CSS's mechanisms, diagnosis and therapy. Although the prototypic concept of CSS was first proposed 116 years ago, we have only begun to study and understand CSS for less than 30 years. Actually, diseases under CSS umbrella include familial/primary and secondary hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), infection-associated hemophagocytic syndrome, cytokine release syndrome (CRS), and cytokine storm (CS). Hematologic malignancies and autoimmune diseases that cause CSS are named malignancy-associated hemophagocytic syndrome (MAHS) and MAS, respectively. In-depth research on the pathogenesis of HLH/CSS has greatly increased the number of patients that were able to be definitively diagnosed with HLH/CSS. However, it should be emphasized that HLH/CSS diagnosis is difficult at the early stages due to the non-specific clinical signs and symptoms, which tends to result in missed and incorrect diagnoses. Therefore, clinicians should not only possess extensive clinical experience to ensure high sensitivity to the characteristics of HLH/CSS but must also be familiar with HLH-2004/2009 diagnostic criteria, and HScore methods. The paper concisely comment evolution of CSS classifications, cytokines associated with CSS, evolution of CSS diagnostic criteria and importance of the correct identification of hemophagocytes in diagnosing CSS, which is timely and may benefit clinicians familiar HLH-2004/2009 diagnostic criteria, and HScore methods. In addition, clinicians must also understand that there are some limitations to these diagnostic criteria. Abbreviations: aBMT: autologous bone marrow transplantation; CAR-T: chimeric antigen receptor-engineered T-cell; COVID-19: coronavirus disease 2019; CSS: cytokine storm syndrome; HLH: hemophagocytic lymphohistiocytosis; MAS: macrophage activation syndrome; CRS: cytokine release syndrome; CS: cytokine storm; MAHS: malignancy-associated hemophagocytic syndrome; IAHS: infection-associated hemophagocytic syndrome; fHLH/pHLH: familial/primary hemophagocytic lymphohistiocytosis; sHLH: secondary hemophagocytic lymphohistiocytosis; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TCR-T, T-cell receptor-engineered T-cell.
175	Disruption of CCR5 signaling to treat COVID-19-associated cytokine storm: Case series of four critically ill patients treated with leronlimab.	33521616	Coronavirus disease 2019 (COVID-19) is associated with considerable morbidity and mortality. The number of confirmed cases of infection with SARS-CoV-2, the virus causing COVID-19 continues to escalate with over 70 million confirmed cases and over 1.6 million confirmed deaths. Severe-to-critical COVID-19 is associated with a dysregulated host immune response to the virus, which is thought to lead to pathogenic immune dysregulation and end-organ damage. Presently few effective treatment options are available to treat COVID-19. Leronlimab is a humanized IgG4, kappa monoclonal antibody that blocks C-C chemokine receptor type 5 (CCR5). It has been shown that in patients with severe COVID-19 treatment with leronlimab reduces elevated plasma IL-6 and chemokine ligand 5 (CCL5), and normalized CD4/CD8 ratios. We administered leronlimab to 4 critically ill COVID-19 patients in intensive care. All 4 of these patients improved clinically as measured by vasopressor support, and discontinuation of hemodialysis and mechanical ventilation. Following administration of leronlimab there was a statistically significant decrease in IL-6 observed in patient A (p=0.034) from day 0-7 and patient D (p=0.027) from day 0-14. This corresponds to restoration of the immune function as measured by CD4+/CD8+ T cell ratio. Although two of the patients went on to survive the other two subsequently died of surgical complications after an initial recovery from SARS-CoV-2 infection.
176	Hemophagocytic Lymphohistiocytosis Associated with Respiratory Syncytial Virus Infection in an Immunocompetent Elderly Patient.	33520319	Hemophagocytic lymphohistiocytosis is a serious and potentially fatal disorder characterized by excessive immune system activation. The disorder is diagnosed mainly based on laboratory, clinical, and pathologic criteria. The spectrum comprises hereditary or "primary" HLH that comprises genetically heterogeneous conditions, occurring during childhood. The secondary form presents later in life and is associated with several conditions mainly malignancy, autoimmune diseases, viral or bacterial infections, and hematological diseases. We present the case of an 80-year-old female patient who initially presented with an acute viral syndrome secondary to respiratory syncytial virus. The hospital course was complicated by disseminated intravascular coagulation and shock with multiorgan failure. Extensive workup revealed that several of the criteria for hemophagocytic lymphohistiocytosis were met. A review of literature fails to identify cases of hemophagocytic lymphohistiocytosis associated with respiratory syncytial virus in immunocompetent adults. This case report provides further insight on RSV as a possible etiologic agent associated with HLH and the importance of early recognition of this fatal disorder in RSV-positive patients who show unpredictable clinical decompensation.
177	Helmet mask and tocilizumab for a patient with hemophagocytic lymphohistiocytosis syndrome and COVID-19: a case report.	33518836	The management of acute hypoxemic respiratory failure and the effect of antiviral drugs in patients with severe COVID-19 have been debated. This case presents the management of a 64-year-old man COVID-19 patient admitted to the Intensive Care Unit with fever, fatigue, shortness of breath and hemophagocytic lymphohistiocytosis syndrome. Helmet mask was successfully used to treat his hypoxemic respiratory failure without any aerosol problems. Tocilizumab, an antagonist interleukin-6, was intravenously infused as an alternative drug. After administration, the high level of IL-6, CRP, ferritin, D-dimer, triglyceride, and H-scores decreased, and the patient observed good clinical and laboratory improvements. In this case report, we describe the effect of noninvasive ventilation delivered by helmet mask and antiviral drugs, and the intravenous administration of tocilizumab in a patient with hemophagocytic lymphohistiocytosis syndrome and COVID-19.
178	A Recurrent Case of Adult-onset Still's Disease with Concurrent Acalculous Cholecystitis and Macrophage Activation Syndrome/Hemophagocytic Lymphohistiocytosis Successfully Treated with Combination Immunosuppressive Therapy.	33518559	We herein report the case of 21-year-old female diagnosed with adult-onset Still's disease (AOSD) three years earlier who presented with fever and right upper abdominal pain. She was diagnosed with acute acalculous cholecystitis (AAC) based on hepatic dysfunction, elevated C-reactive protein, and gallbladder wall thickening on abdominal ultrasound. Based on the presence of pancytopenia, hyperferritinemia, and hemophagocytosis by a bone marrow examination, she was diagnosed with macrophage activation syndrome (MAS)/hemophagocytic lymphohistiocytosis (HLH) which was refractory to glucocorticoid pulse therapy. The combination of intravenous cyclosporine A with glucocorticoids was able to successfully control the disease activity of AOSD-related AAC and MAS/HLH.
179	Haemophagocytic lymphohistiocytosis and liver failure-induced massive hyperferritinaemia in a male COVID-19 patient.	33516166	The authors present the case of a 58-year-old man with the unique combination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and, later on, haemophagocytic lymphohistiocytosis admitted to the intensive care unit. During his ICU stay the patient developed a variety of complications including acute respiratory distress syndrome, pulmonary embolism, right heart failure and suspected HLH leading to multiorgan failure and death. Despite the proven diagnosis of haemophagocytic lymphohistiocytosis, the excessively high ferritin levels of the patient did not seem fully explained by this diagnosis. Therefore, the authors want to highlight different causes of hyperferritinaemia in critically ill patients and underline the importance of differential diagnoses when interpreting continuously rising ferritin levels.
180	RhoG deficiency abrogates cytotoxicity of human lymphocytes and causes hemophagocytic lymphohistiocytosis.	33513601	Exocytosis of cytotoxic granules (CG) by lymphocytes is required for the elimination of infected and malignant cells. Impairments in this process underly a group of diseases with dramatic hyperferritinemic inflammation termed hemophagocytic lymphohistiocytosis (HLH). Although genetic and functional studies of HLH have identified proteins controlling distinct steps of CG exocytosis, the molecular mechanisms that spatiotemporally coordinate CG release remain partially elusive. We studied a patient exhibiting characteristic clinical features of HLH associated with markedly impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell exocytosis functions, who beared biallelic deleterious mutations in the gene encoding the small GTPase RhoG. Experimental ablation of RHOG in a model cell line and primary CTLs from healthy individuals uncovered a hitherto unappreciated role of RhoG in retaining CGs in the vicinity of the plasma membrane (PM), a fundamental prerequisite for CG exocytotic release. We discovered that RhoG engages in a protein-protein interaction with Munc13-4, an exocytosis protein essential for CG fusion with the PM. We show that this interaction is critical for docking of Munc13-4+ CGs to the PM and subsequent membrane fusion and release of CG content. Thus, our study illuminates RhoG as a novel essential regulator of human lymphocyte cytotoxicity and provides the molecular pathomechanism behind the identified here and previously unreported genetically determined form of HLH.
181	Primary Cutaneous Natural Killer/T-Cell Lymphoma: A Posttransplant Lymphoproliferative Disorder Demonstrated by 18F-FDG PET/CT.	33512953	A 24-year-old man with a history of hemophagocytic lymphohistiocytosis (HLH) presented with swelling of the left foot and skin ulcer. The patient received bone marrow transplantation for HLH 3 years ago for his HLH. Biopsy of left foot skin demonstrated primary cutaneous natural killer/T-cell lymphoma: a posttransplant lymphoproliferative disorder. FDG PET/CT images demonstrated multiple foci of abnormal accumulation in the body, especially in the skin. Follow-up PET/CT after chemotherapy demonstrated that most abnormal activities disappeared except for the lesion in the left foot.
182	T-cell activation profiles distinguish hemophagocytic lymphohistiocytosis and early sepsis.	33512385	Hemophagocytic lymphohistiocytosis (HLH) is a fatal disorder of immune hyperactivation that has been described as a cytokine storm. Sepsis due to known or suspected infection has also been viewed as a cytokine storm. Although clinical similarities between these syndromes suggest similar immunopathology and may create diagnostic uncertainty, distinguishing them is critical as treatments are widely divergent. We examined T-cell profiles from children with either HLH or sepsis and found that HLH is characterized by acute T-cell activation, in clear contrast to sepsis. Activated T cells in patients with HLH were characterized as CD38high/HLA-DR+ effector cells, with activation of CD8+ T cells being most pronounced. Activated T cells were type 1 polarized, proliferative, and displayed evidence of recent and persistent activation. Circulating activated T cells appeared to be broadly characteristic of HLH, as they were seen in children with and without genetic lesions or identifiable infections and resolved with conventional treatment of HLH. Furthermore, we observed even greater activation and type 1 polarization in tissue-infiltrating T cells, described here for the first time in a series of patients with HLH. Finally, we observed that a threshold of >7% CD38high/HLA-DR+ cells among CD8+ T cells had strong positive and negative predictive value for distinguishing HLH from early sepsis or healthy controls. We conclude that the cytokine storm of HLH is marked by distinctive T-cell activation whereas early sepsis is not, and that these 2 syndromes can be readily distinguished by T-cell phenotypes.
183	The Dilemma of Diagnosing Hemophagocytic Lymphohistiocytosis in Sickle Cell Disease.	33510976	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder due to uncontrolled activation of macrophage and cytokine release, which can be due to either genetic causes (familial) or secondary to infections malignancy and other less common cause. Parvovirus B19 rarely causes HLH. Diagnosing HLH in sickle cell disease, which inherently has high ferritin levels and pancytopenia, is particularly challenging. We are reporting HLH as a complication with parvovirus B19 infection in the background of sickle beta-thalassemia. Based on our search of available medical literature, this is the first case of HLH complicating parvovirus B19 infection in a pediatric age group with sickle beta-thalassemia.
184	Haemophagocytic Lymphohistiocytosis in an Elderly Patient after Recent Cardiac Surgery.	33510920	The underlying pathophysiology of haemophagocytic lymphohistiocytosis (HLH) is characterised by excessive inflammation and tissue destruction secondary to abnormal immune activation. The term primary HLH refers to a genetic abnormality that predisposes to the condition whereas secondary refers to HLH being triggered by an underlying condition such as infection (often Epstein Barr Virus), autoimmune, or neoplastic disease. Its variable clinical presentation poses an obstacle to prompt diagnosis in the elderly patient.A 70-year-old Caucasian man was admitted to hospital from a convalescence center with symptoms of fatigue, fever, decreased oral intake, and increasing shortness of breath on exertion. The patient was three weeks after coronary artery bypass grafting. Over the next two weeks, the patient continued to deteriorate both clinically and biochemically. The patient met criteria for haemophagocytic lymphohistiocytosis, likely driven by EBV infection. Bone marrow biopsy supported the diagnosis with evidence of active phagocytosis. The patient was commenced on high-dose dexamethasone and reviewed by haematology with further molecular testing confirming the diagnosis. Discussion. LH is becoming more common in older patients. We propose that new guidelines be developed to aid its prompt diagnosis in this age group.
185	The impact of introducing ambulance and delivery fees in a rural hospital in Tanzania.	33509195	Access to health care facilities is a key requirement to enhance safety for mothers and newborns during labour and delivery. Haydom Lutheran Hospital (HLH) is a regional hospital in rural Tanzania with a catchment area of about two million inhabitants. Up to June 2013 ambulance transport and delivery at HLH were free of charge, while a user fee for both services was introduced from January 2014. We aimed to explore the impact of introducing user fees on the population of women giving birth at HLH in order to document potentially unwanted consequences in the period after introduction of fees.Retrospective analysis of data from a prospective observational study. Data was compared between the period before introduction of fees from February 2010 through June 2013 and the period after from January 2014 through January 2017. Logistic regression modelling was used to construct risk-adjusted variable-life adjusted display (VLAD) and cumulative sum (CUSUM) plots to monitor changes.A total of 28,601 births were observed. The monthly number of births was reduced by 17.3% during the post-introduction period. Spontaneous vaginal deliveries were registered less frequently with a decrease of about 17/1000 births in non-cephalic presentations. Labour complications and caesarean sections increased with about 80/1000 births. There was a reduction in newborns with birth weight less than 2500 g. The observed changes were stable over time. For most variables, a significant change could be detected after a few weeks.After the introduction of ambulance and delivery fees, an increase in labour complications and caesarean sections and a decrease in newborns with low birthweight were observed. This might indicate that women delay the decision to seek skilled birth attendance or do not seek help at all, possibly due to financial reasons. Lower rates of births in a safe health care facility like HLH is of great concern, as access to skilled birth attendance is a key requirement in order to further reduce perinatal mortality. Therefore, free delivery care should be a high priority.
186	Bone Marrow and Peripheral Blood Findings in Patients Infected by SARS-CoV-2.	33502434	Coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with diverse clinical, including hematologic, abnormalities. We describe peripheral blood and bone marrow findings in deceased and living patients with COVID-19.We examined bone marrows from 20 autopsies and 2 living patients with COVID-19 using H&E-stained slides and immunohistochemical stains. Clinical history and laboratory values were reviewed. HScore was calculated to estimate risk of hemophagocytic lymphohistocytosis (HLH).The deceased patients included 12 men and 8 women (aged 32 to >89 years; median, 63 years). Hematologic abnormalities included frequent neutrophilic leukocytosis, lymphopenia, anemia, and thrombocytopenia; one patient showed striking erythrocytosis. The bone marrows were all normocellular to hypercellular, most showing maturing trilineage hematopoiesis with myeloid left shift. In all 19 evaluable bone marrows, hemophagocytic histiocytes were identified. The HScore for secondary HLH ranged from 35 to 269 (median, 125; >169 in 5 patients). Coinfections were identified in 6 patients. In 2 living patients, bone marrow showed maturing trilineage hematopoiesis, including one showing few hemophagocytic histiocytes.Peripheral blood from deceased patients with COVID-19 frequently showed neutrophilic leukocytosis, lymphopenia, and, rarely, secondary polycythemia; hemophagocytosis was common in their bone marrow. Consistent with other studies, we provide histopathologic evidence of secondary HLH development in patients with COVID-19.
187	Acute liver injury secondary to hemophagocytic lymphohistiocytosis triggered by Epstein-Barr virus infection.	33490632	We present a 23-year-old man with hemophagocytic lymphohistiocytosis (HLH) triggered by Epstein-Bar virus (EBV) infection. This patient presented with persistent fever and acute liver injury 6 weeks after having an infectious mononucleosis associated with EBV infection. He had hypofibrinogenemia, hyperferritinemia, increased soluble interleukin-2 receptor, elevated prothrombin time, and pancytopenia. Bone marrow examination for evaluation of pancytopenia revealed that macrophages had phagocytosed mature erythrocytes. Based on these findings, we suspected an HLH triggered by EBV infection (EBV-HLH). To distinguish from HLH triggered by malignant lymphomas accompanying EBV infection, we performed a percutaneous liver biopsy, which revealed that atypical T-lymphocytes had infiltrated the liver tissues. The T-lymphocytes were positive for EBV-encoded RNA in situ hybridization, and no distinct monoclonal T-cell receptor chain gene rearrangement was detected. These findings indicated EBV hepatitis and, accordingly, malignant lymphoma was ruled out. We finally made a diagnosis of EBV-HLH. The patient was treated with corticosteroid and etoposide, according to HLH-2004 guideline recommendations, and the patient's symptoms and laboratory values improved. After that, he experienced no recurrence. Prompt recognition and initiation of treatment remains the key to the survival of patients with EBV-HLH, and the liver biopsy was helpful in making the diagnosis.
188	MiT/TFE Family of Transcription Factors: An Evolutionary Perspective.	33490073	Response and adaptation to stress are critical for the survival of all living organisms. The regulation of the transcriptional machinery is an important aspect of these complex processes. The members of the microphthalmia (MiT/TFE) family of transcription factors, apart from their involvement in melanocyte biology, are emerging as key players in a wide range of cellular functions in response to a plethora of internal and external stresses. The MiT/TFE proteins are structurally related and conserved through evolution. Their tissue expression and activities are highly regulated by alternative splicing, promoter usage, and posttranslational modifications. Here, we summarize the functions of MiT/TFE proteins as master transcriptional regulators across evolution and discuss the contribution of animal models to our understanding of the various roles of these transcription factors. We also highlight the importance of deciphering transcriptional regulatory mechanisms in the quest for potential therapeutic targets for human diseases, such as lysosomal storage disorders, neurodegeneration, and cancer.
189	COVID-19: High-JAKing of the Inflammatory "Flight" by Ruxolitinib to Avoid the Cytokine Storm.	33489899	Since SARS-CoV-2 outbreak in December 2019, world health-system has been severely impacted with increased hospitalization, Intensive-Care-Unit (ICU) access and high mortality rates, mostly due to severe acute respiratory failure and multi-organ failure. Excessive and uncontrolled release of proinflammatory cytokines (cytokine release/storm syndrome, CRS) have been linked to the development of these events. The recent advancements of immunotherapy for the treatment of hematologic and solid tumors shed light on many of the molecular mechanisms underlying this phenomenon, thus rendering desirable a multidisciplinary approach to improve COVID-19 patients' outcome. Indeed, currently available therapeutic-strategies to overcome CRS, should be urgently evaluated for their capability of reducing COVID-19 mortality. Notably, COVID-19 shares different pathogenic aspects with acute graft-versus-host-disease (aGVHD), hemophagocytic-lymphohistiocytosis (HLH), myelofibrosis, and CAR-T-associated CRS. Specifically, similarly to aGVHD, an induced tissue damage (caused by the virus) leads to increased cytokine release (TNFα and IL-6) which in turn leads to exaggerated dendritic cells, macrophages (like in HLH) and lymphocytes (as in CAR-T) activation, immune-cells migration, and tissue-damage (including late-stage fibrosis, similar to myelofibrosis). Janus Kinase (JAK) signaling represents a molecular hub linking all these events, rendering JAK-inhibitors suitable to limit deleterious effects of an overwhelming inflammatory-response. Accordingly, ruxolitinib is the only selective JAK1 and JAK2-inhibitor approved for the treatment of myelofibrosis and aGVHD. Here, we discuss, from a molecular and hematological point of view, the rationale for targeting JAK signaling in the management of COVID-19 patients and report the clinical results of a patient admitted to ICU among the firsts to be treated with ruxolitinib in Italy.
190	Outcomes of adult critically ill patients with hemophagocytic lymphohistiocytosis in united states-analysis from an administrative database from 2007 to 2015.	33489441	Severe infections caused by the novel coronavirus 2 display similarities to secondary hemophagocytic lymphohistiocytosis (HLH). However, HLH is a rare disease and has not been well described in critically ill patients.We used the Nationwide Inpatient Sample (NIS), the largest all-payer inpatient care database publicly available in the United States to identify all adult discharges with Hemophagocytic syndrome (ICD-9 CM code 288.4) between 2007 and 2015. Critical illness was considered present if patient had either ICD-9 CM code indicating the requirement of invasive mechanical ventilation or the presence of shock. We used ICD-9-CM codes to identify various infections (inf-HLH), malignancies (mal-HLH) and autoimmune diseases associated with HLH (MAS-HLH) and classified them in their respective groups. Primary outcome was in-hospital mortality in critically ill patients. We developed multivariable regression model to examine variables associated with mortality in critically ill HLH patients. P value was kept at < 0.05.Of the 7420 (95% CI 6959-7881) estimated discharges with HLH, 2313 (31%) were critically ill. Of the critically ill patients, 442 (34%) were mal-HLH, 422 (43.3%) were inf-HLH, 403 (30.7%) were MAS-HLH and 1046 (27.3%) were unable to be classified. In hospital mortality rates were 6.4% in non-critically ill and 48.4% in critically ill patients. Among the subtypes of HLH, in-hospital mortality was 53% in mal-HLH, 49.4% in inf-HLH, 26% in MAS-HLH and 54.6% in unclassified group. On multivariable regression analysis, development of acute renal failure requiring hemodialysis (OR 2.06, 95% CI 1.29-3.3, P=0.002) and acute hepatic failure (OR 2.21, 95% CI 1.38-3.52, P=0.001) were significantly associated with higher mortality.Inpatient mortality of critically ill patients is remarkably high. Patients with MAS-HLH had better outcomes when compared to other groups of HLH.
191	Multicenter Outcome of Hematopoietic Stem Cell Transplantation for Primary Immune Deficiency Disorders in India.	33488609	Hematopoietic stem cell transplantation (HSCT) is the curative option for many primary immune deficiency disorders (PID). In the last 5 years, increased awareness, availability of diagnostics based on flow cytometry, genetic testing, improved supportive care, use of reduced toxicity conditioning, and success of haploidentical donor HSCT have improved access to HSCT for children with PID in India. We present results on children with PID who underwent HSCT across India and the factors that influenced outcome.We collected retrospective data on the outcome of HSCT for PID from seven centers. We analyzed the impact of the type of PID, conditioning regimen, time period of HSCT- before or after January 2016, graft versus host disease prophylaxis, cause of mortality and overall survival.A total of 228 children underwent HSCT for PID at a median age of 12 months (range, 1 to 220 months) with a median follow up of 14.4 months. Infants accounted for 51.3% of the cohort and the male female ratio was 3:1. SCID (25%) and HLH (25%) were the more frequent diagnoses. Matched family donor was available in 36.4% and 44.3% children had a haploidentical HSCT. Reduced and myeloablative conditioning regimens were used with 64% children receiving a treosulfan based conditioning regimen. Peripheral blood stem cells were the predominant graft source at 69.3%. The survival in infants (60.2%) was inferior to children aged over 1 year (75.7% p value = 0.01). Children with Wiskott Aldrich syndrome (74.3%) and chronic granulomatous disease (82.6%) had the best outcomes. The survival was superior in children receiving HSCT from a matched sibling (78%) versus an alternate donor HSCT (61% p value = 0.04). In the cohort transplanted after January 2016 survival improved from 26.8% to 77.5% (p value = 0.00). Infection remains the main cause of mortality at in over 50% children. The 5-year overall survival rate was 68%.Survival of children with PID undergoing HSCT in India has improved dramatically in last 5 years. Alternate donor HSCT is now feasible and has made a therapeutic option accessible to all children with PID.
192	Screening for hemophagocytic lymphohistiocytosis in child abuse evaluations: Twelve years of data.	33486258	Laboratory evaluation is commonly integrated into evaluation of children with suspected physical abuse to identify occult injury and potential mimics of abuse, including hemophagocytic lymphohistiocytosis (HLH). We evaluated the utility of ferritin in laboratory screening panels for physical abuse.To determine if hyperferritinemia is a useful screening marker of HLH in physical abuse diagnostic evaluations.Children being evaluated for physical abuse at a quaternary pediatric referral and level one trauma center in Houston, Texas.We conducted a 12-year (2003 - 2015) retrospective descriptive analysis of all ferritin values obtained as part of routine screening panels for physical child abuse. Medical records were abstracted for patients with ferritin > 500 ng/mL.2954 ferritin levels were obtained in 3091 encounters for suspected physical abuse (median age 6.5 months, interquartile range 2.3-23.5 months). Elevated ferritin was found in 82/2954 (2.8 %); no child evaluated for physical abuse was found to have HLH (95 % CI: 0-4.5%). The child abuse team was consulted in 48/82 (58.5 %) of cases, with the final impression being physical abuse in 33/48 (68.8 %).We found no instances where HLH was identified by institutional screening panels. The inclusion of ferritin in the screening panel was not beneficial. The presence of hyperferritinemia should not cast medical or legal doubt on physical abuse diagnoses when there is a high index of clinical suspicion. Novel hypotheses from case reports and case series should be studied more rigorously before affecting system change.
193	The role of interferon-gamma and its signaling pathway in pediatric hematological disorders.	33484058	Interferon-gamma (IFN-γ) plays a key role in the pathophysiology of hemophagocytic lymphohistiocytosis (HLH), and available evidence also points to a role in other conditions, including aplastic anemia (AA) and graft failure following allogeneic hematopoietic stem cell transplantation. Recently, the therapeutic potential of IFN-γ inhibition has been documented; emapalumab, an anti-IFN-γ monoclonal antibody, has been approved in the United States for treatment of primary HLH that is refractory, recurrent or progressive, or in patients with intolerance to conventional therapy. Moreover, ruxolitinib, an inhibitor of JAK/STAT intracellular signaling, is currently being investigated for treating HLH. In AA, IFN-γ inhibits hematopoiesis by disrupting the interaction between thrombopoietin and its receptor, c-MPL. Eltrombopag, a small-molecule agonist of c-MPL, acts at a different binding site to IFN-γ and is thus able to circumvent its inhibitory effects. Ongoing trials will elucidate the role of IFN-γ neutralization in secondary HLH and future studies could explore this strategy in controlling hyperinflammation due to CAR T cells.
194	Intestinal long non-coding RNAs in response to simulated microgravity stress in Caenorhabditis elegans.	33479427	Long non-coding RNAs (lncRNAs) are important in regulating the response to environmental stresses in organisms. In this study, we used Caenorhabditis elegans as an animal model to determine the functions of intestinal lncRNAs in regulating response to simulated microgravity stress. Among the intestinal lncRNAs, linc-2, linc-46, linc-61, and linc-78 were increased by simulated microgravity treatment, and linc-13, linc-14, linc-50, and linc-125 were decreased by simulated microgravity treatment. Among these 8 intestinal lncRNAs, RNAi knockdown of linc-2 or linc-61 induced a susceptibility to toxicity of simulated microgravity, whereas RNAi knockdown of linc-13, linc-14, or linc-50 induced a resistance to toxicity of simulated microgravity. In simulated microgravity treated nematodes, linc-50 potentially binds to three transcriptional factors (DAF-16, SKN-1, and HLH-30). RNAi knockdown of daf-16, skn-1, or hlh-30 could suppress resistance of linc-50(RNAi) nematodes to the toxicity of simulated microgravity. Therefore, our results provide an important basis for intestinal lncRNAs, such as the linc-50, in regulating the response to simulated microgravity in nematodes.
195	Estimating health state utilities in hemophagocytic lymphohistiocytosis.	33471193	Hemophagocyti.c lymphohistiocytosis (HLH) is a rare and severe disorder characterized by abnormal activation of the immune system. Primary HLH causes prolonged fever, spleen and liver enlargement, and organ dysfunction, usually in infancy and early childhood and is fatal if left untreated. As effective treatment options emerge, such as emapalumab-lzsg, Health Technology Assessment bodies around the world will assess them in terms of cost-effectiveness. This study was designed to estimate quality of life weights (utilities) for such analyses.Vignettes were developed describing HLH treatment related health states. Health states included active HLH, HLH plus neurological symptoms, receiving chemotherapy, undergoing stem cell transplant (SCT), graft versus host disease (GVHD), cure and end of life care. The vignettes were based on information from in depth interviews with clinical specialists; and qualitative research with four parents of children with primary HLH aged between 1 and 18 years old. The vignettes were then assessed in time trade off (TTO) interviews with members of the UK general public in one on one face to face interviews with trained, experienced interviewers. Preference data were analysed using the generalised estimating equations framework.Detailed qualitative data captured the substantial burden of this disease for young children. One hundred participants completed the TTO interviews. The utility score for Active HLH was estimated as 0.32 (95% CI, 0.24 to 0.39). Values for other states were HLH plus neurological symptoms (0.27, 95%CI 0.18-0.35), receiving chemotherapy (0.26, 95%CI 0.17-0.34), undergoing SCT (0.18, 95%CI 0.07-0.28), GVHD (0.07, 95%CI -0.04-0.17), cure (0.72, 95%CI 0.67-0.77) and end of life care (- 0.17, 95%CI -0.27- -0.07).This study has estimated utility weights for seven different HLH related states which are based on detailed input from carers and physicians and have good face validity. There are few other options for collecting these data in an ultra-rare setting.
196	Cytokine adsorption therapy in lymphoma-associated hemophagocytic lymphohistiocytosis and allogeneic stem cell transplantation.	33459910	Lymphoma-associated Hemophagocytic lymphohistiocytosis (HLH) represents a severe complication of disease progression, mediated through cytokine release from the lymphoma cells. Cytokine adsorption may contribute as a supportive treatment to stabilize organ function by reduction of cytokine levels. So far, no experiences of cytokine adsorption and simultaneous stem cell transplantation were published. We report the case of a patient with aggressive lymphoma secondary to chronic lymphocytic leukemia with rapidly progressive HLH (Richter's transformation) upon conditioning chemotherapy prior to allogeneic stem cell transplantation (ASCT). Continuous hemodiafiltration was initiated in the treatment of shock with acute renal failure, lactacidosis and need for high-dose catecholamine therapy, integrating an additional cytokine-adsorbing filter (CytoSorb®) to reduce cytokine levels. This was followed by scheduled allogenic stem cell transplantation. We observed a marked decrease in interleukin-6 plasma levels, associated with a reduced need for vasopressor therapy and organ function stabilization. Hematopoietic engraftment was present at day 14 post-ASCT, leading to disease-free discharge at day 100 post-transplantation. Cytokine adsorption may serve as a safe adjunct to HLH/sepsis treatment during allogeneic stem cell transplantation. Clinical studies are required to make future treatment recommendations.
197	Report of a Fatal Case of Hemophagocytic Lymphohistiocytosis Syndrome and a Review of the Literature.	33457141	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition in children, with a high mortality rate of 41.99%. Often, due to the atypical presentation of HLH, the syndrome is difficult to diagnose. We report a case of an atypical presentation of HLH and the diagnostic dilemma that we faced. An 11-year-old boy was hospitalized with recurrent fever, hepatosplenomegaly, and worsening jaundice. Initial laboratory workup revealed an elevated prothrombin time (PT), high bilirubin, increased alanine transaminase (ALT), and positive Epstein Barr virus (EBV) deoxyribonucleic acid (DNA) polymerase chain reaction (PCR) and EBV immunoglobulin G (IgG). Based on our patient's presentation and initial laboratory findings, further extensive workup was done, which revealed cytopenias, hypofibrinogenemia, hemophagocytosis on biopsy, absent natural killer (NK) cell activity, high serum ferritin level, and high soluble CD25 (sIL-2 receptor); a diagnosis of HLH was made. He was started on broad-spectrum antibiotics, antivirals, antifungals, and cyclosporine. He became hypoxic and hypotensive and hence was intubated and started on vasopressors. However, his coagulation profile continued to deteriorate. He started bleeding from multiple sites and became unresponsive to ventilatory support, eventually dying as a result of complications of HLH. The ambiguous clinical presentation makes the diagnosis of this syndrome difficult. However, due to the high fatality rate, early recognition and prompt treatment are of utmost importance.
198	Age-related miRNome landscape of cumulus oophorus cells during controlled ovarian stimulation protocols in IVF cycles.	33454781	Is the microRNA (miRNA) expression pattern of cumulus oophorus cells (COCs) in women undergoing medically assisted reproduction (MAR) procedures differentially modulated according to patient age and gonadotropin treatment strategy?Maternal age is an independent factor impacting miRNA expression in COCs while gonadotropin treatment may affect follicular miRNA expression and IVF efficacy.Epigenetic mechanisms in female infertility are complex and poorly studied. DNA methylation, histone modifications, miRNAs and nucleosome positioning influence cellular machinery through positive and negative feedback mechanisms either alone or interactively. miRNAs are important regulators during oogenesis, spermatogenesis and early embryogenesis, and are reported to play a role in regulating crosstalk between the oocyte and COCs. Although miRNome analysis has been performed in female human reproductive tissues (endometrium, myometrium, cervix and ovaries), epigenetic modifications in women with infertility have not been explored in detail. In addition, the impact of gonadotropin treatments during MAR on miRNA expression in COCs has not been fully investigated.This study was carried out in 53 COC samples obtained from mature metaphase II (MII) oocytes in 53 women undergoing MAR treatment. A total of 38 samples for assay development were pooled by maternal age and gonadotropin treatment into four predetermined subgroups: ≥36 years and recombinant human FSH (r-hFSH), n = 10; ≥36 years and r-hFSH+ recombinant human-luteinizing hormone (r-hLH), n = 10; ≤35 years and r-hFSH, n = 9; ≤35 years and r-hFSH+r-hLH, n = 9. miRNome profiles were determined and compared between subgroups. Expression of defined miRNAs was validated in the remaining fifteen samples, representative of each subgroup, by quantitative polymerase chain reaction (PCR).COCs were processed for miRNA-enriched total RNA extraction and pooled in homogeneous subgroups to obtain a sufficient amount and quality of starting material to perform the analysis. Each pooled sample underwent miRNA profiling using PCR assay system to examine expression of 752 human miRNAs without pre-amplification. Data were analyzed using the delta-delta Ct method for relative quantitation and prediction of target genes (with at least four algorithms predicting the same miRNA-gene interaction pair (HIT)>4). The miRSystem database provided functional annotation enrichment (raw P-value <0.05) of co-expressed miRNAs.We found distinctive miRNA expression profiles in each subgroup correlating with age and MAR stimulation. In addition, a number of selective and co-expressed miRNAs were revealed by comparative analysis. A cluster of 37 miRNAs were commonly but differentially expressed in all four pools. Significant differences were observed in expression regulation of 37 miRNAs between age groups (≤35 or ≥36) in women receiving r-hFSH+r-hLH compared to those receiving r-hFSH alone. Higher concentrations and increased numbers of miRNAs were recorded in younger than in older patients, regardless of treatment. Functional and expression studies performed to retrieve common miRNome profiles revealed an enrichment of biological functions in oocyte growth and maturation, embryo development, steroidogenesis, ovarian hyperstimulation, apoptosis and cell survival, glucagon and lipid metabolism, and cell trafficking. The highest scored pathways of target genes of the 37 common miRNAs were associated with mitogen-activated protein kinase (MAPK) signaling pathways, G alpha signaling, transcription regulation, tight junctions, RNA polymerase I and III, and mitochondrial transcription. We identified a potential age- and MAR stimulation-dependent signature in the miRNA landscape of COCs.We cannot rule out the possibility that other unknown individual genetic or clinical factors may have interfered with the reported results. Since miRNA profiling was conducted with a predefined array of target probes, other miRNA molecules, potentially modulated by age and hormonal stimulation, may have been missed in this study.miRNA expression in COCs is modulated by gonadotropin treatment and correlates strongly with age. A better understanding of the expression patterns and functions of miRNAs may lead to the development of novel therapeutics to treat ovarian dysfunction and improve fertility in older women.This study was funded by Merck KGaA, Darmstadt, Germany. All authors declared no competing interest, except SL and TD who are fully employed by Merck KGaA.N/A.
199	Characterizing EBV-associated lymphoproliferative diseases and the role of myeloid-derived suppressor cells.	3344355333443554	Chronic active Epstein-Barr virus (CAEBV) typically presents as persistent infectious mononucleosis-like disease and/or hemophagocytic lymphohistocytosis (HLH), reflecting ectopic Epstein-Barr virus (EBV) infection and lymphoproliferation of T and/or NK cells. Clinical behavior ranges from indolent, stable disease through to rapidly progressive, life-threatening disease. Although it is thought the chronicity and/or progression reflect an escape from immune control, very little is known about the phenotype and function of the infected cells vs coresident noninfected population, nor about the mechanisms that could underpin their evasion of host immune surveillance. To investigate these questions, we developed a multicolor flow cytometry technique combining phenotypic and functional marker staining with in situ hybridization for the EBV-encoded RNAs (EBERs) expressed in every infected cell. This allows the identification, phenotyping, and functional comparison of infected (EBERPOS) and noninfected (EBERNEG) lymphocyte subset(s) in patients' blood samples ex vivo. We have characterized CAEBV and HLH cases with monoclonal populations of discrete EBV-activated T-cell subsets, in some cases accompanied by EBV-activated NK-cell subsets, with longitudinal data on the infected cells' progression despite standard steroid-based therapy. Given that cytotoxic CD8+ T cells with relevant EBV antigen specificity were detectable in the blood of the best studied patient, we searched for means whereby host surveillance might be impaired. This revealed a unique feature in almost every patient with CAEBV studied: the presence of large numbers of myeloid-derived suppressor cells that exhibited robust inhibition of T-cell growth. We suggest that their influence is likely to explain the host's failure to contain EBV-positive T/NK-cell proliferation.
200	Hemophagocytic lymphohistiocytosis secondary to Candida albicans and reactivated EBV infections: A case report and review of the literature.	33433441	Hemophagocytic lymphohistiocytosis (HLH) has been recognized as a potentially life-threatening syndrome. This is the first case of acquired HLH caused by dual infections with Candida albicans and reactivated EBV infections, which focuses on the importance of morphological awareness of peripheral blood and bone marrow because sometimes they are the only locations that HLH and fungal microorganisms can be diagnosed. A 29-year-old woman with a history of abdominal distension and 9 months of intermittent fevers ($38.8°C) was admitted to the hematology department with treatment for leukopenia and thrombocytopenia. Severe infection of bilateral pulmonary and marked hepatosplenomegaly were detected by computed tomography. EB virus-CA IgG, EB virus-NA IgG and EB virus-CA IgM were positive. Scattered yeast-like fungi were found on peripheral blood and bone marrow (BM) smears. BM smears indicated prominent hemophagocytosis. Cultures of bronchoalveolar lavage and BM confirmed the growth of C. albicans. A diagnosis of HLH caused by dual infections with Candida albicans and reactivated EBV infections was established based on the clinical features of the patient because 7 of the 8 diagnostic criteria were met. She was treated with etoposide, dexamethasone for HLH, as well as highly active antifungal and antiviral therapies for the underlying etiology of dual infections. The patient eventually recovered following the effective treatment. A timely and accurate diagnosis is crucial to the prognosis of the dangerous disease.
201	Chediak-Higashi syndrome: a review of the past, present, and future.	33424983	Since the initial description of Chediak-Higashi syndrome (CHS), over 75 years ago, several studies have been conducted to underscore the role of the lysosomal trafficking regulator (LYST) gene in the pathogenesis of disease. CHS is a rare autosomal recessive disorder, which is caused by biallelic mutations in the highly conserved LYST gene. The disease is characterized by partial oculocutaneous albinism, prolonged bleeding, immune and neurologic dysfunction, and risk for the development of hemophagocytic lympohistiocytosis (HLH). The presence of giant secretory granules in leukocytes is the classical diagnostic feature, which distinguishes CHS from closely related Griscelli and Hermansky-Pudlak syndromes. While the exact mechanism of the formation of the giant granules in CHS patients is not understood, dysregulation of LYST function in regulating lysosomal biogenesis has been proposed to play a role. In this review, we discuss the clinical characteristics of the disease and highlight the functional consequences of enlarged lysosomes and lysosome-related organelles (LROs) in CHS.
202	Novel Therapeutic Approaches to Familial HLH (Emapalumab in FHL).	33424859	Primary Hemophagocytic lymphohistiocytosis (pHLH) is a rare, life-threatening, hyperinflammatory disorder, characterized by uncontrolled activation of the immune system. Mutations affecting several genes coding for proteins involved in the cytotoxicity machinery of both natural killer (NK) and T cells have been found to be responsible for the development of pHLH. So far, front-line treatment, established on the results of large international trials, is based on the use of glucocorticoids, etoposide ± cyclosporine, followed by allogeneic hematopoietic stem cell transplantation (HSCT), the sole curative treatment for the genetic forms of the disease. However, despite major efforts to improve the outcome of pHLH, many patients still experience unfavorable outcomes, as well as severe toxicities; moreover, treatment-refractory or relapsing disease is a major challenge for pediatricians/hematologists. In this article, we review the epidemiology, etiology and pathophysiology of pHLH, with a particular focus on different cytokines at the origin of the disease. The central role of interferon-γ (IFNγ) in the development and maintenance of hyperinflammation is analyzed. The value of emapalumab, a novel IFNγ-neutralizing monoclonal antibody is discussed. Available data support the use of emapalumab for treatment of pHLH patients with refractory, recurrent or progressive disease, or intolerance to conventional therapy, recently, leading to FDA approval of the drug for these indications. Additional data are needed to define the role of emapalumab in front-line treatment or in combination with other drugs.
203	Effect of Single-Injection Thoracic Paravertebral Block via the Intrathoracic Approach for Analgesia After Single-Port Video-Assisted Thoracoscopic Lung Wedge Resection: A Randomized Controlled Trial.	33420979	Pain is still severe after single-port video-assisted thoracoscopic (SPVAT) lung wedge resection. We observed the effect of single-injection thoracic paravertebral block (TPB) via the intrathoracic approach for analgesia after SPVAT lung wedge resection.Sixty patients undergoing SPVAT lung wedge resection were randomly divided into a control group and an observation group. All patients underwent TPB via the intrathoracic approach at the T4 level with a scalp needle before closing the chest. The patients in the observation group received 20 ml 0.375% ropivacaine at the T4 level, and the patients in the control group received 20 ml of 0.9% saline. A patient-controlled intravenous analgesic (PCIA) pump with sufentanil was attached to all patients after surgery. The sufentanil consumption and number of PCIA presses in the first 24 h after surgery were recorded. The visual analogue scale (VAS) scores (during rest and coughing) were recorded at 6 h, 12 h, 24 h, and 36 h after surgery. The incidence of adverse reactions after surgery were recorded.The sufentanil consumption in the observation group was significantly lower than that in the control group (34.2 ± 1.9 µg vs. 52.3 ± 2.3 µg; P < 0.001). The VAS score at 6, 12, and 24 h after surgery, the incidence of adverse reactions after surgery in the observation group were significantly lower than those in the control group (all P < 0.05). The number of PCIA presses in the observation group was significantly lower than that in the control group [0 (0-0) times vs. 3 (2-4) times, P < 0.001].Single-injection TPB via the intrathoracic approach under thoracoscopic direct vision is easy to perform and can effectively alleviate postoperative pain after SPVAT lung wedge resection, with fewer adverse reactions.ChiCTR2000034726.
204	Can we truly diagnose adult secondary hemophagocytic lymphohistiocytosis (HLH)? A critical review of current paradigms.	33418346	The HLH-2004 criteria were initially conceived as inclusion criteria for a clinical trial investigating therapy for (largely primary) pediatric hemophagocytic lymphohistiocytosis (HLH). These criteria have since been extrapolated to diagnose adult secondary HLH despite their questionable generalizability. It remains unclear whether these diagnostic criteria are truly applicable among adult secondary cases, and rigorous evidence for their use among such patients is lacking. This review critically examines the utility of the HLH-2004 criteria for the diagnosis of adult secondary HLH. It is framed as a reappraisal of each of the criteria's individual components, with an assessment of the relevance of, and/or evidence regarding, each. There are clear limitations to these criteria as they apply to adult secondary HLH, however they may help guide our understanding of the disease to some extent. Some new paradigms are emerging for the diagnosis of adult secondary HLH, however these too are limited by the difficulties inherent in formulating specific criteria for a very non-specific syndrome, which lacks any single gold-standard diagnostic test.
205	Biomarkers for Early Diagnosis of Hemophagocytic Lymphohistiocytosis in Critically Ill Patients.	33417087	Many biomarkers have been proposed for the diagnosis of secondary hemophagocytic lymphohistiocytosis (HLH) in adults, but comparative studies are lacking. We analyzed ferritin, glycosylated ferritin, soluble CD25, CD163 and CD14, IL-6, IFN-γ, IL-18, IL-10, IL-1ß, IL-12p70, IL-17α, IP-10, and CXCL9 levels to differentiate HLH from sepsis in critically ill patients. Of 120 patients, HLH was confirmed for 14 patients. Among the biomarkers tested, ferritin, IL-18, and glycosylated ferritin were the most efficient parameters for early diagnosis of HLH. With a sensitivity set at 85%, ferritin, IL-18, and glycosylated ferritin were the biomarkers with the highest specificity: 84, 79, and 71% respectively. Combining IL-18 with the HScore provided a new score with an increased specificity compared to the HScore alone, 86% compared to 70% with a sensitivity set at 100%. A distinct cytokine pattern was highlighted in patients with malignancy-triggered HLH, with highly increased levels of INF-ɣ and CXCL9, compared to HLH secondary to infection. This is the largest study available to date, comparing diagnostic biomarkers for HLH on a cohort of critically ill adult patients. Serum ferritin was the most discriminating parameter for early diagnosis of secondary HLH. IL18*HScore was identified as a highly potential score.
206	Pregnancy-Induced Hemophagocytic Lymphohistiocytosis: A Case Report and Literature Review.	33413754	Hemophagocytic lymphohistiocytosis (HLH) is a rare but devastating disease characterized by dysregulated immune response and hyperinflammation. To our knowledge, pregnancy-induced HLH has been rarely reported in the literature. A 30-year-old pregnant woman presented persistent fever for 21 days since 17 weeks of pregnancy. The possible etiologies such as infection, autoimmune disorder, and malignancy had been ruled out based on a series of exhaustive examinations. The disease progressed despite the use of broad-spectrum antibiotics and dexamethasone. The patient was diagnosed as pregnancy-induced HLH, and finally recovered completely after termination of pregnancy by caesarean and the continuous use of glucocorticoid which played a crucial part in controlling hyperinflammation. Pregnancy-induced HLH could be fatal if effective treatment was not initiated timely. Further studies are needed to improve early diagnosis and etiology identification of HLH.
207	Clinical warning of hemophagocytic syndrome caused by Epstein-Barr virus.	33413556	This study aimed to compare the clinical features and laboratory tests of infectious mononucleosis (IM) and hemophagocytic syndrome (HLH) caused by Epstein-Barr virus (EBV) in 1-3-year-old children and to explore the risk factor of HLH caused by EBV (EBV-HLH).The clinical data of 92 children with EBV infection admitted in our hospital from 2011 to 2019 were collected; 61 cases were diagnosed as EBV-IM, and 31 cases were diagnosed as EBV-HLH. The subjects' clinical manifestations and laboratory tests were analyzed retrospectively.Compared with EBV-IM patients, EBV-HLH patients had longer durations of fever, both before hospitalization and overall, and a higher probability of hepatomegaly. The levels of ALT, AST, LDH, TG, SF, D-Dimer and the plasma EBV DNA load of EBV-HLH patients were significantly higher than those of EBV-IM patients. The absolute values of CD3+, CD4+, CD8+, NK, and CD3-CD19+ cells and IgA and IgM levels of EBV-HLH patients were significantly lower than those of EBV-IM patients. The plasma EBV DNA load was positively correlated with the PT, TT, α-HBDH, AST, LDH, CK, Scr, BUN, UA, TG, and CRP levels in EBV-HLH patients, and the plasma EBV DNA load was positively correlated with the D-Dimer level in the EBV-IM patients. Among the 10 different potential markers, at the cut-off point of 1721.500 μg/L, the sensitivity and specificity of D-Dimer was 88.90 and 90.20%, respectively.The D-Dimer level may be a good prognostic indicator of EBV-HLH caused by EBV.
208	Lamotrigine-associated hemophagocytic lymphohistiocytosis.	33408106	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening disorder of excessive immune activation. It is mostly seen in the paediatric population and is rarely observed in adults. HLH can be inherited or acquired and is commonly triggered by activation of the immune system by an underlying viral infection or in immune system deficiency such as malignancy or underlying rheumatological disease. HLH is a difficult entity to diagnose due to the rarity of this disorder, variable clinical presentation and non-specific clinical and laboratory findings. HLH carries a high mortality if left untreated, and therefore prompt diagnosis and initiation of immunosuppressive, immunomodulatory and cytostatic medications are critical to improve survival in affected patients. Here, we present a case of lamotrigine-associated HLH. To our knowledge, only eight other cases of lamotrigine-associated HLH have been reported in adult patients.
209	Is neutralization of IFN-γ sufficient to control inflammation in HLH?	33405364	NaN
210	Feasibility and effectiveness of multi-injection thoracic paravertebral block via the intrathoracic approach for analgesia after thoracoscopic-laparoscopic esophagectomy.	33403428	We observed the feasibility and effectiveness of multi-injection thoracic paravertebral block (TPB) via the intrathoracic approach under thoracoscopic direct vision for analgesia after thoracoscopic-laparoscopic esophagectomy (TLE).Sixty patients undergoing TLE were randomly divided into a control group and an observation group. All patients underwent TPB via the intrathoracic approach at the three levels of T2, 5, and 8 with a scalp needle before closing the chest. The patients in the observation group received 10 ml 0.375% ropivacaine at each level, and the patients in the control group received 10 ml of 0.9% saline at each level. A patient-controlled intravenous analgesic (PCIA) pump with sufentanil was attached to all patients after surgery. The sufentanil consumption, number of PCIA presses and use of rescue analgesia in the first 24 h after surgery were recorded. The visual analogue scale (VAS) scores (rest and coughing) were recorded at 2 h, 6 h, 12 h, 24 h, and 48 h after surgery. The duration of postoperative hospital stay, active cough rate, first ambulation, and the incidence of adverse reactions after surgery was recorded.The sufentanil consumption in the observation group was significantly lower than that in the control group (34.7 ± 1.9 µg vs. 52.1 ± 2.1 µg; P < 0.001). The VAS score at each postoperative time point, number of PCIA presses, use of rescue analgesia, and the incidence of adverse reactions in the observation group were significantly lower than those in the control group. The postoperative active cough rate of patients in the observation group was significantly higher than those in the control group, and the times of the first ambulation after surgery and postoperative hospital stay in the observation group were significantly shorter than those in the control group (all P < 0.05).Multi-injection TPB via the intrathoracic approach under thoracoscopic direct vision is easy to perform and can effectively alleviate postoperative pain after TLE with fewer adverse reactions and contributing to improved postoperative recovery.
211	Chondrodysplasia and growth failure in children after early hematopoietic stem cell transplantation for non-oncologic disorders.	33398909	Bone dysplasias (osteochondrodysplasias) are a large group of conditions associated with short stature, skeletal disproportion, and radiographic abnormalities of skeletal elements. Nearly all are genetic in origin. We report a series of seven children with similar findings of chondrodysplasia and growth failure following early hematopoietic stem cell transplantation (HSCT) for pediatric non-oncologic disease: hemophagocytic lymphohistiocytosis or HLH (five children, three with biallelic HLH-associated variants [in PRF1 and UNC13D] and one with HLH secondary to visceral Leishmaniasis), one child with severe combined immunodeficiency and one with Omenn syndrome (both children had biallelic RAG1 pathogenic variants). All children had normal growth and no sign of chondrodysplasia at birth and prior to their primary disease. After HSCT, all children developed growth failure, with standard deviation scores for height at or below -3. Radiographically, all children had changes in the spine, metaphyses and epiphyses, compatible with a spondyloepimetaphyseal dysplasia. Genomic sequencing failed to detect pathogenic variants in genes associated with osteochondrodysplasias. We propose that such chondrodysplasia with growth failure is a novel, rare, but clinically important complication following early HSCT for non-oncologic pediatric diseases. The pathogenesis is unknown but could possibly involve loss or perturbation of the cartilage-bone stem cell population.
212	Haemophagocytic syndrome and COVID-19.	33389315	Primary and secondary haemophagocytic lymphohistiocytosis (HLH) are hyperferritinaemic hyperinflammatory syndromes with a common terminal pathway triggered by different etiopathogenetic factors. HLH is characterised by a decreased capacity of interferon gamma production with an activated NK phenotype profile similar to other hyperinflammatory syndromes. Viruses are closely linked to the development of HLH as infectious triggers, and the break of tolerance to self-antigens is considered a critical mechanism involved in the development of immune-mediated conditions triggered by viral infections. Emerging studies in patients with COVID-19 are suggesting a key role of monocytes/macrophages in the pathogenesis of this viral infection, and there is a significant overlap between several features reported in severe COVID-19 and the features included in the HLH-2004 diagnostic criteria. Therefore, SARS-Cov-2, as other respiratory viruses, may also be considered a potential etiological trigger of HLH. The frequency of HLH in adult patients with severe COVID-19 is lower than 5%, although this figure could be underestimated considering that most reported cases lacked information about some specific criteria (mainly the histopathological criteria and the measurement of NK cell function and sCD25 levels). Because HLH is a multi-organ syndrome, the diagnostic approach in a patient with severe COVID-19 in whom HLH is suspected must be carried out in a syndromic and holistic way, and not in the light of isolated clinical or laboratory features. In COVID-19 patients presenting with persistent high fever, progressive pancytopenia, and hepatosplenic involvement, together with the characteristic triad of laboratory abnormalities (hyperferritinaemia, hypertriglyceridaemia, and hypofibrinogenaemia), the suspicion of HLH is high, and the diagnostic workup must be completed with specific immunological and histopathological studies.
213	Macrophage activation syndrome in systemic lupus erythematosus and systemic-onset juvenile idiopathic arthritis: a retrospective study of similarities and dissimilarities.	33388903	Macrophage activation syndrome (MAS) is a serious complication of rheumatic diseases. Fever and hyperferritinemia are common in active systemic-onset juvenile idiopathic arthritis (sJIA) and cytopenia in active systemic lupus erythematosus (SLE), thus recognizing MAS in them is a challenge. We compared clinical and laboratory parameters, various classification criteria, and outcomes of MAS in SLE and sJIA. Clinical and laboratory data were extracted from case records of patients with clinician diagnosed cases of SLE-MAS (adult and pediatric) and sJIA-MAS, admitted (2004-2018) at a tertiary care hospital. Ravelli, International consensus, HLH-2004, and criteria proposed by Parodi et al. were applied and compared. Among 33 patients (18 females) with MAS, 19 had SLE (7, childhood-onset SLE) and 14 had sJIA. MAS was more likely to be the presenting manifestation of disease in SLE (p < 0.05). There were no differences in the clinical features among them. Patients with SLE-MAS had lower baseline total leucocyte and platelet counts (p < 0.01), whereas patients with sJIA-MAS had significantly higher median CRP (p = 0.002), fall in TLC (p = 0.012), delta ESR/CRP ratio (p = 0.02), and lower fibrinogen level (p = 0.006). Neutrophil-to-lymphocyte ratio, ferritin/CRP ratio, and the number of patients with ferritin/ESR > 80 were similar. Only 6/33(18%) fulfilled the HLH criteria. Criteria meant for sJIA-MAS or SLE-MAS performed well for both diseases and the majority of patients could be diagnosed using them. Two patients died in each group. MAS in SLE and sJIA is more similar than dissimilar in clinical features and outcome. Criteria meant for MAS in sJIA or SLE-MAS performed equally well in both diseases.
214	Characteristics and predictors of post-transplant-associated hemophagocytic lymphohistiocytosis in adults.	33385294	Hemophagocytic lymphohistiocytosis (HLH) is an uncontrolled hyperinflammatory disorder driven by an overactive immune system that results in high mortality. Post-transplant-associated hemophagocytic lymphohistiocytosis (PT-HLH) is a type of secondary HLH that occurs following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical features of PT-HLH remain unclear and diagnostic and prognostic tools have not yet been established. Here, we retrospectively evaluated the clinical manifestations and outcomes of PT-HLH in 94 patients who underwent allo-HSCT. According to our PT-HLH criteria (hyperferritinemia and increased macrophage count in bone marrow), PT-HLH occurred in 12 patients (12.8%). The PT-HLH patients showed splenomegaly (P = .001), a higher risk of engraftment failure (P = .013), and an increased percentage of macrophages and hemophagocytes in bone marrow aspirates (P = .0009 and P = .0006, respectively). Moreover, univariate and multivariate analyses revealed that the survival rate was lower in PT-HLH patients than non-PT-HLH patients (P = .0017 and P = .034, respectively). This study defines the clinical features of PT-HLH and PT-HLH criteria that could be useful tools for diagnosing PT-HLH.
215	Primary dengue infection triggered haemophagocytic lymphohistiocytosis in a neonate.	33384343	Haemophagocytic lymphohistiocytosis (HLH) is an aggressive syndrome which has characteristic symptoms and laboratory findings. Infection is a common trigger of HLH. We report a 2700 g male infant with persistent fever, massive hepatosplenomegaly and severe thrombocytopaenia. Laboratory evidence of primary dengue infection was detected. Investigations revealed hypertriglyceridaemia, hypofibrinogenaemia, hyperferritinaemia and elevated soluble CD25. Bone marrow examination revealed haemophagocytes. The diagnostic criteria for HLH were fulfilled. A diagnosis of secondary HLH triggered by primary dengue infection was considered. Dexamethasone was initiated and continued for 8 weeks. He responded clinically with regression of hepatosplenomegaly, was afebrile and platelet counts normalised. Dengue-associated HLH is often missed clinically as treating physicians focus more on the underlying infection and its treatment. In neonates, HLH should be considered as differential diagnosis of sepsis and other viral infections, particularly in situations of inappropriate response to standard management.
216	Pathology of macrophage activation syndrome in humanized NSGS mice.	33383491	"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.
217	Kikuchi's disease with hemophagocytic lymphohistiocytosis: A case report and literature review.	33371072	Kikuchi's disease (KD) is a rare form of necrotizing lymphadenitis that rarely occurs in association with hemophagocytic lymphohistiocytosis (HLH) in children.We report the case of a 4-year-5-month-old boy who suffered from fever, cervical lymphadenopathy, pancytopenia, hypertriglyceridemia, splenomegaly, low NK cell activity.A diagnosis of KD with HLH was made based on the results of biopsy of cervical lymph node and HLH-2004 trial guidelines.The patient was treated with corticosteroids, cyclosporine, etoposide, continuous hemodiafiltration (HDF), and plasma exchange (PE).He showed a complete response to therapy, and his condition gradually improved. He was discharged on day 45 after admission due to his good recovery status.HLH can be associated with KD, especially in childhood, and may have an aggressive clinical course. Continuous HDF and PE and chemotherapy should be reserved for those patients who fail to respond to IVIG and corticosteroids.
218	Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target the E-box DNA Site.	33370105	Protein-based therapeutics are part of the next-generation arsenal of drugs being developed against proto-oncoprotein Myc. We designed protein MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of Max and Myc, which bind to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. We used phage-assisted continuous evolution (PACE), which uncovered mutations at Arg12 that contact the DNA phosphodiester backbone. The Arg12 mutations improved ME47's stability. We replaced Cys29 with Ala to eliminate potential undesired disulfide formation and fused the designed FosW leucine zipper to mutated ME47 to increase the dimerization interface and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper. Compared with ME47, MEF gives 2-fold stronger binding to E-box and 4-fold increased specificity for E-box over nonspecific DNA. The synergistic combination of rational design and PACE allowed us to make MEF and demonstrates the power and utility of our two-pronged approach toward development of promising protein drugs with robust structure and DNA-binding function.
219	Fatal Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with virus-infected T cells after pediatric multivisceral transplantation: A proof-of-concept case report.	33368911	EBV-associated HLH driven by EBV-infected CD8+ T cells is a rare complication after pediatric solid organ transplantation. The etiology and disease spectrum of post-transplant EBV-HLH are poorly understood, and making a precise diagnosis and providing optimal treatment remain a challenge.We report a 2-year-old multivisceral transplant recipient who developed fever and cytopenia with a persistent high EBV-load state. Repeated tissue examinations and CT scans could not identify a localized mass, which is the key to the diagnosis of PTLD as per the WHO classification. Hence, EBV-HLH was diagnosed by clinical manifestations as well as characterization of EBV-infected cells, pathological examination on cell block of pleural effusion and clonality analysis. This EBV-HLH did not respond to intensive chemotherapy, resulted in the recipient's death, acting similarly to hematological malignancy.Characterization of EBV-infected cells in peripheral blood should be considered when persistent high EBV loads develop with symptoms consistent with PTLD, but no evidence of localized mass, and the tissue diagnosis is unavailable after pediatric solid organ transplantation.
220	Hemophagocytic Lymphohistiocytosis as the Presenting Manifestation of Relapsed Classic Hodgkin's Lymphoma in the Presence of Concurrent Human Immunodeficiency Virus, Genital Herpes, Epstein-Barr Virus and Mycobacterium Avium Complex Infection.	33364090	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of uncontrolled immune activation which is usually divided into two main types. Primary, which is associated with genetic mutation and familial predisposition and secondary, which is usually associated with infections, malignancies and autoimmune conditions. More often multiple risk factors are present at the time of initial presentation. We report a case where HLH was the presenting manifestation of relapsed Classic Hodgkin's Lymphoma in the presence of multiple risk factors of secondary HLH such as human immunodeficiency virus (HIV), active genital herpes, Epstein-Barr virus (EBV) viremia, Mycobacterium avium complex (MAC) infection and prior chemotherapy. A 38-year-old male to female transgender woman presented with one-week history of fever, nausea, vomiting and generalized weakness. The past medical history was significant for HIV and previously treated and positron emission tomography (PET) scan confirmed complete remission of Classic Hodgkin's Lymphoma. Physical examination showed BP 92/40 mmHg, fever of 102.6 F, heart rate of 114 beats per minutes, diffuse abdominal tenderness and male genitalia with multiple small ulcerative lesions. Labs showed pancytopenia, hyponatremia, mildly elevated total and direct bilirubin, transaminitis, CD-4 count 96/mcL, HIV viral load undetectable and COVID-19 polymerase chain reaction (PCR) negative. Imaging showed right middle lung lobe consolidation and hepatosplenomegaly with multiple hypodense lesions. Lymphadenopathy was reported in mediastinum and retroperitoneum. The patient was initially treated with broad spectrum antibiotics, IV fluids, vasopressors and stress dose steroids. After initial improvement, vasopressors and steroids were stopped. The patient again started spiking fever on day 9 despite being on antibiotics. Workup showed EBV viremia, genital herpes and evidence of MAC infection on sputum culture. No improvement noted despite appropriate treatment for genital herpes and MAC. Additional lab work showed hyperferritinemia and elevated soluble Interleukin-2 receptors. The patient was diagnosed with HLH as per HLH-2004 criteria and treated with dexamethasone and etoposide. Bone marrow biopsy confirmed hemophagocytosis and immunoperoxidase staining established the diagnosis of relapsed Classic Hodgkin's Lymphoma. We can conclude that in patients with a history of hematological malignancy presenting with HLH, a high degree of suspicion for relapse should be maintained even in the presence of other risk factors.
221	Purpuric rash in an adolescent with fever, pancytopenia, and an hemophagocytic lymphohistiocytosis-like syndrome due to parvovirus B19.	33363887	A rare case of parvovirus B19 infection associated with fever, hemorrhagic rash, and a clinical course resembling an incomplete HLH syndrome is presented. Parvovirus B19 should be included in the evaluation of febrile purpura and HLH-like syndrome.
222	Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement.	33362801	Griscelli syndrome type 2 (GS-2) is an inborn error of immunity characterized by partial albinism and episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by RAB27A mutations that encode RAB27A, a member of the Rab GTPase family. RAB27A is expressed in many tissues and regulates vesicular transport and organelle dynamics. Occasionally, GS-2 patients with RAB27A mutation display normal pigmentation. The study of such variants provides the opportunity to map distinct binding sites for tissue-specific effectors on RAB27A. Here we present a new case of GS-2 without albinism (GS-2 sine albinism) caused by a novel missense mutation (Val143Ala) in the RAB27A and characterize its functional cellular consequences. Using pertinent animal cell lines, the Val143Ala mutation impairs both the RAB27A-SLP2-A interaction and RAB27A-MUNC13-4 interaction, but it does not affect the RAB27A-melanophilin (MLPH)/SLAC2-A interaction that is crucial for skin and hair pigmentation. We conclude that disruption of the RAB27A-MUNC13-4 interaction in cytotoxic lymphocytes leads to the HLH predisposition of the GS-2 patient with the Val143Ala mutation. Finally, we include a review of GS-2 sine albinism cases reported in the literature, summarizing their genetic and clinical characteristics.
223	Case Report: Secondary Hemophagocytic Lymphohistiocytosis With Disseminated Infection in Chronic Granulomatous Disease-A Serious Cause of Mortality.	33362767	Chronic granulomatous disease (CGD) is a primary immune deficiency due to defects in phagocyte respiratory burst leading to severe and life-threatening infections. Patients with CGD also suffer from disorders of inflammation and immune dysregulation including colitis and granulomatous lung disease, among others. Additionally, patients with CGD may be at increased risk of systemic inflammatory disorders such as hemophagocytic lymphohistiocytosis (HLH). The presentation of HLH often overlaps with symptoms of systemic inflammatory response syndrome (SIRS) or sepsis and therefore can be difficult to identify, especially in patients with a primary immune deficiency in which incidence of infection is increased. Thorough evaluation and empiric treatment for bacterial and fungal infections is necessary as HLH in CGD is almost always secondary to infection. Simultaneous treatment of infection with anti-microbials and inflammation with immunosuppression may be needed to blunt the hyperinflammatory response in secondary HLH. Herein, we present a series of X-linked CGD patients who developed HLH secondary to or with concurrent disseminated CGD-related infection. In two patients, CGD was a known diagnosis prior to development of HLH and in the other two CGD was diagnosed as part of the evaluation for HLH. Concurrent infection and HLH were fatal in three; one case was successfully treated, ultimately receiving hematopoietic stem cell transplantation. The current literature on presentation, diagnosis, and treatment of HLH in CGD is reviewed.
224	Hemophagocytic lymphohistiocytosis caused by STAT1 gain-of-function mutation is not driven by interferon-γ: A case report.	33344614	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyper-inflammatory syndrome caused by many genetic defects. STAT1 is a DNA-binding factor that regulates gene transcription. HLH caused by STAT1 gain-of-function (GOF) mutations has rarely been reported and its clinical manifestations and mechanisms are not clearly defined.A 2-year-old boy presented to our hospital with recurrent fever for > 20 d. The patient had a personal history of persistent oral candidiasis and inoculation site infection during the past 2 years. Hepatosplenomegaly was noted. Complete blood cell count showed severe anemia, thrombocytopenia and neutropenia. Other laboratory tests showed liver dysfunction, hypertriglyceridemia and decreased fibrinogen. Hemophagocytosis was found in the bone marrow. Chest computed tomography showed a cavitary lesion. Tests for fungal infection were positive. Serum T helper (Th) 1/Th2 cytokine determination demonstrated moderately elevated levels of interleukin (IL)-6 and IL-10 with normal interferon (IFN)-γ concentration. Mycobacterium bovis was identified in bronchoalveolar lavage fluid by polymerase chain reaction. Genetic testing identified a heterozygous mutation of c.1154C>T causing a T385M amino acid substitution in STAT1. Despite antibacterial and antifungal therapy, the febrile disease was not controlled. The signs of HLH were relieved after HLH-94 protocol administration, except fever. Fever was not resolved until he received anti-tuberculosis therapy. Hematopoietic stem cell transplantation was refused and the patient died six months later due to severe pneumonia.Patients with STAT1 GOF mutation have broad clinical manifestations and may develop HLH. This form of HLH presents with normal IFN-γ level without cytokine storm.
225	Hemophagocytic Lymphohistiocytosis Hospitalizations in Adults and Its Association With Rheumatologic Diseases: Data From Nationwide Inpatient Sample.	33337810	Hemophagocytic lymphohistiocytosis (HLH) is a rare potentially fatal multisystem inflammatory condition that is often triggered by an underlying medical condition. Epidemiologic data of HLH in adults with rheumatologic diseases are limited. The aim of our study was to characterize HLH hospitalizations in the US adult population with a special focus on patients with concomitant rheumatologic diseases.We conducted a medical records review of hospitalizations in the United States during 2016 and 2017 with a diagnosis of HLH. Hospitalizations were selected from the National Inpatient Sample. International Classification of Diseases, Tenth Revision codes were used to identify rheumatologic diseases. A multivariate logistic regression analysis was used to calculate adjusted odds ratios (ORadj) for the association of HLH and rheumatologic diseases.Seven hundred fifty hospitalizations had a principal billing diagnosis of HLH. The median age of our study population was 47.5 years, and males made up 55% of the population. Overall mortality was 17%, and the median length of stay was 12 days. Twenty-five percent of the HLH cases had a concomitant rheumatologic diagnosis. Multivariate logistic regression analysis showed systemic lupus erythematosus (SLE) with nephritis (ORadj, 5.7), SLE without nephritis (ORadj, 9.2), adult-onset Still disease (ORadj, 338.9), and ankylosing spondylitis (ORadj, 10.7) were significantly associated with HLH.This analysis represents the largest sample to date to assess HLH hospitalizations. Our study showed that SLE, adult-onset Still disease, and ankylosing spondylitis were strongly associated with HLH.
226	Continuous Renal Replacement Therapy with oXiris® Membrane in Severe Ebstein-Barr Virus-Mediated Hemophagocytic Lymphohistiocytosis: A Case Report.	33333505	Hemophagocytic lymphohistiocytosis (HLH), a life-threatening disease with uncontrolled immune activation and inflammatory reaction, often leads to a deadly cytokine storm. In severe Ebstein-Barr virus-triggered HLH receiving standard immunosuppression, continuous renal replacement therapy (CRRT) with oXiris® blood purification membrane resulted in a timely reduction of inflammatory markers and discontinuation of vasopressors. To our knowledge, this is the first report of successful use of the oXiris® membrane in HLH.
227	Hemophagocytic Lymphohistiocytosis as the Initial Presentation of Subcutaneous Panniculitis-Like T-Cell Lymphoma: A Rare Case Responding to Cyclosporine A and Steroids.	33331177	Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare peripheral cytotoxic T-cell lymphoma, clinically resembling panniculitis. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of immune overactivation, triggered by underlying conditions. SPTCL presenting with HLH may represent a severe and rapidly progressive disease course. Currently, there is no standardized approach to treatment of HLH secondary to underlying SPTCL. A 34-year-old Asian male presented with a several months history of high fevers, weight loss, and nonpruritic purple discoloration of the skin. He had a skin biopsy showing atypical lymphohistiocytic panniculitis with dermal mucinosis and erythrophagocytosis consistent with SPTCL. The patient was initiated on treatment with dexamethasone and cyclosporine A. Almost immediate improvement of his skin lesions was noted and laboratory abnormalities trended toward baseline within 2 weeks. He noted complete symptom resolution after 3 months on therapy. SPTCL may be treated effectively with cyclosporine A and steroids to achieve rapid clinical and symptom management of this rare malignancy.
228	Chediak-Higashi Syndrome With Epstein-Barr Virus Triggered Hemophagocytic Lymphohistiocytosis: A Case Report.	33329964	Chediak-Higashi syndrome (CHS) is a rare, autosomal-recessive disorder characterized by oculocutaneous albinism, recurrent bacterial infections, progressive neurologic abnormalities, coagulation defects and a high risk of developing hemophagocytic lymphohistiocytosis characterized by pancytopenia, high fever, and lymphohistiocytic infiltration of liver, spleen, and lymph nodes. Treatment of accelerated-phase CHS is difficult with poor prognosis. Here, we report a two-and-a-half-year-old male child who was diagnosed with Chediak-Higashi Syndrome based on silvery hair, pathognomonic hair microscopy and giant azurophilic granules in granulocytes. The patient was in advanced stage of HLH induced by an Epstein-Barr virus (EBV) infection and given etoposide, cyclosporine and dexamethasone according to hemophagocytic lymphohistiocytosis (HLH)-2004 protocol but did not survive.
229	A conserved cysteine-based redox mechanism sustains TFEB/HLH-30 activity under persistent stress.	33314217	Mammalian TFEB and TFE3, as well as their ortholog in Caenorhabditis elegans HLH-30, play an important role in mediating cellular response to a variety of stress conditions, including nutrient deprivation, oxidative stress, and pathogen infection. In this study, we identify a novel mechanism of TFEB/HLH-30 regulation through a cysteine-mediated redox switch. Under stress conditions, TFEB-C212 undergoes oxidation, allowing the formation of intermolecular disulfide bonds that result in TFEB oligomerization. TFEB oligomers display increased resistance to mTORC1-mediated inactivation and are more stable under prolonged stress conditions. Mutation of the only cysteine residue present in HLH-30 (C284) significantly reduced its activity, resulting in developmental defects and increased pathogen susceptibility in worms. Therefore, cysteine oxidation represents a new type of TFEB post-translational modification that functions as a molecular switch to link changes in redox balance with expression of TFEB/HLH-30 target genes.
230	Id(entifying) the inhibitor of DNA binding 3 in the brain of Nothobranchius furzeri upon aging.	33314133	Inhibitors of DNA (Id) are key transcription factors (TFs) regulating neurogenic processes. They belong to the helix-loop-helix (HLH) TF family and are dominant negative regulators of basic HLH proteins (bHLHs). Specifically, they inhibit cell differentiation and enhance cell proliferation and motility. The Id family includes four members, Id1, Id2, Id3, and Id4, which have been identified in nearly all vertebrates. The transcript catalog of the African turquoise killifish, Nothobranchius furzeri, contains all four TFs and has evolved showing positive selection for Id3. N. furzeri, a teleost, is the short-lived vertebrate and is gaining increasing scientific interest as a new model organism in aging research. It is characterized by embryonic diapause, explosive sexual maturation, and rapid aging. In this study, we investigated both the expression and the role of Id3 in the brain of this model organism. Interestingly, Id3 was upregulated age-dependently along with a distribution pattern resembling that of other vertebrates. Additionally, the gene has undergone positive selection during evolution and shows a high degree of conservation relative to that of other vertebrates. These features make N. furzeri a valid tool for aging studies and a potential model in translational research.
231	Etiologies and management of haemophagocytic lymphohistiocytosis: is it time for an updated protocol and targeted treatments?	33313881	The objective of this study was to analyse the features, therapeutic approaches, and outcomes for adult patients with haemophagocytic lymphohistiocytosis (HLH) at a single centre.This study was a retrospective chart review of all patients >18 years of age diagnosed with HLH according to HLH-2004 or H-score criteria at Ochsner Medical Center-New Orleans between 2013 and 2019.A total of 29 patients with HLH met inclusion criteria. A total of 7 patients had an underlying malignancy, 12 had an autoimmune disease, 2 were transplant patients, and 2 had a combination of malignancy, autoimmune disease, or immunodeficiency. A total of 6 patients developed HLH precipitated by infection alone. All 29 patients presented with fever. A total of 28 (97%) patients met H-score criteria, and only 20 (67%) met HLH-2004 criteria. Fifteen patients were treated with the HLH-2004 protocol. Of those treated with the HLH-2004 protocol, 73% (11/15) died, 8% (1/15) had recurrence of HLH, and 20% (3/15) had resolution of HLH. A total of 14 patients were treated with targeted therapy. Of those treated with targeted therapy, 93% (13/14) had resolution of HLH and 1 died. Targeted therapy included pulse steroids, tocilizumab, anakinra, IVIG, CSA, rituximab, and/or CYC in addition to antiviral or antibiotic therapy.Our findings suggested that the rheumatologic patient population responded well to a targeted therapeutic approach and poorly to the HLH-2004 protocol. Whether the poor outcomes found with the use of the HLH-2004 protocol are secondary to the protocol itself or the aggressive nature of malignancy-associated HLH is unclear. Further studies are needed to develop tailored therapeutic regimens.
232	Co-Occurrence of Familial Hemophagocytic Lymphohistiocytosis Type 2 and Chronic Active Epstein-Barr Virus in Adulthood.	33309387	We report, to the best of our best knowledge, the oldest individual to ever be diagnosed with Familial Hemophagocytic Lymphohistiocytosis (FHL) Type 2 from homozygous c.1349C>T (p.T450M) missense variants in the PRF1 gene. This rare case advanced in complexity with a simultaneous diagnosis of Chronic Active Epstein-Barr Virus (CAEBV) - a distinct clinical entity from acute EBV infections and a well-described trigger of Hemophagocytic Lymphohistiocytosis (HLH). This is, to the best of our knowledge, the only individual to ever be diagnosed with CAEBV in the setting of this specific variant and the oldest to be diagnosed with a coexisting perforin variant. This case provides understanding of EBV, human genetics, and lymphoproliferative disorders while adding a unique differential diagnosis to adults who present with fever of unknown origin and diffuse lymphadenopathy without evidence of malignancy. This report explores the diagnosis and treatment of both HLH and CAEBV, encouraging discussion regarding current clinical management and future research needs.
233	Dengue and Plasmodium falciparum Coinfection With Secondary Hemophagocytic Lymphohistiocytosis in a 3-Year-Old Boy: A Clinical Conundrum.	33306604	Hemophagocytic lymphohistiocytosis (HLH) is a multisystem disease wherein there is an exaggerated immune system activation following a trigger such as infection, malignancy, or autoimmune diseases. Here we report a case of a 3-year-old boy who presented to us with fever, was diagnosed with dengue fever, and treatment started for the same. Clinical response was poor to treatment and high-grade fever persisted. Subsequent evaluation showed Plasmodium falciparum malaria and treatment was initiated with antimalarial drugs. Further clinical deterioration with poor trend of laboratory values over the next few days prompted evaluation for HLH; workup was positive satisfying the HLH-2004 criteria and IV dexamethasone was started. The child gradually improved and was discharged with normal counts on follow-up over the next 3 months. This article emphasizes on the importance of high degree of suspicion, early workup, and initiation of treatment for HLH for a better outcome.
234	Hemophagocytic Lymphohistiocytosis as a Manifestation of Underlying Visceral Leishmaniasis.	33304709	Hemophagocytic lymphohistiocytosis (HLH), or hemophagocytic syndrome (HS) is a severe syndrome involving an extreme participation of the immune system, resulting in a cascade of cytokines, hyperinflammation and extensive hemophagocytosis in the bone marrow (BM) and affecting the peripheral blood (PB) lineages. Fever, splenomegaly, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia are often encountered in this disease. The syndrome can be seen in all ages and it is either primary due to genetic defects or secondary because of malignancies, immune deficiencies, rheumatic diseases, and infections. Bacteria, viruses, protozoa, and fungi are often implicated. Visceral leishmaniasis (VL) is among the infectious causes of HLH. We describe a patient with a successful treatment of HLH after the initiation of liposomal amphotericin B, due to VL, even though there was a delay in diagnosing the leishmaniasis. The exact precipitating pathophysiological events triggering HLH remain unknown and provide their clear impact for future research. An instructive, critical review of the literature related to the presented case is provided. Distinguishing secondary HS from primary HS is essential for the application of suitable treatment. Improper use of corticosteroids could cover up an underlying possible malignancy or infection and delay the initiation of the etiologic therapeutic strategy.
235	Clinical Characteristics of Hemophagocytic Lymphohistiocytosis Associated with Non-Hodgkin B-Cell Lymphoma: A Multicenter Retrospective Study.	33303420	Hemophagocytic lymphohistiocytosis (HLH) associated with B-cell lymphoma is a highly aggressive disease with unclear clinical features and has no standard treatment.We analyzed the clinical characteristics of 31 patients from two individual centers.The median overall survival was only 1.5 months. Both univariate and multivariate analyses, based on lymphoma or HLH-related characteristics, revealed that patients with high Epstein-Barr virus (EBV) DNA load and ≥ 2 extranodal lesions, or hypofibrinogenemia, respectively, showed significantly poorer overall survival. Interestingly, some patients with high EBV DNA load had EBV-positive natural killer (NK) and/or T cells, which may be related to the coexistence of immunodeficiency and/or chronic active EBV infection. Molecular genetics examination confirmed that 47.4% (9/19) of patients had complex karyotypes, 37.5% (3/8) of patients had TP53 deletions, and 21.34% (3/14) of patients had TP53 mutation or alteration of malignancy-related pathways, including BCR/NF-κB, JAK-STAT, and epigenetic regulatory pathways, which may provide clues to choose targets for therapy. Treatment regimens containing etoposide, anti-CD20 monoclonal antibodies, or anthracyclines improved patient prognosis (P = .0183, .025, and .0436, respectively). Patients with infections had significantly shorter survival than those without infections (P = .00019).The patients' performance status, number of extranodal lesions, high EBV DNA load, and hypofibrinogenemia are poor prognostic factors for HLH associated with B-cell lymphoma. Molecular genetic high-risk factors are of particular importance because these factors can provide information for prognosis prediction, treatment decisions, and disease surveillance.
236	Suspected Primary Adrenal Lymphoma (PAL) Associated With Hemophagocytic Lymphohistiocytosis (HLH).	33294760	Adrenal incidentalomas, masses noted on imaging performed for other purposes, are common, with 10% to 15% presenting as bilateral adrenal masses. These cases can be challenging as the differential diagnosis is broad, including metastatic disease, primary adrenal lymphoma (PAL), or infection, and often requiring a biopsy if initial biochemical workup is unrevealing. We present here a relevant case description, laboratory and radiologic imaging studies, and discussion of literature. A 62-year-old Korean woman presented with altered mental status and fevers. She was found to have bilateral adrenal incidentalomas and retained acupuncture needles. Adrenal workup did not show biochemical evidence of hormonal excess. Infectious workup was unrevealing, as was a metal/toxin workup due to retained acupuncture needles. Fevers and episodes of hypotension persisted which prevented the patient from obtaining an adrenal biopsy. Bone marrow biopsy was obtained for pancytopenia and revealed B-cell lymphoma with large cell morphology and few histiocytes with hemophagocytosis, raising concern for lymphoma-induced hemophagocytic lymphohistiocytosis (HLH). PAL associated with HLH was highly suspected in our patient, given the large (7 cm) bilateral adrenal masses and bone marrow biopsy findings of lymphoma. The patient was treated for diffuse large B-cell lymphoma, with clinical improvement. PAL is a rare but aggressive lymphoma with few reported cases. It should be considered in the differential for both unilateral and bilateral adrenal masses. An early diagnosis is crucial as the main treatment is chemotherapy rather than surgery and it confers a significant survival benefit.
237	T cell gene therapy to treat immunodeficiency.	33280098	The application of therapeutic T cells for a number of conditions has been developed over the past few decades with notable successes including donor lymphocyte infusions, virus-specific T cells and more recently CAR-T cell therapy. Primary immunodeficiencies are monogenetic disorders leading to abnormal development or function of the immune system. Haematopoietic stem cell transplantation and, in specific candidate diseases, haematopoietic stem cell gene therapy has been the only definitive treatment option so far. However, autologous gene-modified T cell therapy may offer a potential cure in conditions primarily affecting the lymphoid compartment. In this review we will highlight several T cell gene addition or gene-editing approaches in different target diseases with a focus on what we have learnt from clinical experience and promising preclinical studies in primary immunodeficiencies. Functional T cells are required not only for normal immune responses to infection (affected in CD40 ligand deficiency), but also for immune regulation [disrupted in IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, X-Linked) due to dysfunctional FOXP3 and CTLA4 deficiency] or cytotoxicity [defective in X-lymphoproliferative disease and familial haemophagocytic lymphohistiocytosis (HLH) syndromes]. In all these candidate diseases, restoration of T cell function by gene therapy could be of great value.
238	Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation.	33276448	The PACLOBUTRAZOL-RESISTANCE (PRE) gene family encodes a group of atypical helix-loop-helix (HLH) proteins that act as the major hub integrating a wide range of environmental and hormonal signals to regulate plant growth and development. PRE1, as a positive regulator of cell elongation, activates HBI1 DNA binding by sequestering its inhibitor IBH1. Furthermore, PRE1 can be phosphorylated at Ser-46 and Ser-67, but how this phosphorylation regulates the functions of PRE1 remains unclear. Here, we used a phospho-mutant activity assay to reveal that the phosphorylation at Ser-67 negatively regulates the functions of PRE1 on cell elongation. Both of mutations of serine 46, either to phospho-dead alanine or phospho-mimicking glutamic acid, had no significant effects on the functions of PRE1. However, the mutation of serine 67 to glutamic acid (PRE1S67E-Ox), but not alanine (PRE1S67A-Ox), significantly reduced the promoting effects of PRE1 on cell elongation. The mutation of Ser-67 to Glu-67 impaired the interaction of PRE1 with IBH1 and resulted in PRE1 failing to inhibit the interaction between IBH1 and HBI1, losing the ability to induce the expression of the subsequent cell elongation-related genes. Furthermore, we showed that PRE1-Ox and PRE1S67A-Ox both suppressed but PRE1S67E-Ox had no strong effects on the dwarf phenotypes of IBH1-Ox. Our study demonstrated that the PRE1 activity is negatively regulated by the phosphorylation at Ser-67.
239	Role of inhibitor of differentiation 3 gene in cellular differentiation of human corneal stromal fibroblasts.	33273801	Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGFβ)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGFβ-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation.Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGFβ1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGFβ-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (α-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis.The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGFβ1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in α-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGFβ1 showed the expected low expression of α-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGFβ-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGFβ-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein.Id3 regulates TGFβ-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.
240	Dengue Fever Associated Haemophagocytic Lymphohistiocytosis: A Report of Two Children.	33269160	Hemophagocytic lymphohistiocytosis (HLH) is a rare, fatal complication of dengue infection and often leads to multi-system involvement and failure. Early recognition is crucial in improving the outcome. We report two children who developed secondary haemophagocytic lymphohistiocytosis following dengue fever. A 14-year-old girl who was diagnosed with beta-thalassemia major presented with dengue hemorrhagic fever and developed a continuous very high fever, persistent thrombocytopenia, and anemia despite several transfusions of blood and blood products. The other child was a 12-year old girl who, following initial confirmation of dengue fever, presented with persistent fever and hepatosplenomegaly. The diagnosis of HLH in both children was confirmed by clinical and laboratory features supported by the demonstration of haemophagocytes in the bone marrow. Both children were treated with steroids and supportive care and made a gradual recovery with treatment. Second-line immune-suppressive treatment was not needed. Whilst sepsis is a priority differential diagnosis in children with persistent fever following recovery from dengue fever, HLH should always be suspected early in these patients. Early, appropriate immunosuppressive treatment is likely to improve long-term outcomes and prevent mortality.
241	A Fatal Case of Kaposi Sarcoma Immune Reconstitution Syndrome (KS-IRIS) Complicated by Kaposi Sarcoma Inflammatory Cytokine Syndrome (KICS) or Multicentric Castleman Disease (MCD): A Case Report and Review.	33268763	BACKGROUND Kaposi Sarcoma Inflammatory Cytokine Syndrome (KICS) is a relatively new syndrome described in patients co-infected with Human Immunodeficiency Virus (HIV) and Kaposi Sarcoma (KS) Herpes Virus (KSHV). KICS clinically resembles Multicentric Castleman disease (MCD) and both present with various degrees of lymphadenopathy, pancytopenia, HIV and KSHV viremia, and signs of systemic inflammatory syndrome (SIRS). KICS has higher mortality than MCD and is rarely recognized. Lymph node, bone marrow, or splenic biopsy can help differentiate between the 2 entities. CASE REPORT We present a case of a 28-year-old African American man with advanced acquired immunodeficiency syndrome (AIDS) who was diagnosed with disseminated pulmonary and cutaneous KS. Following initiation of combined antiretroviral therapy (cART), rapid immunologic recovery occurred followed by rapid clinical deterioration (IRIS) with multiorgan failure, overwhelming SIRS, and ultimately death. The patient's symptoms, signs, and laboratory findings during this episode could not be solely explained by KS-IRIS, and MCD versus KICS was diagnosed. CONCLUSIONS SIRS in patients with uncontrolled HIV viremia and CD4 lymphopenia has a broad differential diagnosis, including infectious and noninfectious causes. It encompasses sepsis due to common bacterial pathogens, various HIV-specific opportunistic infections, immunological conditions such as hemophagocytic lymphohistiocytosis (HLH), and IRIS, malignancies such as primary effusion lymphoma (PEL) and MCD, and finally KCIS. Clinicians involved in treatment of these patients should have a high index of suspicion for less-known and recently described syndromes such as KICS to recognize it early and initiate timely treatment, which might improve the high mortality associated with KICS.
242	Hemophagocytic Lymphohistiocytosis in Children with Chronic Granulomatous Disease-Single-Center Experience from North India.	33259975	Chronic granulomatous disease (CGD) is an inherited defect in components of the nicotinamide adenine dinucleotide phosphate oxidase complex that results in potential life-threatening infective and noninfective complications. Hemophagocytic lymphohistiocytosis (HLH) is an unusual but important inflammatory complication of CGD. Optimal management strategies have not yet been identified in children with CGD who develop HLH.To analyze clinical and laboratory features of HLH in CGD from a tertiary-care center in North India.A retrospective review of medical records of children with CGD diagnosed in the last 20 years was performed. Clinical and laboratory features of children with CGD who developed HLH were analyzed.Of 80 patients diagnosed with CGD, 5 (6.25%) had evidence of HLH. All 5 were males; 4 had X-linked CGD and 1 had autosomal recessive CGD (NCF2 defect). Two children with CGD had HLH as the predominant presenting manifestation mimicking the clinical presentation of congenital HLH. Infectious triggers identified were bloodstream infections (n = 3) (Candida albicans, Burkholderia cenocepacia, Francisella noatuensis), pneumonia (n = 4), and splenic abscess (n = 1). We document the first human infection with a fish pathogen, F. noatuensis, in a child with X-linked CGD. Although mortality was seen in 3 children who received only intravenous (IV) immunoglobulin therapy, the other 2 who received IV methylprednisolone pulse therapy survived.HLH can be a presenting manifestation of CGD, and workup for CGD must be considered in children with HLH. Early recognition with optimal management of both infectious trigger and HLH is very important to prevent mortality.
243	The CIB1 transcription factor regulates light- and heat-inducible cell elongation via a two-step HLH/bHLH system.	33258952	Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4-PAR1/HFR1 and CIB1-PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA binding small HLH proteins in the transcriptional regulation of cell elongation in plants.
244	[The diagnostic value of flow cytometry in patients with lymphoma associated hemophagocytic syndrome].	33256339	Objective: To evaluate the reliability of flow cytometry (FCM) for diagnosing lymphoma associated hemophagocytic syndrome (LAHS). Method: The clinical data in 57 patients with hemophagocytic lymphohistiocytosis (HLH)were retrospective analyzed at Peking University Shenzhen Hospital from July 2010 to July 2019. All patients were performed bone marrow FCM and bone marrow pathological examination before final diagnoses were made. The golden diagnosis criterion was based on clinical, biochemical and histopathological evidence, which was regarded as the standard to evaluate the sensitivity and specificity of FCM analysis in diagnosing LAHS. Results: Among 57 cases, 36 cases were eventually diagnosed with LAHS, including 15 B-cell lymphoma(14 diffuse large B-cell lymphoma, 1 B-cell lymphoma with reactive T-cell hyperplasia), 13 aggressive NK/T cell lymphoma/leukemia, 2 cases of gamma-delta T-cell lymphoma, 4 angioimmunoblastic T-cell lymphoma, 1 enteropathy-associated peripheral T-cell lymphoma and 1 anaplastic T-cell lymphoma. Lymphoma cells in bone marrow were detected in all patients by FCM except one ENTCL patient. The sensitivity and the specificity of FCM in LASH compared to bone marrow biopsy were 97.2%(P=0.014)and 90.5%(P=0.488) respectively. In the other 21 non-LAHS patients, T cell receptor Vβ (TCRVβ) rearrangement was detected in 2 patients with Epstein-Barr virus (EBV) associated primary HLH. Conclusions: FCM effectively detects lymphoma cells in bone marrow of lymphoma patients with LHL, suggesting that FCM could be an important indicator for the diagnosis of LAHS. FCM also has the advantage in differentiating LAHS from other HLH.目的： 应用流式细胞术（FCM）对骨髓淋巴瘤细胞特征性免疫表型进行分析，评估FCM对初诊以噬血细胞综合征（HLH）为首发临床表现的非霍奇金淋巴瘤的快速鉴别诊断价值。 方法： 回顾性分析2010年7月至2019年7月在北京大学深圳医院血液科收治的57例已经明确病因诊断的HLH患者的临床资料，并且所有患者在起病时至少完成1次骨髓流式细胞检测和骨髓病理检查，以同期患者的临床、生化及组织病理为金标准，评估FCM分析骨髓淋巴瘤细胞的免疫表型特征对淋巴瘤相关的噬血细胞综合征（LAHS）的诊断效能。 结果： 57例符合HLH诊断标准的患者，36例患者经临床、生化和组织病理最终明确诊断为LAHS，其中B细胞淋巴瘤15例（弥漫大B细胞淋巴瘤14例，B细胞淋巴瘤伴反应性T细胞增生1例）；侵袭性NK细胞淋巴瘤/白血病13例；肝脾γδT细胞淋巴瘤2例；血管免疫母T细胞淋巴瘤（AILT）4例；肠病性外周T细胞淋巴瘤（ENTCL）1例；间变性T细胞淋巴瘤（小细胞型）（ATCL）1例。除1例ENTCL外，FCM均在上述病例骨髓中发现有特征性免疫表型的淋巴瘤细胞，与骨髓疾病比较，FCM对本组LAHS病例诊断的敏感度为97.2%（P=0.014），特异度为90.5%（P=0.488）。在21例非LAHS患者中，FCM在2例原发EB病毒相关噬血细胞综合征检测出有TCRVβ重排，其他均未显示有异常特征性免疫表型。 结论： FCM对骨髓淋巴瘤细胞特征性免疫表型分析可作为诊断LAHS的重要指标，具有快速鉴别LAHS和非LAHS的优势，缩短了确诊时间，有利于临床作出早期诊断和快速处理。.
245	A novel case of homozygous IFNAR1 deficiency with haemophagocytic lymphohistiocytosis.	33252644	We present a case of complete deficiency of the Interferon alpha/beta receptor alpha chain (IFNAR1) in a child with fatal systemic hyperinflammation, apparently provoked by live-attenuated viral vaccination. Such pathologic hyperinflammation, fulfilling criteria for haemophagocytic lymphohistiocytosis, is an emerging phenotype accompanying inborn errors of type I interferon immunity.
246	Intrahepatic cholangiocarcinoma: MRI texture signature as predictive biomarkers of immunophenotyping and survival.	33245493	Clinical evidence suggests that the response to immune checkpoint blockade depends on the immune status in the tumor microenvironment. This study aims to predict the immunophenotyping (IP) and overall survival (OS) of intrahepatic cholangiocarcinoma (ICC) patients using preoperative magnetic resonance imaging (MRI) texture analysis.A total of 78 ICC patients were included and divided into inflamed (n = 26) or non-inflamed (n = 52) immunophenotyping based on the density of CD8+ T cells. The enhanced T1-weighted MRI in the arterial phase was employed with texture analysis. The logistic regression analysis was applied to select the significant features related to IP. The OS-related feature was determined by Cox proportional-hazards model and Kaplan-Meier analysis. IP and OS predictive models were developed using the selected features, respectively.Three wavelets and one 3D feature have favorable ability to discriminate IP, a combination of which performed best with an AUC of 0.919. The inflamed immunophenotyping had a better prognosis than the non-inflamed one. The 5-year survival rates of the two groups were 48.5% and 25.3%, respectively (p < 0.05). The only wavelet-HLH_firstorder_Median feature was associated with OS and used to build the OS predictive model with a C-index of 0.70 (95% CI, 0.57, 0.82), which could well stratify ICC patients into high- and low-risk groups. The 1-, 3-, and 5-year survival probabilities of the stratified groups were 62.5%, 30.0%, and 24.2%, and 89.5%, 62.2%, and 42.1%, respectively (p < 0.05).The MRI texture signature could serve as a potential predictive biomarker for the IP and OS of ICC patients.• The MRI texture signature, including three wavelets and one 3D feature, showed significant associations with immunophenotyping of ICC, and all have favorable ability to discriminate immunophenotyping; a combination of the above features performed best with an AUC of 0.919. • The only wavelet-HLH_firstorder_Median feature was associated with the OS of ICC and used to build the OS predictive model, which could well stratify ICC patients into high- and low-risk groups.
247	A retrospective analysis of etiology and outcomes of hemophagocytic lymphohistiocytosis in children and adults.	33242384	Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe, life-threatening inflammatory condition if untreated. We aimed to investigate the etiologies, outcomes, and risk factors for death in children and adults with HLH.The medical records of patients who met the HLH criteria of two regional university hospitals in Korea between January 2001 and December 2019 were retrospectively investigated.Sixty patients with HLH (35 children and 25 adults) were included. The median age at diagnosis was 7.0 years (range, 0.1-83 years), and the median follow-up duration was 8.5 months (range, 0-204 months). Four patients had primary HLH, 48 patients had secondary HLH (20 infection-associated, 18 neoplasm-associated, and 10 autoimmune-associated HLH), and eight patients had HLH of unknown cause. Infection was the most common cause in children (14/35, 40.0%), whereas neoplasia was the most common cause in adults (13/25, 52.0%). Twenty-eight patients were treated with HLH-2004/94 immunochemotherapy. The 5-year overall survival (OS) rate for all HLH patients was 59.9%. The 5-year OS rates for patients with primary, infection-associated, neoplasm-associated, autoimmune-associated, and unknown cause HLH were 25.0%, 85.0%, 26.7%, 87.5%, and 62.5%, respectively. Using multivariate analysis, neoplasm-induced HLH (p=0.001) and a platelet count <50×109/L (p=0.008) were identified as independent risk factors for poor prognosis in patients with HLH.Infection was the most common cause of HLH in children, while it was neoplasia in adults. The 5-year OS rate for all HLH patients was 59.9%. HLH caused by an underlying neoplasm or a low platelet count at the time of diagnosis were risk factors for poor prognosis.
248	An Unusual Presentation of Adult-Onset Still's Disease as Hemophagocytic Lymphohistiocytosis in a Male Patient.	33240728	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and potentially fatal condition characterized by immune activation and multi-organ dysfunction. HLH can be inherited in an autosomal recessive fashion, but can also be secondary to infections, malignancy, immunosuppression, and autoimmune conditions. Adult-onset Still's disease (AOSD) is an autoimmune disorder that can predispose patients to HLH. AOSD, similar to other autoimmune conditions, is more common in females than males. However, the occurrence of AOSD in males and subsequent predisposition to HLH is rarely reported. We report the case of a 23-year-old male patient who presented with fever, joint pain, and rash for 20 days. On evaluation, he fulfilled the diagnostic criteria for AOSD and HLH, and a diagnosis of HLH secondary to AOSD was made. He was treated with pulse dose steroids and gradually tapered. AOSD and HLH have overlapping clinical and laboratory features and hence their co-occurrence poses diagnostic challenges. The mortality rate of HLH is high and hence prompt initiation of treatment is of utmost importance.
249	Clinical characteristics of Epstein-Barr virus infection in the pediatric nervous system.	33238935	To investigate the clinical characteristics of Epstein-Barr virus (EBV) infection in the pediatric nervous system (NS).We retrospectively analyzed the clinical data and follow-up results of 89 children with neurological damage caused by EBV who were hospitalized in the children's hospital of Chongqing Medical University from January 2008 to April 2019.EBV infection of the NS can occur at any time of the year. The highest incidence was seen in the age group of 0-4 years. Fever is the main clinical feature (74/89, 83.1%). The main clinical types were encephalitis/meningoencephalitis (64/89, 71.9%), acute myelitis (2/89, 2.2%), acute disseminated encephalomyelitis (ADEM) (3/89, 3.4%), Guillain-Barré Syndrome (GBS) (15/89, 16.9%), neurological damage caused by EBV-hemophagocytic lymphohistiocytosis (EBV-HLH) (4/89, 4.5%), and NS-post-transplant lymphoproliferative disorder (NS-PTLD) (1/89, 1.1%). Anti-N-methyl-D-aspartate receptor encephalitis was found during the convalescence of EBV encephalitis. EBV encephalitis/meningitis showed no symptoms of tonsillitis, lymph node enlargement, skin rash, hepatosplenomegaly. Acute motor axonal neuropathy is the chief complication in GBS caused by EBV.There were significant differences in neurological complications caused by EBV. The prognosis of EBV infection in the NS is generally good. These illnesses are often self-limiting. A few cases may show residual sequelae.
250	Stimulated by retinoic acid gene 8 (STRA8) interacts with the germ cell specific bHLH factor SOHLH1 and represses c-KIT expression in vitro.	33236849	STRA8 (Stimulated by Retinoic Acid Gene 8) controls the crucial decision of germ cells to engage meiotic division up and down-regulating genes involved in the meiotic programme. It has been proven as an amplifier of genes involved in cell cycle control and chromosome events, however, how STRA8 functions as negative regulator are not well understood. In this study, we demonstrate that STRA8 can interact with itself and with other basic Helix-Loop-Helix (bHLH) transcription factors through its HLH domain and that this domain is important for its ability to negatively interfere with the Ebox-mediated transcriptional activity of bHLH transcription factors. Significantly, we show that STRA8 interacts with TCF3/E47, a class I bHLH transcription factors, and with SOHLH1, a gonadal-specific bHLH, in male germ cells obtained from prepuberal mouse testis. We demonstrated that STRA8, indirectly, is able to exert a negative control on the SOHLH1-dependent stimulation of c-KIT expression in late differentiating spermatogonia and preleptotene spermatocytes. Although part of this results were obtained only 'in vitro', they support the notion that STRA8 interacting with different transcription factors, besides its established role as 'amplifier' of meiotic programme, is able to finely modulate the balance between spermatogonia proliferation, differentiation and acquisition of meiotic competence.
251	HLH-11 modulates lipid metabolism in response to nutrient availability.	33235199	The ability of organisms to sense nutrient availability and tailor their metabolic states to withstand nutrient deficiency is critical for survival. To identify previously unknown regulators that couple nutrient deficiency to body fat utilization, we performed a cherry-picked RNAi screen in C. elegans and found that the transcription factor HLH-11 regulates lipid metabolism in response to food availability. In well-fed worms, HLH-11 suppresses transcription of lipid catabolism genes. Upon fasting, the HLH-11 protein level is reduced through lysosome- and proteasome-mediated degradation, thus alleviating the inhibitory effect of HLH-11, activating the transcription of lipid catabolism genes, and utilizing fat. Additionally, lipid profiling revealed that reduction in the HLH-11 protein level remodels the lipid landscape in C. elegans. Moreover, TFAP4, the mammalian homolog of HLH-11, plays an evolutionarily conserved role in regulating lipid metabolism in response to starvation. Thus, TFAP4 may represent a potential therapeutic target for lipid storage disorders.
252	Hemophagocytic lymphohistiocytosis of a melanoma patient under BRAF/MEK-inhibitor therapy following anti-PD1 inhibitor treatment: a case report and review to the literature.	33234845	Hemophagocytic lymphohistiocytosis (HLH) is a rare but life-threatening condition. HLH in infants and young children is usually inherited, which is then classified as primary HLH. Secondary HLH, in contrast, is caused by many different conditions such as infections, cancer or medication and affects mostly adults. HLH is a hyperinflammatory condition, which may mimic an acute septic shock. We report on a 68-year-old patient with malignant melanoma with lymph node metastases. Due to the lymphogenic progression, treatment was switched from nivolumab to dabrafenib and trametinib. Twenty-one days after initiation of BRAF/MEK inhibitor therapy, the patient presented to our emergency department with clinical signs of infection such as fever and fatigue. Laboratory tests showed excessive inflammation levels without identifying an underlying pathogen. Two days later, the patient developed an increasing pancytopenia. After extending the diagnosis, we found very high ferritin levels, hypertriglyceridemia, hypofibrinogenemia and a soluble CD25 receptor. Based on the laboratory results, prolonged fever and splenomegaly, we were able to diagnose HLH as the underlying condition. We immediately initiated treatment with intravenous prednisone, which remarkably improved the clinical symptoms. After full recovery, we reinitiated anti-tumor treatment with vemurafenib and cobimetinib, which was tolerated without side effects. Due to the relatively nonspecific nature of the clinical signs and symptoms and the significant overlap with other diseases such as sepsis, the diagnosis of HLH is often delayed. This explains, in part, the high morbidity and mortality rate. Our case shows that early treatment with steroids is effective. However, much work remains in order to raise awareness and improve the understanding of the pathophysiology of this condition.
253	X-linked Lymphoproliferative Disease (XLP1) Presenting as Non-Epstein Barr Virus (EBV) - Related Hemophagocytic Lymphohistiocytosis (HLH).	33231181	NaN
254	The DEP regimen is superior to the HLH-1994 regimen as first-line therapy for lymphoma-associated haemophagocytic lymphohistiocytosis.	33225788	Lymphoma-associated haemophagocytic lymphohistiocytosis (LA-HLH) has a poor prognosis. Currently, there is no unified, effective first-line treatment regimen for it. We retrospectively analyzed the clinical data of 50 patients who received the DEP regimen and 30 patients who received the HLH-1994 regimen. After 2 weeks of treatment, the ORR of the DEP group was higher than that of the HLH-1994 group (p = 0.024). After 4 weeks, the CR and ORR of the DEP group were higher than those of the HLH-1994 group (p < 0.05). The recurrence rate of the HLH-1994 group within 4 weeks (20.0%) was higher than that of the DEP group (2.1%) (p < 0.05). The median survival of patients with NK/T and T-cell lymphoma in the DEP group (10.1 months) was longer than the median survival of the HLH-1994 group (2.6 months) (p = 0.017). Our study suggests that the DEP regimen can improve Week 2 and Week 4 ORR, Week 4 CR, and reduce Week 4 recurrence rate for LA-HLH more than the HLH-1994 regimen.
255	A Rare Case of Glycogen Storage Disease Type 1a Presenting with Hemophagocytic Lymphohistiocytosis (HLH).	33224545	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome characterized by fever, respiratory distress, massive hepatomegaly, and bicytopenia. It is classified into primary (congenital) and secondary (acquired) types. There are many diseases associated with secondary HLH, but glycogen storage disease is a novel cause of secondary HLH. Case Presentation. In this case, we present a five-month-old female infant with recurrent fever, poor feeding, pallor, and prolonged diarrhea for two months. With a diagnosis of HLH, the patient was treated with IVIG and prednisolone. After treatment was initiated, the patient's general condition improved. All metabolic workup was normal. We did whole-exome sequencing that confirmed glycogen storage disease (GSD) type 1.Metabolic diseases are one of the severe causes of secondary HLH in infants; hence, complete metabolic assessment is mandatory in these patients, and GSD must be included in the differential diagnosis of HLH metabolic causes.
256	Clinico-laboratory profile and perforin gene mutations of pediatric hemophagocytic lymphohistiocytosis cases: a five-year single center study.	33224420	hemophagocytic lymphohistiocytosis (HLH) is an immunological disease characterized by hemophagocytosis of blood cells and proliferation of T-cells and histiocytes in the spleen and bone marrow then infiltration into body organs. Familial HLH (FHL) is a fatal disorder and determining gene mutations is a good guide for predicting the prognosis and choosing treatment options. This study aimed to illustrate the clinical, laboratory characteristics, including perforin gene mutation screening, treatment and survival outcome of pediatric HLH patients.we conducted this cross-sectional study on pediatric patients who were diagnosed with HLH using the revised HLH-2004 criteria, from January 2014 to February 2019 at Zagazig University Children's Hospital, Egypt. We collected demographic, clinical and laboratory data and screened for the presence of mutations in perforin (PRF1) gene by polymerase chain reaction (PCR) amplification. We treated the patients according to HLH-2004 treatment protocol and documented their survival outcome.the total number of cases were 18; eight males and ten females, the age range was between three months and 12 years. Of the eight HLH-2004 diagnostic criteria, all patients met at least five criteria. We detected PRF1 gene mutation in 38.9% (7 patients) with nine previously unreported mutations. Sixteen patients (88.9%) received HLH-2004 treatment protocol and the remaining two patients died before initiation of treatment. The overall mortality was 72.2% (13 patients).our results increase the awareness of clinical and laboratory characterizations of pediatric HLH patients and the prevalence of PRF1 gene mutations among those patients.
257	Outcomes and prognostic factors associated with 180-day mortality in Taiwanese pediatric patients with Hemophagocytic Lymphohistiocytosis.	33218852	Hemophagocytic lymphohistiocytosis (HLH), a rarely occurring syndrome with various triggers, is associated with early mortality. Owing to a lack of sufficient corresponding data in Taiwan, this study aimed to identify the outcome and potential factors associated with 180-day mortality in pediatric HLH.This retrospective study analyzed clinical and laboratory data on pediatric patients diagnosed with HLH at our institute (1995-2019). Logistic regression analysis was conducted to determine the associations between various factors and 180-day mortality.Overall, 48 patients had HLH; their median age at diagnosis was 5 years (interquartile range: 2-11 years). Clinical presentations and laboratory parameters required for diagnosis included fever (98%), splenomegaly (79%), hyperferritinemia (98%), hemophagocytosis (94%), thrombocytopenia (90%), anemia (63%), hypertriglyceridemia (68%), and neutropenia (57%). The 5-year overall survival (OS) rate was 49%. Of 22 patients who had died at the last follow-up, 15 (68%) died within 180 days after diagnosis. In the multivariate analysis, hemoglobin (odds ratio [OR]: 0.564, p = 0.024) and triglyceride (OR: 1.004, p = 0.049) were significantly associated with 180-day mortality. Higher triglyceride levels at diagnosis were related to significantly lower 180-day OS rates (52.9% vs. 86.1%, p = 0.018).The overall outcome in our cohort was similar to that reported in some of the largest international cohorts. Hypertriglyceridemia and anemia may be indicative of poor prognoses in pediatric HLH patients independently and may be used to guide treatment strategy formulations for better outcomes.
258	Development of Mast Cell and Eosinophil Hyperplasia and HLH/MAS-Like Disease in NSG-SGM3 Mice Receiving Human CD34+ Hematopoietic Stem Cells or Patient-Derived Leukemia Xenografts.	33208054	Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD scid gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)-like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied.
259	Systemic autoimmune abnormalities complicated by cytomegalovirus-induced hemophagocytic lymphohistiocytosis: A case report.	33195665	Hemophagocytic lymphohistiocytosis (HLH) is a rare but life-threatening disorder, characterized by a hyperimmune response. The mortality is high despite progress being made in the diagnosis and treatment of the disease. HLH is traditionally divided into primary (familial or genetic) and secondary (reactive) according to the etiology. Secondary HLH (sHLH), more common in adults, is often associated with underlying conditions including severe infections, malignancies, autoimmune diseases, or other etiologies.The case involves a 31-year-old woman, presented with a high persistent fever, rash, and splenomegaly. She met the diagnostic criteria of the HLH-2004 guideline and thus was diagnosed with HLH, with positive anti-nuclear antibody (ANA) and positive cytomegalovirus (CMV)-DNA. The patient responded well to a combination of immunomodulatory, chemotherapy, and supportive treatments. When her PCR evaluation for CMV turned negative, her serum ferritin also dropped significantly. Her clinical symptoms improved dramatically, and except for ANA, the abnormal laboratory findings associated with HLH returned to normal. Our previous study has shown that the median overall survival of HLH patients is only 6 mo; however, our patient has been cured and has not presented with any relapse of the disease for 6 years.This case emphasizes that thorough early removal of the CMV infection is significant for the prognosis of this HLH patient.
260	Tuberculosis-Associated HLH in an 8-Month-Old Infant: A Case Report and Review.	33194891	Hemophagocytic lymphohistiocytosis (HLH) is a rare immunological disease, which can be mistaken for sepsis easily. Among the infectious causes that may trigger secondary HLH, tuberculosis (TBC), a rather rare pathogen nowadays, is typical. To our knowledge, this is the first case report of an infant suffering from TBC-associated HLH-induced acute respiratory failure who was treated successfully using extracorporeal membrane oxygenation. An 8-month-old boy with fever (over the last 8 wk) and pancytopenia was transferred to our institution with acute respiratory failure and for extracorporeal membrane oxygenation therapy. Bone marrow biopsy revealed hemophagocytosis. Immunological work-up for familial HLH was negative. In a desperate search for the cause of secondary HLH, an interferon-gamma release assay for TBC returned positive. However, microscopy for acid-fast bacteria as well as polymerase chain reaction for TBC were initially negative. Despite this, the child was treated with tuberculostatic therapy. TBC was finally confirmed. The child remained on extracorporeal membrane oxygenation for 28 d. Further work-up showed typical lesions of disseminated TBC. The mother was identified as the source of TBC. The boy presents with mild sequelae (fine motor skills). In infants with suspected septicemia, TBC should be considered as differential diagnosis even if the results are initially negative.
261	Anaplastic Large Cell Lymphoma Presenting as Haemophagocytic Lymphohistiocytosis with Underlying Coxiella burnetii and Bartonella henselae Seropositivity.	33194860	A 44-year-old woman with no significant medical history presented with a 3-week history of high-grade fevers, fatigue and shortness of breath. Laboratory investigation was significant for lymphopenia and thrombocytopenia which progressively worsened during her hospital stay, along with new-onset anaemia, and elevated ferritin, transaminase and triglycerides. A computerized tomography (CT) scan of the abdomen revealed retroperitoneal lymphadenopathy. A bone marrow biopsy confirmed the diagnosis of haemophagocytic lymphohistiocytosis (HLH). Extensive infectious work-up revealed high IgG titres for Bartonella henselae and Coxiella burnetii. Interestingly, the left supraclavicular node was negative for both microbes by polymerase chain reaction (PCR), but the biopsy revealed anaplastic large T-cell lymphoma.Haemophagocytic lymphohistiocytosis (HLH) is an important differential diagnosis to consider for fever of unknown origin in adults, especially in the setting of pancytopenia and hyperferritinaemia.Q fever resulting from Coxiella burnetii can cause HLH and is also postulated to increase the risk of lymphoma.Bartonella henselae infection can also trigger HLH, but the risk of lymphoma following infection by B. henselae is unknown.
262	IFNAR2 Deficiency Causing Dysregulation of NK Cell Functions and Presenting With Hemophagocytic Lymphohistiocytosis.	33193576	We describe a 2 year old boy with two previously undescribed frameshift mutations in the interferon (IFN)α/β receptor 2 (IFNAR2) gene presenting with hemophagocytic lymphohistiocytosis (HLH) following measles-mumps-rubella vaccination. Functional analyses show the absence of response to type I IFN in the patient's cells, as revealed by the lack of phosphorylation of STAT1 and the lack of induction of interferon-stimulated genes upon ex vivo stimulation with IFNα. HLH has been reported in patients with inborn errors of type I IFN-mediated immune responses following vaccination with live-attenuated viruses. The relation between HLH and defective type I IFN-mediated responses is unclear. We show that in patient's natural killer (NK) cells stimulated with IFNα the expected increase in degranulation and inhibition of IFNγ production were affected. These data support a role for NK cell function dysregulation and lack of inhibition of IFNγ production as contributors to the development of HLH in patients with impaired type I IFN signaling.
263	Clinical and laboratory signs of haemophagocytic lymphohistiocytosis associated with pandemic influenza A (H1N1) infection in patients needing extracorporeal membrane oxygenation: A retrospective observational study.	33186307	Severe pandemic influenza has been associated with the hyperinflammatory condition secondary haemophagocytic lymphohistiocytosis (HLH).To determine the frequency, degree, character and possible cause of influenza-associated HLH in critically ill patients with severe acute respiratory distress syndrome due to influenza A (H1N1) infection requiring extracorporeal membrane oxygenation (ECMO) support at our hospital.A retrospective observational study.Medical data were retrieved retrospectively from 11 consenting patients of thirteen adults infected with pandemic influenza A (H1N1) 2009 requiring ECMO between July 2009 and January 2010 at the ECMO Centre of Karolinska University Hospital, Stockholm, Sweden. All patients were evaluated for HLH using HLH-2004 criteria and HScore.Eleven patients (median age 31 years) were included in the study and all survived. All patients showed signs of multiple organ dysfunction and pronounced inflammation, more severe in the four patients with HLH who had significantly higher peak serum concentrations of ferritin (P = 0.024), alkaline phosphatase (P = 0.012) and gamma-glutamyl transferase (P = 0.024), lower concentration of albumin (P = 0.0086) and more frequently hepatomegaly (P = 0.048). Abnormal lymphocyte cytotoxicity (lytic units <10) and a low proportion of natural killer (NK) cells were observed in three of four patients with HLH. Notably, we found a significant inverse correlation between serum ferritin concentration and NK cell and cytotoxic T lymphocyte percentages (rs = -0.74, P = 0.0013 and rs = -0.79, P = 0.0025, respectively). One HLH patient received HLH-directed cytotoxic therapy, another intravenous immunoglobulin and the other two no specific HLH-directed therapy.Critically ill patients, including healthy young adults, with pandemic influenza may develop HLH and should be monitored for signs of hyperinflammation and increasing organ dysfunction, and evaluated promptly for HLH because HLH-directed therapy may then be beneficial. The association of low NK percentages with hyperferritinaemia may suggest a role for reduced NK cell numbers, possibly also cytotoxic T lymphocytes, and subsequently reduced lymphocyte cytotoxicity, in the pathogenesis of hyperinflammation and secondary HLH.
264	A Rare Case of Hemophagocytic Lymphohistiocytosis Triggered by Sepsis Due to Methicillin-Resistant Staphylococcus aureus Bacteremia.	33185139	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease that occurs due to unregulated immune system activation induced by various causes including infection and cancer. In this article, we report a case of a 67-year-old male with history of small cell lung cancer who developed HLH triggered by methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. The patient was initially admitted for septic shock and gastrointestinal bleed. Further workup showed that the patient met criteria for HLH diagnosis as he was positive for 5 of the 8 parameters. Unfortunately, the patient's condition worsened and he eventually expired. With this case, we wish to draw attention to the fact that sepsis due to MRSA bacteremia can be a trigger for HLH.
265	Haemophagocytic lymphohistiocytosis that spontaneously resolved: a case of EBV.	33184051	A 23-year-old Caucasian woman, presented with recurrent fevers, elevated liver function tests and pancytopenia. Her labs at presentation were white blood cells 1.5 ×109/L, haemoglobin 8 g/L, platelets 59 k/mcl, lactate dehydrogenase (LDH) over 2000 U/L, aspartate aminotransferase 593 U/L, alanine aminotransferase 1321 U/L, alkaline phosphatase 223 U/L and ferritin 7665 µg/L. Epstein-Barr virus (EBV) IgM and IgG antibodies were positive in serum. A soluble interleukin 2 receptor was elevated at 2458. A bone marrow biopsy revealed scattered macrophages containing erythrocytes and other cellular elements. Immunohistochemistry for CD68 highlighted macrophages with erythrophagocytosis and in situ hybridisation was positive for EBV. She met the diagnostic criteria for haemophagocytic lymphohistiocytosis (HLH). She was initially treated with broad spectrum antibiotics which were eventually discontinued once the diagnosis was established. Over a period of 2-3 weeks her fever, transaminitis, ferritin and LDH improved spontaneously. She continued to improve clinically and was subsequently discharged. HLH is an aggressive, life-threatening hyper-inflammatory syndrome which, if not promptly recognised and treated, can be fatal. Treatment involves etoposide-based chemotherapy and possible stem-cell transplantation. This patient showed signs of improvement spontaneously and a decision was made to not treat her. This was a rare case of EBV-associated HLH which resolved spontaneously without any intervention. This young patient was not subjected to unnecessary chemotherapy. So far only few cases of spontaneous resolution of EBV-associated HLH have been reported.
266	Immunological assessment of pediatric multisystem inflammatory syndrome related to COVID-19.	33180935	Recently, cases of multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19 have been reported worldwide. Negative RT-PCR testing associated with positive serology in most cases suggests a post-infectious syndrome. Because the pathophysiology of this syndrome is still poorly understood, extensive virological and immunological investigations are needed.We report a series of four pediatric patients admitted to Geneva University Hospitals with persistent fever and laboratory evidence of inflammation meeting published definition of MIS-C related to COVID-19, to whom an extensive virological and immunological workup was performed.RT-PCRs on multiple anatomical compartments were negative whereas anti-SARS-CoV-2 IgA and IgG were strongly positive by ELISA and immunofluorescence. Both pseudo- and full virus neutralization assays showed the presence of neutralizing antibodies in all children, confirming a recent infection with SARS-CoV-2. Analyses of cytokine profiles revealed an elevation in all cytokines, as reported in adults with severe COVID-19. Although differing in clinical presentation, some features of MIS-C show phenotypic overlap with haemophagocytic lymphohistiocytosis (HLH). In contrast to patients with primary HLH, our patients showed normal perforin expression and NK cell degranulation. The levels of soluble IL-2 receptor (sIL-2R) correlated with the severity of disease, reflecting recent T-cell activation.Our findings suggest that MIS-C related to COVID-19 is caused by a post-infectious inflammatory syndrome associated with elevation in all cytokines, and markers of recent T-cell activation (sIL-2R) occurring despite a strong and specific humoral response to SARS-CoV2. Further functional and genetic analyses are essential to better understand the mechanisms of host-pathogen interactions.
267	A novel mutation of adenosine deaminase causing SCID presenting as hemophagocytic lymphohistiocytosis with acute kidney injury.	33174709	NaN
268	Development of a dendrimer PAMAM‑based gold biochip for rapid and sensitive detection of endogenous IFN‑γ and anti‑IFN‑γ IgG in patients with hemophagocytic lymphohistiocytosis.	33173980	Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe disease characterized by immune hyperactivation and cytokine storm. Given the high mortality rate of HLH, there is a need for more effective diagnostic tools and treatments. The present study developed a dendrimer‑based protein biochip for rapid, sensitive and simultaneous detection of serum interferon (IFN)‑γ and endogenous anti‑IFN‑γ antibody (Ab) in patients with HLH. A gold biochip was modified with 1, 4‑phenylene diisothiocyanate (PDITC), polyamidoamine (PAMAM) or PDITC‑activated PAMAM. The optimal immobilization concentration for Ab capture and the reaction concentration for detecting Ab on the PDITC‑activated PAMAM‑modified biochip were 6.25 and 3.12 µg/ml, respectively; the limit of detection of IFN‑γ protein was 50 pg/ml. The efficiency of the protein‑probed biochip in detecting IFN‑γ and anti‑IFN‑γ Ab in serum samples from 77 patients with HLH was evaluated; the positive rates for IFN‑γ and anti‑IFN‑γ IgG Ab were 63.6% (49/77) and 61.0% (47/77), respectively. The present results demonstrated that the PDITC‑activated PAMAM‑modified biochip might be a sensitive tool for the specific detection of IFN‑γ and anti‑IFN‑γ Ab in serum, and might have clinical applicability for the diagnosis of HLH.
269	Hemophagocytic lymphohistiocytosis and miliary tuberculosis in a previously healthy individual: a case report.	33172493	Hemophagocytic lymphohistiocytosis (HLH) is a rare heterogenous genetic or acquired hyperinflammatory syndrome associated with a high degree of morbidity and mortality. HLH has clinical manifestations related to abnormal prolonged activation of T lymphocytes and macrophages with an excess of proinflammatory cytokines. The main causes of secondary HLH are malignancies and infectious diseases.The patient was a 54-year-old man, originally from Eastern Africa, who had lived in Northern Europe for 30 years. Here we describe the clinical features, laboratory parameters, diagnostic workup, management and outcome data of a previously healthy 54-year-old man diagnosed with HLH secondary to tuberculosis. The patient was initially treated for a community-acquired pneumonia. He developed multiorgan failure with acute respiratory distress syndrome, hypertransaminasemia, and kidney and bone marrow dysfunction. The clinical course together with a simultaneous increase in serum ferritin raised the suspicion of HLH. The patient fulfilled seven out of eight diagnostic criteria for HLH. A thorough diagnostic workup with respect to HLH and a potential underlying disease was initiated. Cultivation of bronchoalveolar lavage fluid, stool and urine, and polymerase chain reaction of epithelioid cell granulomas in the bone marrow were all positive for Mycobacterium tuberculosis. He was treated for both HLH and tuberculosis, and he survived without any sequelae.We present one of few published cases of a patient who survived HLH triggered by miliary tuberculosis. The current case illustrates the need for awareness of these two diagnoses, and the timely initiation of specific and supportive treatment to reduce mortality.
270	Hemophagocytic lymphohistiocytosis with recurrent Kikuchi-Fujimoto disease.	33171575	Kikuchi-Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis, is a self-limiting lymphadenitis. It is a benign disease mainly characterized by high fever, lymph node swelling, and leukopenia. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease with clinical symptoms similar to those of KFD, but it requires a significantly more aggressive treatment. A 19-year-old Korean male patient was hospitalized for fever and cervical lymphadenopathy. Variable-sized lymph node enlargements with slightly necrotic lesions were detected on computed tomography. Biopsy specimen from a cervical lymph node showed necrotizing lymphadenitis with HLH. Bone marrow aspiration showed hemophagocytic histiocytosis. The clinical symptoms and the results of the laboratory test and bone marrow aspiration met the diagnostic criteria for HLH. The patient was diagnosed with macrophage activation syndrome-HLH, a secondary HLH associated with KFD. He was treated with dexamethasone (10 mg/m2/day) without immunosuppressive therapy or etoposide-based chemotherapy. The fever disappeared within a day, and other symptoms such as lymphadenopathy, ascites, and pleural effusion improved. Dexamethasone was reduced from day 2 of hospitalization and was tapered over 8 weeks. The patient was discharged on day 6 with continuation of dexamethasone. The patient had no recurrence at the 18-month follow-up.
271	Hereditary haemochromatosis, haemophagocytic lymphohistiocytosis and COVID-19.	33163955	Syndromes of iron overload have been shown to increase the risk of severe clinical disease in viral infections. Immune dysfunction is similarly described in hereditary haemochromatosis (HH). We present here the case of a 51-year-old man who developed severe coronavirus disease 2019 (COVID-19) complicated by suspected haemophagocytic lymphohistiocytosis (HLH). He was found to have HH post-mortem and we propose a link between his iron overload and the development of severe COVID-19.The initial clinical presentation consisted of cough, shortness of breath and fever. Pancytopenia, markedly elevated ferritin and d-dimer were present. Computed tomography (CT) showed bilateral ground glass changes consistent with COVID-19, widespread lymphadenopathy and splenomegaly. A subsequent combined nose and throat swab was positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). HLH was suspected based upon the H-score and Anakinra, an IL-1 receptor antagonist, was commenced. Liver function acutely worsened and magnetic resonance cholangiopancreatography (MRCP) revealed hepatic haemosiderosis. Intense splenic and cervical lymph node uptake were seen on a positron emission tomography (PET) scan and high doses of intravenous steroids were administered due to concerns over haematological malignancy.Day fourteen of admission heralded the start of progressive clinical deterioration with rapid increase in oxygen demands. Continuous positive airway pressure (CPAP) was trialled without success and the patient unfortunately died seventeen days into admission. Results returned after his death showed homozygous C282Y mutation of the HFE gene consistent with a diagnosis of HH. Post-mortem examination revealed widespread haemosiderin deposition in the liver along with lung pathology in keeping with severe COVID-19 and widespread splenic infarctions.An association between HH and COVID-19 is not currently described in the literature. What does exist, however, is an evidence base for the detrimental impacts iron overload has on viral infections in general and the negative effects of HH on the immune system. We therefore postulate that the underlying metabolic and immune disturbances seen in HH should be considered a potential risk factor for the development of severe COVID-19. This case also adds to the evidence that hyperinflammation appears to be a unique and interesting characteristic of this novel viral disease.
272	STXBP2-R190C Variant in a Patient With Neonatal Hemophagocytic Lymphohistiocytosis (HLH) and G6PD Deficiency Reveals a Critical Role of STXBP2 Domain 2 on Granule Exocytosis.	33162974	Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH), which is caused by gene mutations interfering with the cytolytic pathway of cytotoxic T-lymphocytes (CTLs) and natural killer cells. Here we describe a male newborn who met the HLH diagnostic criteria, presented with profound cholestasis, and carried a maternally inherited heterozygous mutation in syntaxin-binding protein-2 [STXBP2, c.568C>T (p.Arg190Cys)] in addition to a severe pathogenic variant in glucose 6-phosphate dehydrogenase [G6PD, hemizygous c.1153T>C (Cys385Arg)]. Although mutations in STXBP2 gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity outcompeting the effect of endogenous wild-type STXBP2. Interestingly, arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that may act in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied G6PD mutation may have compounded the clinical symptoms; however, the extent by which G6PD deficiency has contributed to HLH in our patient remains unclear.
273	Malignancy-associated hemophagocytic lymphohistiocytosis in children: a 10-year experience of a single pediatric hematology center.	33161882	Objective: Malignancy-associated hemophagocytic lymphohistiocytosis (M-HLH) in children is a relatively rare but life-threatening secondary hemophagocytic lymphohistiocytosis (sHLH). Until now, only a limited number of cases regarding children with M-HLH has been reported. Methods: We conducted a retrospective study of 27 children with M-HLH, who admitted to our center between July 2007 and October 2019. The clinical data and laboratory data were analyzed. Results: The median age of the children with M-HLH was 7 years. Underlying diseases included myeloid malignancy (n = 6), lymphoid malignancy (n = 18) and unknown type lymphoma (n = 3). The one-year mortality rate was 56%. All patients had persistent fever. The clinical manifestations included hepatomegaly (89%), splenomegaly (67%) and central nervous system symptoms (56%). Thirteen children (48%) had Epstein-Barr virus (EBV) infection. No significant differences were observed between EBV-positive and negative M-HLH patients in terms of most clinical indicators. However, EBV-positive M-HLH patients showed prolonged activated partial thromboplastin time (APTT) and more hemophagocytosis in the bone marrow (BM) in contrast to EBV-negative patients. Eighteen patients (67%) received the HLH-94/04 regimen as the initial treatment. There were no significant differences in the overall survival (OS) between EBV-positive and negative patients. Patients with prolonged APTT had a significantly poorer OS than other patients (p = 0.012). Conclusions: The M-HLH children with EBV infection are more likely to have prolonged APTT and more hemophagocytosis in BM. The M-HLH children had a poor prognosis, especially those with prolonged APTT.
274	Atypical neuroimaging characteristics of hemophagocytic lymphohistiocytosis in infants: a case series of hemorrhagic brain lesions in the deep grey matter.	33156371	Hemophagocytic lymphohistiocytosis (HLH) is a rare multisystem condition associated with uncontrolled overproduction and infiltration of lymphocytes and histiocytes predominantly in liver, lymph nodes, spleen, and central nervous system. Neuroimaging findings on MRI are fairly nonspecific and classically include periventricular white matter signal abnormalities and diffuse atrophy. Focal parenchymal lesions may demonstrate post contrast ring or nodular enhancement and calcification. However, the MR imaging characteristics can be highly variable. Here, we present two cases of HLH in infants with multiple hemorrhagic lesions mostly depicted in both thalami and basal ganglia regions. Thalamic, basal ganglia, and brain stem involvement with hemorrhagic changes in HLH are rarely described in literature. Early diagnosis of HLH may be lifesaving. Awareness of the disease is necessary to investigate its characteristic findings and avoiding a delay in diagnosis.
275	Clinical Characteristics and Outcomes of 101 Children with Hemophagocytic Lymphohistiocytosis: A Four-Year Single-Center Experience from Egypt.	33150824	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition, arising either due to genetic mutations or from a variety of underlying diseases. This prospective observational study reports the clinical and laboratory data as well as the outcome of pediatric HLH in Egypt. HLH was diagnosed according to the Histiocyte Society HLH-2004 diagnostic criteria conducted at Alexandria University Children's Hospital over four years. One-hundred-one patients were enrolled (44 males and 57 females), and the median age at presentation was 13.1 months (range: 1 day - 181.4 months). Almost 75% of the patients had consanguineous parents, and one-third had a history of an affected family member (HLH or HLH-like illness). The median time interval between the first HLH symptom and diagnosis was 34 days. At diagnosis, patients were preliminarily classified as having primary HLH in 61% of patients and secondary HLH in 39% of patients. The diagnosis was confirmed genetically in 57 patients. Seventy-eight percent of patients received the HLH-94 and HLH-2004 protocols, only seven patients have undergone hematopoietic stem cell transplantation. The overall survival was 26.4% by the end of the study; the most common cause of death was uncontrolled disease activity. This descriptive study, on a large cohort of pediatric HLH in Africa and the Middle East, sheds some light on the epidemiological characteristics of HLH patients and the available diagnostic and therapeutic tools. The mortality rate was considerably high, highlighting the importance of early diagnosis and initiation of appropriate therapy.
276	Coronavirus disease 2019: investigational therapies in the prevention and treatment of hyperinflammation.	33146561	Introduction: The mortality of coronavirus disease 2019 (COVID-19) is frequently driven by an injurious immune response characterized by the development of acute respiratory distress syndrome (ARDS), endotheliitis, coagulopathy, and multi-organ failure. This spectrum of hyperinflammation in COVID-19 is commonly referred to as cytokine storm syndrome (CSS). Areas covered: Medline and Google Scholar were searched up until 15th of August 2020 for relevant literature. Evidence supports a role of dysregulated immune responses in the immunopathogenesis of severe COVID-19. CSS associated with SARS-CoV-2 shows similarities to the exuberant cytokine production in some patients with viral infection (e.g.SARS-CoV-1) and may be confused with other syndromes of hyperinflammation like the cytokine release syndrome (CRS) in CAR-T cell therapy. Interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha have emerged as predictors of COVID-19 severity and in-hospital mortality. Expert opinion: Despite similarities, COVID-19-CSS appears to be distinct from HLH, MAS, and CRS, and the application of HLH diagnostic scores and criteria to COVID-19 is not supported by emerging data. While immunosuppressive therapy with glucocorticoids has shown a mortality benefit, cytokine inhibitors may hold promise as 'rescue therapies' in severe COVID-19. Given the arguably limited benefit in advanced disease, strategies to prevent the development of COVID-19-CSS are needed.
277	Etoposide combined with ruxolitinib for refractory hemophagocytic lymphohistiocytosis during pregnancy: a case report and literature review.	33138732	Hemophagocytic lymphohistiocytosis (HLH) is an immune-mediated disorder caused by uncontrolled inflammatory responses and the activation of T lymphocytes. This life-threatening disease, characterized by fever, cytopenia and hepatosplenomegaly, is extremely rare during pregnancy with high mortality. Despite the improvement of treatment regimen in recent years, HLH is still a great challenge for clinicians. Here, we described a 26-year-old woman who admitted to our hospital at her first pregnancy with pyrexia. Her condition continued to deteriorate after receiving broad-spectrum antimicrobials, presenting with fever, pancytopenia, hepatosplenomegaly, ferritin ≥ 500 μg/L, hemophagocytosis and low NK-cell activity. HLH was eventually diagnosed by clinical manifestation and laboratory examination results. Then the patient recovered well after treatment with etoposide combined with ruxolitinib therapy and underwent successful induced-labor operation. Additionally, we summarized similar cases from the literature to improve the management of HLH during pregnancy. In conclusion, this study highlights the challenges and difficulties in the diagnosis and management of patients with HLH during pregnancy. Moreover, this is the first case report of etoposide combined with ruxolitinib in the treatment of patients with refractory secondary HLH during pregnancy.
278	Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition.	33134924	The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies.Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro.The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone.Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.
279	Hemophagocytic lymphohistiocytosis syndrome associated with Epstein-Barr infection in an immunocompetent patient. A case study.	33134207	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening rare disease resulting from the uncontrolled activation of the immune system, leading to unrestrained cytokine release and macrophage activation. It can be either hereditary or acquired due to infections, hematological disease or malignancy.We present the case of a 19-year old woman that presented with high fever and acute cholestatic hepatitis. She was initially admitted to the Gastroenterology department and the following days she developed respiratory distress and multiorgan insufficiency that necessitated intubation and support in the Intensive Care Unit. Fever, splenomegaly, hypertriglyceridemia, increased ferritin levels and hemophagocytosis in the bone marrow were found, thus, fulfilling the criteria of hemophagocytic lymphohistiocytosis. Laboratory examination was notable for positive serology (IgM and IgG) and PCR for EBV in the serum. An extensive workup including virology and immunologic workup, blood cultures, a CT of the thorax and the abdomen and a bone marrow biopsy did not reveal any cause of secondary HLH other than the EBV infection. The patient was treated with high dose corticosteroids and intravenous immunoglobulins with slow resolution of her symptoms.In patients with EBV infection who exhibit persistent high fever and unresponsiveness to antibiotics, the possibility of HLH should be considered. Early diagnosis and rapid initiation of appropriate treatment may avert an unfavorable outcome.
280	Tuberculosis-associated HLH in a patient with chronic kidney disease on haemodialysis.	33133616	Haemophagocytic lymphohistiocytosis (HLH) is a rare immunological disorder that is accompanied by a high mortality rate when the underlying aetiology is miliary tuberculosis. We report a case of tuberculosis (TB)-associated HLH in a haemodialysis patient, from a TB-endemic region, who missed two sessions of dialysis before developing the primary symptoms of HLH. The patient presented with non-specific findings including pancytopenia, coagulopathy and transaminitis. Computer-tomography imaging and microbiology from bronchoalveolar lavage evidenced miliary tuberculosis. Further testing revealed the TB-associated-HLH characteristic pattern of thrombocytosis, leukopenia, transaminitis, hyperferritinemia and elevated fibrinogen. The patient initially demonstrated improvement after initiation of anti-TB therapy. However, soon thereafter began to paradoxically deteriorate and then expire from apparent tuberculosis-immune reconstitution inflammatory syndrome. This case highlights the importance of early diagnosis and treatment, and consequently of the utility of diagnostic systems such as the HScore in cases of high clinical suspicion.
281	The role of interleukin-18 in the diagnosis and monitoring of hemophagocytic lymphohistiocytosis/macrophage activation syndrome - a systematic review.	33128796	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, hyperinflammatory disorder, characterized by multiorgan failure, fever and cytopenias. The diagnosis of HLH and its subtype Macrophage Activation Syndrome (MAS) remains a challenge. Interleukin 18 (IL-18) is emerging as a potential biomarker for HLH/MAS but is currently not a part of diagnostic criteria. This systematic review aimed to assess the potential role of IL-18 in the diagnosis and monitoring of HLH and MAS, and was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed and Embase were searched on 30 January 2020. Studies included all subtypes of HLH and a range of underlying disorders in both children and adults. A total of 14 studies were included. Generally, serum IL-18 was elevated in both primary and secondary HLH (> 1000 pg/ml) compared with other inflammatory conditions and with healthy individuals; thus, serum IL-18 may be able to discriminate between HLH and other inflammatory conditions. Significantly increased IL-18 (> 10 000 pg/ml) was also consistently described in MAS compared with other subtypes of HLH. The ability of IL-18 to distinguish MAS from systemic juvenile idiopathic arthritis (JIA) is less unambiguous, as IL-18 levels > 100 000 pg/ml were described in sJIA patients both with and without MAS. IL-18 may help to differentiate between HLH subtypes and other inflammatory conditions. As HLH and MAS are rare disorders, only few and relatively small studies exist on the subject. Larger, prospective multi-center studies are called for to assess the diagnostic precision of IL-18 for HLH and MAS.
282	Haemophagocytic lymphohistiocytosis in human immunodeficiency virus: a systematic review of literature.	33116036	Diagnosis and management of hemophagocytic lymphohistiocytosis (HLH) in patients with human immunodeficiency virus (HIV) infection are scarcely described in the published literature. The aim of this systematic review was to delineate the triggers of HLH in patients with HIV and understand the role of steroids in the management. We conducted a comprehensive search of English medical literature via the Medline ⁄ PubMed database using different synonyms of "HIV" AND "HLH". The review was registered in PROSPERO (CRD42018099987). The titles and abstracts of the 185 articles between January 1986 and April 2018 were reviewed. The final analysis was done from 42 articles with 52 patients. HLH was associated with malignancy in 17 patients, while infection was found in 25 patients. No cause was identified in eight patients, out of which four had acute HIV infection. Death was reported in 21 patients. Presence of either malignancy (p = 0.051) or opportunistic infection (p = 0.69) was not associated with increased chances of death by univariate analysis. A total of 26 patients were treated with steroids, while etoposide was used in only four patients. Administration of steroids as a treatment of HLH was associated with more chances of death (p = 0.048). Malignancy and Opportunistic infections are important triggers for HLH in patients with HIV. Acute HIV by itself can act as a trigger for HLH. Evidence on the use of steroids as a treatment of HLH in patients with HIV is not convincing.
283	Autophagy Plays a Role in the Prolongation of the Life Span of Caenorhabditis elegans by Astaxanthin.	33115330	Astaxanthin (AST), a xanthophyll belonging to the family of carotenoids, is a potent antioxidant. The effect of AST on longevity and its physiological and molecular mechanism are still unclear. In this study, we proved that AST could prolong the life span of Caenorhabditis elegans. To uncover whether AST could delay aging by upregulating autophagy, we measured the expression of autophagy gene and the life span of autophagy gene bec-1 mutant nematodes, and the results showed that the expression of autophagy gene was upregulated after AST intervention and the disruption of bec-1 weakened the extension of the life span. To explore the molecular mechanism of AST-induced autophagy upregulation, we knocked out the daf-16 or hlh-30 (key genes of insulin/insulin growth factor-1 [IGF-1] signal pathway or target of rapamycin [TOR] signal pathway) by RNA interference, and the expression of autophagy gene lgg-1 decreased. Collectively, our results strongly suggest that autophagy, which is both the insulin/IGF-1 signal pathway dependent and TOR signal pathway dependent, plays a role in the prolongation of the life span of Caenorhabditis elegans by AST.
284	Title: Cytokine release syndrome is not usually caused by secondary hemophagocytic lymphohistiocytosis in a cohort of 19 critically ill COVID-19 patients.	33106497	Severe COVID-19 associated respiratory failure, poses the one challenge of our days. Assessment and treatment of COVID-19 associated hyperinflammation may be key to improve outcomes. It was speculated that in subgroups of patients secondary hemophagocytic lymphohistiocytosis (sHLH) or cytokine release syndrome (CRS) with features of macrophage activation syndrome might drive severe disease trajectories. If confirmed, profound immunosuppressive therapy would be a rationale treatment approach. Over a median observation period of 11 (IQR: 8; 16) days, 19 consecutive confirmed severe COVID-19-patients admitted to our intensive-care-unit were tested for presence of sHLH by two independent experts. HScores and 2004-HLH diagnostic criteria were assessed. Patients were grouped according to short-term clinical courses: discharge from ICU versus ongoing ARDS or death at time of analysis. The median HScore at admission was 157 (IQR: 98;180), without the key clinical triad of HLH, i.e. progressive cytopenia, persistent fever and organomegaly. Independent expert chart review revealed the absence of sHLH in all cases. No patient reached more than 3/6 of modified HLH 2004 criteria. Nevertheless, patients presented hyperinflammation with peripheral neutrophilic signatures (neutrophil/lymphocyte-ratio > 3.5). The latter best paralleled their short-term clinical courses, with declining relative neutrophil numbers prior to extubation (4.4, [IQR: 2.5;6.3]; n = 8) versus those with unfavourable courses (7.6, [IQR: 5.2;31], n = 9). Our study rules out virus induced sHLH as the leading cause of most severe-COVID-19 trajectories. Instead, an associated innate neutrophilic hyperinflammatory response or virus-associated-CRS appears dominant in patients with an unfavourable clinical course. Therapeutic implications are discussed.
285	In the Model Host Caenorhabditis elegans, Sphingosine-1-Phosphate-Mediated Signaling Increases Immunity toward Human Opportunistic Bacteria.	33105563	Sphingosine-1-phophate (S1P) is a sphingolipid-derived signaling molecule that controls diverse cellular functions including cell growth, homeostasis, and stress responses. In a variety of metazoans, cytosolic S1P is transported into the extracellular space where it activates S1P receptors in a concentration-dependent manner. In the free-living nematode Caenorhabditis elegans, the spin-2 gene, which encodes a S1P transporter, is activated during Gram-positive or Gram-negative bacterial infection of the intestine. However, the role during infection of spin-2 and three additional genes in the C. elegans genome encoding other putative S1P transporters has not been elucidated. Here, we report an evolutionally conserved function for S1P and a non-canonical role for S1P transporters in the C. elegans immune response to bacterial pathogens. We found that mutations in the sphingosine kinase gene (sphk-1) or in the S1P transporter genes spin-2 or spin-3 decreased nematode survival after infection with Pseudomonas aeruginosa or Enterococcus faecalis. In contrast to spin-2 and spin-3, mutating spin-1 leads to an increase in resistance to P. aeruginosa. Consistent with these results, when wild-type C. elegans were supplemented with extracellular S1P, we found an increase in their lifespan when challenged with P. aeruginosa and E. faecalis. In comparison, spin-2 and spin-3 mutations suppressed the ability of S1P to rescue the worms from pathogen-mediated killing, whereas the spin-1 mutation had no effect on the immune-enhancing activity of S1P. S1P demonstrated no antimicrobial activity toward P. aeruginosa and Escherichia coli and only minimal activity against E. faecalis MMH594 (40 µM). These data suggest that spin-2 and spin-3, on the one hand, and spin-1, on the other hand, transport S1P across cellular membranes in opposite directions. Finally, the immune modulatory effect of S1P was diminished in C. eleganssek-1 and pmk-1 mutants, suggesting that the immunomodulatory effects of S1P are mediated by the p38 MAPK signaling pathway.
286	The great masquerader: Hemophagocytic lymphohistiocytosis secondary to cytomegalovirus infection in an immunocompetent young man.	33102367	Hemophagocytic lymphohistiocytosis (HLH) is a rare, poorly recognized and underdiagnosed syndrome of excessive immune activation, which is rapidly fatal. HLH can occur as a familial or secondary disorder. Secondary HLH is most commonly associated with infections, malignancies, or autoimmune diseases. It primarily affects patients with a compromised immune system and rarely presents in immunocompetent individuals. Acute cytomegalovirus (CMV) associated HLH in the immunocompetent individual is extremely rare and only documented in five case reports till date. We, hereby, report a case of 18 years old young immunocompetent man who was diagnosed to have HLH secondary to CMV infection.
287	Post-transplant Hemophagocytic Lymphohistiocytosis in Benign Hematological Disorders: Experience of 4 Cases with Review of Literature.	33100709	Post transplant Hemophagocytic lymphohistiocytosis (HLH) is a form of secondary HLH, which can be either early onset or late onset and is associated with significant morbidity and mortality. With the increasing popularity of post transplant cyclophosphamide based haploidentical stem cell transplantation (SCT), post transplant HLH is becoming a significant complication especially in benign hematological disorders. Methods: We present 4 cases of post transplant HLH occurring in 2 cases of severe aplastic anemia (post haploidentical SCT) and 2 cases of thalassemia major (post matched sibling SCT). All 4 cases had early onset variety with dismal prognosis. Conclusion: Post-transplant HLH is an important entity in benign hematological disorders, which needs to be identified early and treated promptly with steroids, monoclonal agents or immunosuppressive therapy. Serum ferritin levels are an important biomarker and help in monitoring response.
288	HLH Masquerading Lymphoma: Diagnostic Dilemma and Treatment Outcomes.	33100704	A case series to illustrate difficulties faced in diagnosis, management and subsequent therapeutic approach patients presenting with HLH secondary to lymphoma. A retrospective review of patients treated for HLH and lymphoma in Clinical Hematology department of a tertiary care hospital in North India, was performed from Jan 2017 to April 2019. Follow up was included till September 2019. Diagnosis of HLH was made using HLH 2004 criteria along with H score. Only patients who fulfilled HLH 2004 criteria were included. Nine patients were treated during above period, three patients with Hodgkins lymphoma, two patients had DLBCL and four patients had T-cell lymphoma. All patients presented with features of HLH and underlying lymphoma was detected on further evaluation. All patients had H score above the cut off value for diagnosis of HLH. Out of 9 patients, 6 received lymphoma directed chemotherapy and 1 was given only steroids, 1 received IVIG with steroids. 1 died early, before institution of therapy. Out of the 6 patients who received chemotherapy, all attained remission status but two patients had early relapse. In the remaining 3 patients who could not be started on chemotherapy, all died within 3 weeks of presentation. Underlying lymphoreticular malignancy should be actively searched in adult patients presenting with HLH. Early diagnosis and initiation of disease specific therapy with or without specific HLH directed treatment can improve the historical poor prognosis.
289	Diagnostic strategy for trigger identification in severe reactive hemophagocytic lymphohistiocytosis: A diagnostic accuracy study.	33099794	Reactive hemophagocytic lymphohistiocytosis (rHLH) management requires early recognition, trigger identification, and adequate treatment in order to reduce mortality. We assessed the diagnostic yield of tissue biopsies to identify trigger in severe rHLH. We included all consecutive patients presenting an rHLH diagnosis (HLH-2004 criteria) admitted to the intensive care unit (ICU) of a tertiary hospital. This retrospective diagnostic accuracy study was conducted according to the Standards for Reporting Diagnostic Accuracy Statement. Among the 134 included patients (median age 47 years [IQR 47-56]), an underlying immunodeficiency was previously known in 61.2%. rHLH trigger was identified in 127 patients (94.8%) (hematological disorder 75%, infection 16%, systemic disease 4%). Diagnostic yield of tissue biopsies was as follows: lymph node 75% (95% confidence interval [CI], 61-85), skin 50% (95% CI, 27-73), bone marrow 44% (95% CI, 34-55), liver 30% (95% CI, 15-49). Splenectomy (yield 77%; 95% CI, 46-95) was reserved to cases of diagnostic deadlock. Procedural severe adverse events included two cases of reversible hemorrhagic shock. Seventy-eight percent of patients received etoposide regarding to the rHLH severity, and 68% could receive trigger-specific treatment in the ICU. A comprehensive diagnostic workup led to an rHLH trigger identification in 95% of patients, allowing prompt initiation of appropriate therapy. Prospective studies to validate a standardized diagnostic approach are warranted.
290	HAEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS IN PREGNANT AND POSTPARTUM WOMEN.	33099527	Introduction: Haemophagocytic lymphohistiocytosis (HLH) is an extremely rare, life-threatening disease, caused by uncontrolled activation of lymphocytes T and macrophages. This situation leads to cytokine storm, infiltration and internal organs failure. HLH can be categorised into either primary (familiar) or secondary which may be associated with infections, immunodeficiency syndromes, autoimmune diseases and malignancy. The secondary HLH is difficult to diagnose due to nonspecific symptoms and complicated differential diagnostics. The aim: To conduct a comparative analysis of pregnant and puerperal patients diagnosed with HLH.Material and methods: Review of available literature on haemophagocytic lymphohistiocytosis during pregnancy and the puerperium.Results: Review of the latest literature shows that HLH can occur at any time during pregnancy and in the puerperium. Symptoms of the disease are non-specific: fever not responding to antibiotic therapy, sometimes hectic, hepatosplenomegaly, swelling, lymphadenopathy, disseminated intravascular coagulation, multi-organ failure and death. In laboratory tests, worsening bicytopenia or pancytopenia, increasing indicators of organ damage, hypertriglyceridemia, hypofibrinogenemia and abnormally high serumferritin levels are observed.Conclusions: HLH, due to non-specific symptoms and rarity, is often overlooked in the diagnostic process. Due to the high mortality and morbidity rates of HLH during pregnancy for mother and foetus, timely diagnosis and the inclusion of specialist treatment are particularly important. An interdisciplinary approach to the patient is necessary to make an accurate diagnosis. The assessment of serum ferritin concentrations facilitates diagnosis. The bone marrow is essential to diagnosis and should be performed as early as possible.
291	HLH caused by an HSV-2 infection: a case report and review of the literature.	33093253	Haemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory condition that can be triggered by infections, malignancies, or auto-immune diseases. Here, we present a patient with rapidly progressive HLH triggered by a herpes simplex virus type 2 (HSV-2) primary infection. The patient was successfully treated with intravenous high-dose acyclovir, immunoglobulins, and dexamethasone. This is the first report of HSV-2-associated HLH in an immunocompetent adult patient.
292	Successful treatment of refractory acute lupus haemophagocytic syndrome using rituximab: a case report.	33087000	Systemic lupus erythematosus (SLE)-associated haemophagocytic lymphohistiocytosis (HLH) is called acute lupus haemophagocytic syndrome (ALHS), which is relatively rare but life-threatening. We present the case of a 43-year-old woman diagnosed with SLE with panniculitis, pleuritis, and autoimmune hepatitis. She was treated with high-dose glucocorticoids. Although disease activity temporarily improved, she developed fever, elevation of liver enzymes, hyperferritinemia, severe inflammatory response, and thrombocytopenia a month after starting glucocorticoids. Bone marrow biopsy was performed and haemophagocytosis was observed. She was diagnosed with ALHS on day 49. Since she developed ALHS during administration of glucocorticoids, her ALHS was determined to be refractory to glucocorticoid monotherapy; therefore, additional immunosuppressive agents were needed. She was treated with methylprednisolone pulse, plasma exchange and cyclosporine A (CyA). However, CyA was discontinued on day 54 because CyA-induced hypertensive encephalopathy was suspected. Subsequently, rituximab (RTX) was introduced to treat refractory ALHS on day 56; the disease activity subsequently reduced. After four courses of RTX, her ferritin levels and platelet counts were within the normal range and the glucocorticoid dose could be tapered to betamethasone 2.0 mg/day on day 132. No subsequent recurrence of SLE and ALHS was observed until day 132. RTX might therefore be an effective therapeutic option for refractory ALHS.
293	Successful use of short-term add-on tocilizumab for refractory adult-onset still's disease with macrophage activation syndrome despite treatment with high-dose glucocorticoids, cyclosporine, and etoposide.	33086994	Macrophage activation syndrome (MAS) is a form of secondary hemophagocytic lymphohistiocytosis (HLH) and is a life-threatening complication of adult-onset Still disease. MAS has been usually treated with high-dose glucocorticoid with additional immunosuppressive agents, such as cyclosporine. Etoposide has been used for the treatment of severe refractory MAS based on the successful results of HLH-2004 protocol in patients with mostly primary form of HLH. We herein describe a case of severe refractory MAS secondary to adult-onset Still disease in an elderly woman that inadequately responded to etoposide but remarkably responded to additional tocilizumab. Furthermore, short-term tocilizumab led her into remission and enabled tapering off glucocorticoids after 15 months. Tocilizumab may be effective for the treatment of refractory HLH after the failure of the etoposide-containing induction regimen.
294	The Lazarus effect of very high-dose intravenous anakinra in severe non-familial CNS-HLH.	33083789	NaN
295	Activated Phosphoinositide 3-Kinase Delta Syndrome 1: Clinical and Immunological Data from an Italian Cohort of Patients.	33080915	Activated phosphoinositide 3-kinase delta syndrome 1 (APDS-1) is a recently described inborn error of immunity caused by monoallelic gain-of-function mutations in the PIK3CD gene. We reviewed for the first time medical records and laboratory data of eight Italian APDS-1 patients. Recurrent sinopulmonary infections were the most common clinical feature at onset of disease. Seven patients presented lymphoproliferative disease, at onset or during follow-up, one of which resembled hemophagocytic lymphohistiocytosis (HLH). Genetic analysis of the PIK3CD gene revealed three novel mutations: functional testing confirmed their activating nature. In the remaining patients, the previously reported variants p.E1021K (n = 4) and p.E525A (n = 1) were identified. Six patients were started on immunoglobulin replacement treatment (IgRT). One patient successfully underwent hematopoietic stem cell transplantation (HSCT), with good chimerism and no GVHD at 21 months post-HSCT. APDS-1 is a combined immune deficiency with a wide variety of clinical manifestations and a complex immunological presentation. Besides IgRT, specific therapies targeting the PI3Kδ pathway will most likely become a valid aid for the amelioration of patients' clinical management and their quality of life.
296	A feedback loop governs the relationship between lipid metabolism and longevity.	33078707	The relationship between lipid metabolism and longevity remains unclear. Although fat oxidation is essential for weight loss, whether it remains beneficial when sustained for long periods, and the extent to which it may attenuate or augment lifespan remain important unanswered questions. Here, we develop an experimental handle in the Caenorhabditis elegans model system, in which we uncover the mechanisms that connect long-term fat oxidation with longevity. We find that sustained β-oxidation via activation of the conserved triglyceride lipase ATGL-1, triggers a feedback transcriptional loop that involves the mito-nuclear transcription factor ATFS-1, and a previously unknown and highly conserved repressor of ATGL-1 called HLH-11/AP4. This feedback loop orchestrates the dual control of fat oxidation and lifespan, and shields the organism from life-shortening mitochondrial stress in the face of continuous fat oxidation. Thus, we uncover one mechanism by which fat oxidation can be sustained for long periods without deleterious effects on longevity.
297	MRI features of intra-axial histiocytic brain mass lesions.	33077156	To describe MRI features, including diffusion-weighted imaging (DWI), magnetic resonance spectroscopy (MRS), and perfusion-weighted imaging (PWI), of intra-axial tumour-like presentations of four different subtypes of histiocytosis.The brain MRI findings of 23 patients with histologically proven histiocytosis were reviewed retrospectively (11 Langerhans cell histiocytosis [LCH], eight Erdheim-Chester disease [ECD], one overlap form LCH/ECD, two Rosai-Dorfman disease [RDD], and one haemophagocytic lymphohistiocytosis [HLH]) with single or multiple enhancing intraparenchymal brain lesions.Histiocytic brain mass lesions show some similar MRI features including Supra and/or infratentorial and/or paraventricular subcortical well-delineated masses, linear ependymal enhancement along the ventricles and brain stem lesions. Masses always present with mixed hyper- and hypointense signal on T2-weighted imaging (WI). Their enhancement is often homogeneous. Apparent diffusion coefficient (ADC) values are often normal or elevated.The presence of multiple periventricular and subcortical enhancing lesions with mixed signal intensity on T2WI and normal or high ADC values should lead radiologists to consider the diagnosis of histiocytic lesions and search for associated systemic lesions.
298	Hemophagocytic Lymphohistiocytosis During HIV Infection in Cayenne Hospital 2012-2015: First Think Histoplasmosis.	33072627	Introduction: Haemophagocytic Lymphohistiocytosis (HLH), during HIV infection is a rare complication with a poor prognosis. There are few data on HLH within the Amazon region. The objective was to describe epidemiological, clinical and therapeutic features of HIV-related HLH in French Guiana. Methods: A retrospective analysis of adult HIV patients at Cayenne hospital with HLH between 2012 and 2015. A diagnosis of HLH was given if the patient presented at least 3 of 8 criteria of the HLH-2004 classification. Results: Fourteen cases of HLH were tallied during the study period. The mean age was 46 years with a sex ratio of 1.8. The most frequent etiology of HLH was an associated infection (12/14). Confirmed disseminated histoplasmosis, was found in 10 of 14 cases, and it was suspected in 2 other cases. The CD4 count was below 200/mm3 in 13/14 cases. An HIV viral load >100,000 copies/ml was observed in 13/14 cases. An early treatment with liposomal amphotericin B was initiated in 12/14 cases. The outcome was favorable in 12/14 of all cases and in 10/12 cases involving histoplasmosis. Case fatality was 2/14 among all cases (14.3%) et 1/10 among confirmed disseminated histoplasmosis with HLH (10%). During the study period 1 in 5 cases of known HIV-associated disseminated histoplasmosis in French Guiana was HLH. Conclusion: Histoplasmosis was the most frequent etiology associated with HLH in HIV-infected patients in French Guiana. The prognosis of HLH remains severe. However, a probabilistic empirical first line treatment with liposomal amphotericin B seemed to have a favorable impact on patient survival.
299	Experience with a Reduced Toxicity Allogeneic Transplant Regimen for Non-CGD Primary Immune Deficiencies Requiring Myeloablation.	33067658	A need exists for reduced toxicity conditioning regimens that offer less toxicity while maintaining myeloablation, especially for primary immune deficiencies where myeloablation or high donor myeloid chimerism is required to achieve cure. We adapted a busulfan and fludarabine regimen by Gungor et al. for children and young adults undergoing allogeneic HCT for non-CGD primary immune deficiencies requiring myeloablation or high donor myeloid chimerism, and herein report our experience.We retrospectively reviewed records of 41 consecutive patients who underwent allogeneic HCT for Wiskott-Aldrich syndrome (n = 12), primary HLH/XLP (n = 10), CD40L deficiency (n = 7), or other (n = 12) primary immune deficiencies with a conditioning regimen containing pharmacokinetic-guided busulfan dosing which achieved a cumulative AUC between 57 and 74 mg/L × h (65-80% of conventional myeloablative exposure), along with fludarabine and alemtuzumab or anti-thymocyte globulin at 3 transplant centers between 2014 and 2019.Forty-one patients underwent a first (n = 33) or second (n = 8) allogeneic HCT. Median age was 2.3 years (range, 0.3 years-19.8 years). All but one patient (97.5%) achieved neutrophil recovery at a median of 14 days (range, 11-34 days). One patient developed sinusoidal obstruction syndrome and two patients developed diffuse alveolar hemorrhage. Four patients developed grades II-IV acute GVHD. Three patients developed chronic GVHD. One-year overall survival was 90% (95% confidence interval [CI] 81-99%) and event-free survival was 83% (95% CI 71-94%).Our experience suggests that a reduced toxicity busulfan-fludarabine regimen offers low toxicity, low incidence of grades 2-4 GVHD, durable myeloid engraftment, and excellent survival, and may be considered for a variety of primary immune deficiencies where myeloablative HCT is desired.
300	Primary Cutaneous Gamma/Delta T-cell Lymphoma and Hemophagocytic Lymphohistiocytosis Associated With AIDS.	33062507	Primary cutaneous gamma delta T-cell lymphoma (PCGD-TCL) is a rare lymphoma that makes up less than 1% of all cutaneous T-cell lymphomas. Patients with PCGD-TCL typically present with rapidly progressing plaques and ulceronecrotic nodules most frequently located on extremities without lymph node or bone marrow involvement. The overall prognosis is poor with a median overall survival of approximately 15 months. This case highlights a patient with concomitant PCGD-TCL, hemophagocytic lymphohistiocytosis, and human immunodeficiency virus-1-acquired immunodeficiency syndrome. There is a paucity of case reports describing PCGD-TCL and there are no evidence-based treatment recommendations. Further studies are needed to optimize strategies to treat patients with these diseases.
301	Hemophagocytic lymphohistiocytosis in a patient with COVID-19 treated with tocilizumab: a case report.	33054818	The understanding of coronavirus disease 2019 (COVID-19) is rapidly evolving. Although it is primarily a respiratory illness, other manifestations, such as Guillain-Barré syndrome, immune thrombocytopenia, and immune-mediated thrombotic thrombocytopenic purpura, have been described. We present a case of a patient with hemophagocytic lymphohistiocytosis secondary to COVID-19 treated with tocilizumab with a marked biochemical improvement.In this case report we present a Caucasian patient with COVID-19 who developed a marked elevation of inflammatory parameters with ferritin 36,023 μg/L, but also elevated C-reactive protein 334 mg/L and lactate dehydrogenase 1074 U/L, 1 week after admission to the intensive care unit. He met five of eight criteria for hemophagocytic lymphohistiocytosis, but he lacked the high fever and cytopenia seen in the majority of cases. He was treated with tocilizumab, a monoclonal antibody targeting the interleukin-6 receptor, and over the next days, a rapid decrease in ferritin and C-reactive protein levels was observed. However, his respiratory failure only improved gradually, and he was weaned off the respirator 11 days later.COVID-19 may induce a hyperinflammatory clinical picture and in some cases develop into hemophagocytic lymphohistiocytosis. In our patient's case, therapeutic interleukin-6 blockade abrogated signs of hyperinflammation but did not seem to improve pulmonary function. Measurement of ferritin and C-reactive protein, as well as quantification of interleukin-6 on indication, should be performed in patients with severe COVID-19. Specific treatment in such patients must also be contemplated, preferably in randomized controlled trials.
302	A Gas-and-Brake Mechanism of bHLH Proteins Modulates Shade Avoidance.	33051265	Plants detect proximity of competitors through reduction in the ratio between red and far-red light that triggers the shade avoidance syndrome, inducing responses such as accelerated shoot elongation and early flowering. Shade avoidance is regulated by PHYTOCHROME INTERACTING FACTORs, a group of basic helix-loop-helix (bHLH) transcription factors. Another (b)HLH protein, KIDARI (KDR), which is non-DNA-binding, was identified in de-etiolation studies and proposed to interact with LONG HYPOCOTYL IN FAR-RED1 (HFR1), a (b)HLH protein that inhibits shade avoidance. Here, we established roles of KDR in regulating shade avoidance in Arabidopsis (Arabidopsis thaliana) and investigated how KDR regulates the shade avoidance network. We showed that KDR is a positive regulator of shade avoidance and interacts with several negative growth regulators. We identified KDR interactors using a combination of yeast two-hybrid screening and dedicated confirmations with bimolecular fluorescence complementation. We demonstrated that KDR is translocated primarily to the nucleus when coexpressed with these interactors. A genetic approach confirmed that several of these interactions play a functional role in shade avoidance; however, we propose that KDR does not interact with HFR1 to regulate shade avoidance. Based on these observations, we propose that shade avoidance is regulated by a three-layered gas-and-brake mechanism of bHLH protein interactions, adding a layer of complexity to what was previously known.
303	Ferulic acid delayed amyloid β-induced pathological symptoms by autophagy pathway via a fasting-like effect in Caenorhabditis elegans.	33045309	The amyloid β (Aβ) generation or aggregation plays a crucial role in Alzheimer's disease (AD). Autophagy agonists, which function as the clearance of Aβ, could be the potential drug candidates against AD. In staple food crops, ferulic acid (FA) is an enormously copious and almost ubiquitous phenolic antioxidant. In the present study, FA significantly inhibited Aβ-induced pathological symptoms of paralysis and hypersensitivity to exogenous serotonin, meanwhile restrained Aβ monomers, oligomers, and deposits in AD C. elegans. FA increased the expression of autophagy reporter LGG-1 and enhanced autophagy flux. However, the autophagy inhibitors abolished the restrictive action of FA on the worm paralysis phenotype. According to these results, FA triggered autophagy and ameliorated Aβ-induced pathological symptoms by the autophagy pathway. Moreover, FA activated the HLH-30 transcription factor to nuclear localization, which acts upstream of autophagy in fasted animals, reduced the level of lipids, but affected nor the growth of E. coli OP50, neither animal food intake behavior. These suggest that FA induced a fasting-like effect to activate the autophagy pathway. Additionally, FA ameliorated poly Q aggregations in Huntington's disease worm. Thus, FA could not only affect AD, broadly but also neurodegenerative diseases characterized by misfolded or aggregated proteins.
304	Bile Cast Nephropathy Secondary to Hemophagocytic Lymphohistiocytosis With Liver Failure.	33042669	Acute kidney injury (AKI) is a common complication seen in patients with hemophagocytic lymphohistiocytosis (HLH). More than half of patients with HLH require renal replacement therapy (RRT). There are four main causes of kidney dysfunction in HLH, which include acute tubular necrosis (ATN), hypoperfusion, tumor lysis syndrome (TLS), and HLH-related glomerulopathies. Bile cast nephropathy (BCN) is a known cause of kidney injury in patients with liver failure and hyperbilirubinemia. We present the case of a 58-year-old man who presented to the hospital with painless jaundice, choluria, acholia, and generalized malaise and was found to have hyperbilirubinemia and kidney injury in the setting of HLH, who underwent a renal biopsy showing bile salt casts with degenerating tubular lining cells consistent with BCN. This case highlights the importance of considering BCN as a cause of kidney injury when a patient with HLH presents with liver failure and elevated bilirubin levels.
305	Aggressive diffuse large B-cell lymphoma with hemophagocytic lymphohistiocytosis: report of one case.	33042349	Hemophagocytic lymphohistiocytosis (HLH) is a rare fatal hyperinflammatory syndrome resulting in cytokine storm and secondary multi-organ impairment. The natural killer (NK)/T-cell lymphoma is the predominant subtype in patients with lymphoma-associated hemophagocytic syndrome (LAHS) in Asia. Yet the non-Hodgkin's B-cell lymphoma is a relatively uncommon trigger of HLH. We report a case of a 64-year-old woman who had a bone marrow-spleen type of diffuse large B-cell lymphoma (DLBCL) associated with HLH. The patient presented with EBV-positive infection, significantly increased inflammatory cytokines (IL-6, IL-8, IL-10), and dramatically increased aspartate aminotransferase (AST) and total bilirubin (TB), resulting the patient's aggressive clinical course and early death. This case may not only illustrate the nonspecific manifestation and rapidly progressive characteristics of HLH but also highlight the necessity of anti-inflammatory therapy for the treatment of lymphoma-associated HLH.
306	MRI findings of central nervous system involvement in children with haemophagocytic lymphohistiocytosis: correlation with clinical biochemical tests.	33036779	To investigate the brain magnetic resonance imaging (MRI) features of children with haemophagocytic lymphohistiocytosis (HLH) with central nervous system (CNS) involvement, and to investigate the correlation with clinical biochemical tests.Clinical and MRI data were collected from 118 children with HLH-CNS between January 2012 and June 2019. Patients were grouped according to their MRI findings, and statistical methods were used to test for correlations between the MRI findings and biochemical variables.Patients were divided into three groups, including normal appearance (Group 1, 17/118), diffuse parenchymal volume loss (Group 2, 44/118), and brain parenchyma lesions (Group 3, 57/118) containing three subtypes of brain lesions and HLH-CNS complications. Comparing biochemical values among the three groups revealed a significant difference for all values (p<0.05), except for cell counts in the cerebrospinal fluid (CSF). A pairwise comparison further showed significant inter-group differences for most of the variables. Spearman's rank correlation coefficient also demonstrated that CSF cell counts (r=0.193, p=0.036), CSF microprotein content (r=0.379, p<0.001), serum aspartate aminotransferase (AST; r=0.521, p<0.001), serum lactate dehydrogenase (LDH; r=0.514, p<0.001) and activated partial thromboplastin time (APTT; r=0.326, p<0.001) correlated positively with the MRI groups, while platelet count (PLT; r=-0.633, p<0.001) and plasma fibrinogen (FIB; r=-0.258, p=0.005) correlated negatively.Classification of brain MRI findings of HLH-CNS correlates well with the results of several key biochemical tests. Brain MRI is a promising method to elucidate illness severity and clinical outcomes.
307	A Switch between Two Intrinsically Disordered Conformational Ensembles Modulates the Active Site of a Basic-Helix-Loop-Helix Transcription Factor.	33030907	We report a conformational switch between two distinct intrinsically disordered subensembles within the active site of a transcription factor. This switch highlights an evolutionary benefit conferred by the high plasticity of intrinsically disordered domains, namely, their potential to dynamically sample a heterogeneous conformational space housing multiple states with tailored properties. We focus on proto-oncogenic basic-helix-loop-helix (bHLH)-type transcription factors, as these play key roles in cell regulation and function. Despite intense research efforts, the understanding of structure-function relations of these transcription factors remains incomplete as they feature intrinsically disordered DNA-interaction domains that are difficult to characterize, theoretically as well as experimentally. Here we characterize the structural dynamics of the intrinsically disordered region DNA-binding site of the vital MYC-associated transcription factor X (MAX). Integrating nuclear magnetic resonance (NMR) measurements, molecular dynamics (MD) simulations, and electron paramagnetic resonance (EPR) measurements, we show that, in the absence of DNA, the binding site of the free MAX2 homodimer samples two intrinsically disordered conformational subensembles. These feature distinct structural properties: one subensemble consists of a set of highly flexible and spatially extended conformers, while the second features a set of "hinged" conformations. In this latter ensemble, the disordered N-terminal tails of MAX2 fold back along the dimer, forming transient long-range contacts with the HLH-region and thereby exposing the DNA binding site to the solvent. The features of these divergent substates suggest two mechanisms by which protein conformational dynamics in MAX2 might modulate DNA-complex formation: by enhanced initial recruitment of free DNA ligands, as a result of the wider conformational space sampled by the extended ensemble, and by direct exposure of the binding site and the corresponding strong electrostatic attractions presented while in the hinged conformations.
308	Dengue Hemorrhagic Fever Complicated With Hemophagocytic Lymphohistiocytosis in an Adult With Diabetic Ketoacidosis.	33029452	Dengue infection can cause a wide spectrum of presentations extending from simple self-limiting febrile illness to severe dengue, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue associated hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening condition characterized by the uncontrolled activation of macrophages and T cells, eliciting clusters of symptoms and signs and abnormal biochemical parameters. Herein we report a 28-year-old Sri Lankan female with no past medical history who presented with dengue hemorrhagic fever and diabetic ketoacidosis complicated with hemophagocytic lymphohistiocytosis. She was treated with a three-day course of intravenous methylprednisolone in addition to standard care for diabetic ketoacidosis and dengue hemorrhagic fever. She made an uneventful recovery.
309	Using etoposide + dexamethasone-based regimens to treat nasal type extranodal natural killer/T-cell lymphoma-associated hemophagocytic lymphohistiocytosis.	33025280	Nasal type extranodal natural killer/T-cell lymphoma (ENKTL) can be associated with hemophagocytic lymphohistiocytosis (NK/T-LAHLH), which is a rare and fatal disease with no effective therapy. We evaluated whether etoposide + dexamethasone-based chemotherapy regimens might be useful for treating NK/T-LAHLH.This retrospective single-center study evaluated clinical data from 37 patients with NK/T-LAHLH who were treated between May 2008 and January 2020.Among 363 patients with ENKTL, the cumulative incidence of HLH was 11.9%. Among 43 patients with NK/T-LAHLH, 37 patients received etoposide + dexamethasone-based chemotherapy regimens, with an overall response rate of 45.9% for the HLH. The overall response rate was substantially higher for newly diagnosed NK/T-LAHLH than it was for relapsed or refractory NK/T-LAHLH (66.7% vs. 18.8%). The median overall follow-up time was 4 months, with overall survival rates of 81.1% at 1 month, 62.2% at 2 months, 56.8% at 3 months, and 34.4% at 6 months. Significantly better overall survival (all P < 0.05) was observed for patients with newly diagnosed NK/T-LAHLH (vs. relapsed/refractory disease), stage I/II disease (vs. stage III/IV disease), and nasal disease (vs. non-nasal disease). Patients who responded to the ENKTL treatment also experienced response in their HLH; 8 patients experienced continued complete response for both HLH and ENKTL. Multivariate analysis revealed that a poor prognosis among patients with NK/T-LAHLH was independently related to relapsed/refractory ENKTL and non-nasal disease.Although patients with NK/T-LAHLH generally experienced poor outcomes, etoposide + dexamethasone-based chemotherapy regimens were associated with good outcomes among select patients with newly diagnosed or stage I/II NK/T-LAHLH.
310	Cytomegalovirus pneumonitis-induced secondary hemophagocytic lymphohistiocytosis and SIADH in an immunocompetent elderly male literature review.	33024698	Hemophagocytic lymphohistiocytosis (HLH) is also known as hemophagocytic syndrome. It is a lethal hematologic condition due to a dysregulated immune response which results in inappropriately activated macrophages damaging host tissues. Based on the etiology, HLH can be primary (genetic) or secondary (acquired). The most common cause of a secondary HLH is an infection. Viral infections are the most common cause of secondary HLH. Among the viral causes of secondary HLH, Epstein-Barr virus is the most common etiologic agent. Cytomegalovirus (CMV) is a common causative pathogen in the immunocompromised host but is rare in an immunocompetent adult. In infection- associated secondary HLH, treatment includes antimicrobial therapy. HLH carries a high mortality and morbidity rate as it is an underdiagnosed clinical condition. Successful early diagnosis allows for adequate time for curative therapy. Treatment for HLH includes chemotherapy, immunomodulators, and a hematopoietic stem-cell transplant. The 2004 diagnostic criteria set by the Histiocyte Society serves as a guide to make an earlier clinical diagnosis. A review of PubMed literature revealed only five reported cases of CMV-induced HLH. We describe the sixth case of CMV pneumonitis-induced HLH and syndrome of inappropriate antidiuretic hormone secretion in a 72-year-old White male. He was treated successfully with oral valganciclovir and corticosteroids.
311	Distinct Clinical Features and Novel Mutations in Taiwanese Patients With X-Linked Agammaglobulinemia.	33013854	Background: X-linked agammaglobulinemia (XLA) is caused by a mutation of the Bruton's tyrosine kinase (BTK) gene and is the most common genetic mutation in patients with congenital agammaglobulinemia. The aim of this study was to analyze the clinical features, genetic defects, and/or BTK expression in patients suspected of having XLA who were referred from the Taiwan Foundation of Rare Disorders (TFRD). Methods: Patients with recurrent bacterial infections in the first 2 years of life, serum IgG/A/M below 2 standard deviations of the normal range, and ≦2% CD19+B cells were enrolled during the period of 2004-2019. The frequency of infections, pathogens, B-lymphocyte subsets, and family pedigree were recorded. Peripheral blood samples were sent to our institute for BTK expression and genetic analysis. Results: Nineteen (from 16 families) out of 29 patients had BTK mutations, including 7 missense mutations, 7 splicing mutations, 1 nonsense mutation, 2 huge deletions, and 2 nucleotide deletions. Six novel mutations were detected: c.504G>T [p.K168N], c.895-2A>G [p.Del K290 fs 23*], c.910T>G [p.F304V], c.1132T>C [p.T334H], c.1562A>T [p.D521V], and c.1957delG [Del p.D653 fs plus 45 a.a.]. All patients with BTK mutations had obviously decreased BTK expressions. Pseudomonas sepsis developed in 14 patients and led to both Shanghai fever and recurrent hemophagocytic lymphohistiocytosis (HLH). Recurrent sinopulmonary infections and bronchiectasis occurred in 11 patients. One patient died of pseudomonas sepsis and another died of hepatocellular carcinoma before receiving optimal treatment. Two patients with contiguous gene deletion syndrome (CGS) encompassing the TIMM8A/DDP1 gene presented with early-onset progressive post-lingual sensorineural Deafness, gradual Dystonia, and Optic Neuronopathy syndrome (DDON) or Mohr-Tranebjaerg syndrome (MTS). Conclusion: Pseudomonas sepsis was more common (74%) than recurrent sinopulmonary infections in Taiwanese XLA patients, and related to Shanghai fever and recurrent HLH, both of which were prevented by regular immunoglobulin infusions. Approximately 10% of patients belonged to CGS involving the TIMM8A/DDP1 gene and presented with the DDON/MTS phenotype in need of aggressive psychomotor therapy.
312	Epstein-Barr virus-related hemophagocytic lymphohistiocytosis complicated with coronary artery dilation and acute renal injury in a boy with a novel X-linked inhibitor of apoptosis protein (XIAP) variant: a case report.	33008347	X-linked lymphoproliferative disease (XLP) is a rare inherited X-linked primary immunodeficiency diseases (PID). One such disease, X-linked inhibitor of apoptosis protein (XIAP) deficiency, is characterized by Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH). However, EBV-HLH with coronary artery dilation and acute renal injury (AKI) in children is unusual.We report the case of a young boy aged 17 months with a novel XIAP variant. He was initially diagnosed with EBV-HLH based on the HLH-2004 diagnostic criteria and the condition was accompanied by coronary artery dilation and acute renal injury. The comprehensive genetic analysis of peripheral blood-derived DNA revealed a hemizygous variant of the XIAP gene [c.116G > C(p.G39A)], which was inherited from his mother (heterozygous condition). After combined treatment with rituximab, intravenous immunoglobulin, corticosteroids, antiviral drugs, and mycophenolate mofetil (MMF) in addition to supportive therapy, his clinical manifestations and laboratory indexes were improved. The patient achieved complete remission with MMF treatment in the 8-month follow-up.We report the [c.116G > C(p.G39A)] variant in the XIAP gene for the first time in a case of XLP-2 associated with EBV-HLH. For male patients with severe EBV-HLH, the possibility of XLP should be considered and molecular genetic testing should be used early in auxiliary diagnosis. Reports of EBV-HLH with coronary artery dilation and AKI in children are rare. In the patients with EBV-HLH, color Doppler echocardiography and urine tests should be monitored regularly. If necessary, renal biopsy can be performed to clarify the pathology. Treatment with rituximab, immunosuppressors and supportive therapy achieved a good effect, but long-term follow-up is required.
313	The Storm Beneath the Storm: MAS-HLH in Inflammatory Myopathies.	3300448031575703	NaN
314	Hemophagocytic syndrome associated with Mycobacterium bovis in a patient with X-SCID: a case report.	32993535	Mycobacterium bovis could infect patients with immunodeficiency or immunosuppressive conditions via Bacillus Calmette-Guérin (BCG) vaccination. Tuberculosis-related hemophagocytic syndrome (HPS) is reported, but not HPS caused by Mycobacterium bovis in children.A 4-month Chinese boy presented fever and cough. The initial laboratory investigation showed the lymphocyte count of 0.97 × 109/L, which decreased gradually. HPS was diagnosed based on the test results that fulfilled the HLH-2004 criteria. In addition, Mycobacterium tuberculosis complex was detected from his peripheral blood via metagenomic next-generation sequencing (mNGS) and M. bovis was identified by polymerase chain reaction-reverse dot blot (PCR-RDB). Thus, the patient was treated with Isoniazid, Rifampin, and Pyrazinamide, but not improved. However, parents refused to accept further therapy, and was discharged on the day 12 of admission. To confirm the pathogenesis, genetic analysis was performed. Mutation in the interleukin-2 receptor subunit gamma gene: Exon 6: c.854G > A; p. Arg285Gln was detected in the patient and the mother, which could underlie X-linked severe combined immunodeficiency.A boy with X-SCID was diagnosed with M. bovis-associated HPS, emphasizing that X-SCID should be considered when M. bovis is detected in a male infant with low lymphocyte counts.
315	Acquired hemophagocytic lymphohistiocytosis as initial manifestation of multiple myeloma: A case report and literature review.	32991435	Hemophagocytic lymphohistiocytosis (HLH) is a condition characterized by a hyperinflammatory state and persistent macrophage activation, resulting in reactive phagocytosis of the hematopoietic elements. In children, it is usually a hereditary disorder, while in adults it is usually acquired secondary to viral infections, collagenoses, or tumors. Although accounting for 10% of hematologic malignancies, HLH is rarely associated with multiple myeloma (MM) and other plasmacytic dyscrasias.A 64-year-old Brazilian man seeked medical care with a 3-month history of intermittent fever, weight loss, night sweats, and progressive anemic symptoms.Total blood count showed severe bicytopenia (normocytic-normochromic anemia and thrombocytopenia), biochemical exams showed elevation of creatinine, as well as monoclonal peak in serum protein electrophoresis, high IgA dosage, and serum immunofixation with IgA kappa paraprotein. Bone marrow biopsy showed 30% of monoclonal and phenotypically anomalous plasmocytes, confirming the diagnosis of MM. Diagnosis of HLH was established by the presence of clinical and laboratory criteria: fever, splenomegaly, cytopenias, hypofibrinogenemia, hyperferritinemia, elevation of triglycerides, and several figures of erythrophagocytosis in bone marrow aspirate.The patient experienced pulse therapy with methylprednisolone for hemophagocytic lymphohistiocytosis, followed by initial therapy for multiple myeloma with cyclophosphamide and dexamethasone.Once the diagnosis of MM and secondary hemophagocytic syndrome was established, the patient had a rapid clinical deterioration despite the established therapeutic measures, evolving with cardiovascular failure, acute liver failure, acute disseminated intravascular coagulation, worsening renal dysfunction requiring dialysis support, respiratory dysfunction, and lowering of consciousness, characterizing rapid multiple organ dysfunction, ultimately leading to the death of the patient.Here, we aimed to describe the sixth reported case of HLH associated with MM, according to cases cataloged in the PubMed database, and the first case evaluated by 18-fluordeoxyglucose positron emission tomography (18-FDG-PETCT).Our case report seeks to provide support for a better clinical and laboratory characterization of this rare paraneoplastic entity associated with MM, and aims to call the attention of hematologists and intensivists to this condition that falls within the scope of the differential diagnosis of rapid onset multiple organ failure in patients with plasmacytic neoplasms.
316	Diagnosis of Chediak Higashi disease in a 67-year old woman.	32990340	Chediak-Higashi disease is a rare disease caused by bi-allelic mutations in the lysosomal trafficking regulator gene, LYST. Individuals typically present in early childhood with partial oculocutaneous albinism, a bleeding diathesis, recurrent infections secondary to immune dysfunction, and risk of developing hemophagocytic lymphohistiocytosis (HLH). Without intervention, mortality is high in the first decade of life. However, some individuals with milder phenotypes have attenuated hematologic and immunologic presentations, and lower risk of HLH. Both classic and milder phenotypes develop progressive neurodegeneration in early adulthood. Here we present a remarkable patient diagnosed with Chediak-Higashi disease at age 67, many decades after the diagnosis is usually established. Diagnosis was suspected by observing the pathognomonic granules within leukocytes, and confirmed by identification of bi-allelic mutations in LYST, reduced LYST mRNA expression, enlarged lysosomes within fibroblasts, and decreased NK cell lytic activity. This case further expands the phenotype of Chediak-Higashi disease and highlights the need for increased awareness. Individuals with milder phenotypes may escape early diagnosis, but identification is important for close monitoring of potential complications, and to further our understanding of the function of LYST.
317	Assessment of the Haemophagocytic lymphohistiocytosis HScore in patients with COVID-19.	32985664	The clinical manifestation of moderate to severe COVID-19 has parallels to secondary haemophagocytic lymphohistiocytosis (HLH) both clinically and based on molecular inflammatory response. We found no evidence to support the utility of risk stratifying COVID-19 patients using risk scoring methodology designed for HLH.
318	Consequences of treatment for hemophagocytic lymphohistiocytosis in a patient with undiagnosed Gaucher disease Type 1.	32985097	Gaucher disease, a lysosomal storage disorder and hemophagocytic lymphohistiocytosis (HLH), a disorder of the immune system, have several overlapping clinical features including cytopenias, elevated serum ferritin, and splenomegaly. Prior reports of acute infantile neuronopathic, Type 2 Gaucher disease manifesting with signs of HLH have been published. Here we describe an adult patient who was initially suspected of having HLH, and was treated with a 10-day course of etoposide and a 5-day course alemtuzumab for presumptive HLH, only to later to have his presentation be determined to be due to Type 1 Gaucher disease. HLH chemotherapy treatment appeared to trigger a severe Gaucher acute pain crisis and extensive bony disease including avascular necrosis. Prolonged immunosuppression, and recurrent infections further complicated a lengthy hospitalization. We discuss the clinical overlap between Gaucher and HLH and the iatrogenic consequences of HLH-directed therapy on underlying Type 1 Gaucher disease.
319	Ruxolitinib for hematopoietic cell transplantation-associated hemophagocytic lymphohistiocytosis.	32979171	Hemophagocytic lymphohistiocytosis (HLH) is a severe complication after allogeneic hematopoietic cell transplantation (HCT) and can cause graft failure or multi-organ failure. Here, we report two children with refractory HCT-associated HLH treated with ruxolitinib. In the first patient, ruxolitinib resolved fever, cytopenia and hyperferritinemia. In another patient, although severe hepatic failure, which developed and worsened before the administration of ruxolitinib, was irreversible, rapid improvement in fever, leukopenia and hyperferritinemia was observed. Of note, multiplex cytokine profiling showed amelioration of cytokine storm in both patients. Ruxolitinib may be an encouraging option for HCT-associated HLH.
320	The Conserved ASCL1/MASH-1 Ortholog HLH-3 Specifies Sex-Specific Ventral Cord Motor Neuron Fate in Caenorhabditis elegans.	32973001	Neural specification is regulated by one or many transcription factors that control expression of effector genes that mediate function and determine neuronal type. Here we identify a novel role for one conserved proneural factor, the bHLH protein HLH-3, implicated in the specification of sex-specific ventral cord motor neurons in C. elegans Proneural genes act in early stages of neurogenesis in early progenitors, but here, we demonstrate a later role for hlh-3 First, we document that differentiation of the ventral cord type C motor neuron class (VC) within their neuron class, is dynamic in time and space. Expression of VC class-specific and subclass-specific identity genes is distinct through development and is dependent on the VC position along the A-P axis and their proximity to the vulva. Our characterization of the expression of VC class and VC subclass-specific differentiation markers in the absence of hlh-3 function reveals that VC fate specification, differentiation, and morphology requires hlh-3 function. Finally, we conclude that hlh-3 cell-autonomously specifies VC cell fate.
321	Secondary hemophagocytic lymphohistiocytosis versus cytokine release syndrome in severe COVID-19 patients.	32972235	Severe COVID-19 associated pneumonia and acute respiratory distress syndrome has recently been described with life-threatening features of cytokine storm and loosely referred to as hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS). Although a recent report indicated favorable responses to the interleukin-1 receptor antagonist, anakinra in eight patients with COVID-19 secondary HLH diagnosed using the HScore calculation, others have suggested that the diagnosis of secondary HLH is uncommon and that the use of the HScore has limited value in guiding immunomodulatory therapy for COVID-19. Here, we provide additional perspective on this important controversy based upon comparisons between 14 COVID-19 cytokine storm patients and 10 secondary HLH patients seen immediately prior to the pandemic. We hypothesize that identification of HLH may relate to the severity or timing of cytokine release and suggest distinguishing between cytokine release syndrome and secondary HLH, reserving the latter term for cases fulfilling diagnostic criteria.
322	E-cigarette or Vaping-Associated Acute Lung Injury and Hemophagocytic Lymphohistiocytosis.	32968029	In this report, we describe the case of a 17-year-old boy with progressive respiratory failure requiring extracorporeal support who met clinical criteria for a presumptive diagnosis of electronic cigarette or vaping-associated acute lung injury (EVALI), with clinical, pathologic, and laboratory evidence of hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS). The patient in our report had a history of tetrahydrocannabinol and nicotine electronic cigarette use for months leading up to his presentation of fever, headache, emesis, and weight loss with respiratory distress. Multiple potential diagnoses were explored, and the patient's respiratory status improved, and he was initially discharged from the hospital. Roughly one week later, the patient was readmitted for worsening respiratory distress. The patient then met sufficient criteria for a potential diagnosis of HLH and MAS (elevated ferritin level, inflammatory markers, and cytopenia) to warrant a bone marrow aspirate, which revealed rare hemophagocytic cells. Given the severity of his symptoms and laboratory evidence of HLH and MAS, the patient was started on a course of steroids and anakinra. Although laboratory markers improved after treatment, the patient's respiratory failure worsened, ultimately progressing to a need for mechanical ventilation and extracorporeal support and leading to worsening multiorgan system failure and, ultimately, death. To the best of our knowledge, this is the first report of a patient with a presumptive diagnosis of EVALI with evidence of HLH and MAS, raising the possibility that macrophage activation may play a role in the pathogenesis of EVALI.
323	Overlap between hemophagocytic lymphohistiocytosis and drug reaction and eosinophilia with systemic symptoms: a review.	32964443	Drug reaction and eosinophilia with systemic symptoms (DRESS), also known as drug-induced hypersensitivity syndrome (DIHS), shares features with hemophagocytic lymphohistiocytosis (HLH), most notably fever, rash, and internal organ involvement. However, there is increasing recognition of drug-induced (secondary) HLH and biopsy-proven hemophagocytosis in DRESS, suggesting that HLH and DRESS not only overlap but also may be diseases on the same spectrum of immune dysfunction. To characterize existing literature on HLH/DRESS overlap, we queried the PubMed/MEDLINE database for 23 cases of HLH-DRESS codiagnosis. Average time-to-onset of rash after exposure to inciting drug was 2.7 weeks. Fourteen cases (61%) clinically worsened despite initial therapy, prompting a workup with diagnosis of HLH on average 2.3 weeks after diagnosing DRESS. Nine cases met HLH diagnostic criteria and had a RegiSCAR score ≥4. Nine cases met one set of criteria with a presentation suggestive of the other. Five cases met neither criteria. A patient presenting with fever, generalized rash, bicytopenia, and internal organ involvement after drug exposure was most predictive of meeting diagnostic criteria for both HLH and DRESS. Treatment was highly variable, although most initiated systemic corticosteroids with/without IVIG, plasmapheresis, or etoposide. Patients with poor outcomes in this review were treated using steroid monotherapy and had viral reactivation. Dermatologists should consider the possibility of HLH in any patient presenting with fever, rash, internal organ involvement, and cytopenia. Additional studies will be necessary to further characterize HLH and DRESS overlap and determine optimal management.
324	Contribution of clonal hematopoiesis to adult-onset hemophagocytic lymphohistiocytosis.	32961550	Adult-onset hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease of immune hyperactivation. Unlike pediatric HLH, adult HLH is rarely driven by germline genetic variants. Although numerous precipitating etiologies have been identified, the reason that HLH occurs in only a subset of individuals and how other factors contribute to the disease remains unknown. We hypothesized that clonal hematopoiesis (CH), a state in which somatic mutations in blood cells cause an expanded population of mutant hematopoietic cells and drive an aberrant inflammatory state, could contribute to adult-onset HLH. In a highly annotated cohort of older adults with HLH we found that CH was more prevalent than in control cohorts. Using the adult-onset HLH mouse model in which repeated treatments of the TLR9 agonist, ODN1826, was delivered to the mouse, we observed that macrophages carrying mutations in Tet2, one of the most commonly mutated genes in CH, have an enhanced inflammatory response to TLR9 agonism. Finally, mice carrying Tet2 mutations in the hematopoietic compartment (a common model for CH) displayed an exaggerated response to TLR9 agonism, including worse splenomegaly and anemia. Our data suggest that CH is more common in individuals with adult-onset HLH and can contribute to the pathophysiology of this disease.
325	Case Report: Ceftriaxone-Resistant Invasive Salmonella Enteritidis Infection with Secondary Hemophagocytic Lymphohistiocytosis: A Contrast with Enteric Fever.	32959766	In contrast to enteric fever, reports of secondary hemophagocytic lymphohistiocytosis (HLH) in invasive non-typhoidal salmonellosis are scarce. We report a child with ceftriaxone-resistant invasive Salmonella Enteritidis infection with secondary HLH, who was successfully managed with intravenous meropenem. Secondary HLH in the context of S. Enteritidis has not been described before.
326	Hemophagocytic Lymphohistiocytosis Associated with Histiocytic Necrotizing Lymphadenitis: A Clinical Study of 13 Children and Literature Review.	32956695	To analyze the clinical characteristics and prognosis of pediatric hemophagocytic lymphohistiocytosis (HLH) associated with histiocytic necrotizing lymphadenitis (HNL).We retrospectively collected the clinical data of all children with HNL-HLH enrolled in Beijing Children's Hospital from 2007 to 2019. The control patients with Epstein-Barr virus-associated HLH and simple HNL (not associated with HLH) were case matched (1:2). The clinical features and prognosis were analyzed by case-control study. Cases of HNL-HLH in the literature were reviewed.The male-to-female ratio of the 13 patients in our center was 9:4. The mean age of the patients at disease onset was 8.1 ± 1.2 years, younger than that of the 16 patients in the literature (P = .017). Clinical presentations, especially rash and splenomegaly, and laboratory examination of HNL-HLH group were statistically different from Epstein-Barr virus-HLH group, simple HNL group, and patients reported in the literature (P < .05). Three patients were treated with immunosuppressive drugs or chemotherapy owing to poor control of HLH. One patient died, and all 12 remaining patients survived, 2 of which developed autoimmune diseases. Kaplan-Meier survival curves showed no statistical difference among the 3 groups (P > .05).HNL-HLH is more common in school- and preschool-age children. Most patients have a favorable prognosis. Some patients suffer from relapses or develop autoimmune diseases. Prolonged follow-up should be carried out for patients with HNL-HLH.
327	Ferritin index is a strong prognostic marker in adult hemophagocytic lymphohistiocytosis.	32931059	The relationship between ferritin levels and survival in adult hemophagocytic lymphohistiocytosis (HLH) has been evaluated in previous studies. However, Admission-to-discharge percentage ferritin reduction (named as ferritin index) level in adult patients with HLH has never been evaluated to predict 6-month survival.The demographic, laboratory and clinical information of 102 newly diagnosed adult HLH patients were collected. Regression analysis, receiver operating curve and Kaplan-Meier curves were analysed to explore the performance of ferritin levels.Ferritin index and discharge ferritin level were significantly different between survivour and non-survivour group (all P < .001). Ferritin index had the highest area under the curve (AUC) for predicting the survival (AUC = 0.802, P < .001) followed by discharge ferritin (AUC = 0.746, P < .001). Kaplan-Meier analysis showed a significant difference in survival according to optimum cutoff values of ferritin index ≥ 10.19% (P < .001) or discharge ferritin ≤ 1056.1 μg/L (P < .001). Multivariate analysis confirmed that ferritin index and discharge ferritin are independent predictors of 6-month survival (ferritin index: odds ratio (HR) 6.237, 95% confidence interval (CI) 2.075-18.774, P = .001; discharge ferritin: HR 6.024, 95% CI 1.894-19.231, P = .002). In addition, the combination of a ferritin index ≥ 10.19% and discharge ferritin ≤ 1056.1 μg/L had a significantly higher 6-month survival (P < .001).Ferritin index is a better predictor of 6-month survival than admission and discharges ferritin levels in adult patients with HLH.
328	Hemophagocytic Lymphohistiocytosis Secondary to Ehrlichia Chaffeensis in Adults: A Case Series From Oklahoma.	32928497	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of pathologic immune activation that occurs as either a familial disorder or as a sporadic condition in association with a variety of triggers. Infections are the most common cause of HLH in adults and should be searched for as early treatment usually results in a favorable outcome. Human monocytotropic ehrlichiosis (HME) is a very rare cause of HLH. Failure to consider ehrlichiosis can result in misdiagnosis and an increased length of hospitalization and healthcare cost as described in our report. Treatment for secondary HLH is aimed at reducing hypercytokinemia and eradicating inflammatory and infected cells. It is important to promptly initiate doxycycline when tick-borne diseases are being entertained as a possible trigger, as the antibiotic is effective, safe and inexpensive.
329	Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients.	32925169	Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both CD8+ and CD4+ CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1β and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights.
330	Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with probable mesentery involvement with associated hemophagocytic syndrome (HPS) - how to treat it?	32924664	Subcutaneous panniculitis-like T-cell lymphoma (SPTL) is a rare, cutaneous lymphoma involving subcutaneous adipose tissue. SPTL is associated in less than 20% with hemophagocytic syndrome (HPS). A 5-year overall survival rate is inferior in pts with SPTLand HPS (46%) as compared with 91% in pts without HPS. No standardized therapy for SPTCL has yet been established. A case of 35-year-old Caucasian man with a one-month history of B symptoms with the suspicion of Still's disease. At admission with leucopenia, high LDH, ferritin, sIl-R2, and triglycerides levels, hepatosplenomegaly, small right supraclavicular nodule, and irregular thickening of trunk subcutaneous tissue. The abdomen MRI showed generalized thickening of mesentery and colonic mucosa. In the patient diagnosis of SPTCL was established with secondary HPS. CHOEP chemotherapy and modified HLH 2014 protocol were applied with subsequent high dose chemotherapy (BEAM) supported by autologous stem cells transplantation. Treatment was complicated by pancytopenia and pneumonia. The outcome of the disease treated by intensive protocol seems to be good.
331	AKT-mediated phosphorylation of TWIST1 is essential for breast cancer cell metastasis.	32922123	Previously, it was shown that human TWIST1 (basic helix-loop-helix (b-HLH) is phosphorylated by Akt kinase at S42, T121, and S123. To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. We hypothesized that alanine mutants would inhibit the metastatic ability of 4T1 cells while glutamic acid mutants would convert nonmetastatic 67NR cells into metastatic phenotype. To confirm this hypothesis, we created metastatic 4T1 and nonmetastatic 67NR cells expressing alanine mutants and glutamic acid mutants mouse TWIST1, respectively. Then, we injected 1 × 106 67NR and 1 × 105 4T1 cells overexpressing mutants of TWIST1 into the breast tissue of BALB/c mice. At the end of the 4th week, we sacrificed the animals, determined the numbers of tumors at lungs and liver. Although 67NR cells overexpressing wild-type TWIST1 did not show any metastasis, cells overexpressing S42E and T125E mutants showed 15-30 macroscopic metastasis to liver and lungs. Parallel to this, 4T1 cells expressing S42A and T125A mutants of TWIST1 showed no macroscopic metastasis. Our results indicate that phosphorylation of S42 and T125 by AKT is essential for TWIST1-mediated tumor growth and metastasis.
332	Dengue associated haemophagocytic lymphohystiocytosis: An often-missed complication of a common infection.	32918433	Hemophagocytic lymphohistiocytosis (HLH) is a severe and life-threatening condition causing multisystem involvement such as cytopenia, hepatosplenomegaly, and death. Dengue infection is one of the leading causes of HLH. We reviewed three cases of children at HRH Princess Maha Chakri Sirindhorn Medical Center, Faculty of Medicine, Srinakharinwirot University, Nakhon Nayok, Thailand, with dengue fever who subsequently developed HLH, based on the HLH-2004 diagnostic criteria. Following treatment with dexamethasone and intravenous immunoglobulin, there was a dramatic response including defervescence and improvement of cytopenia, hyperfibrinogenemia, and hyperferritinemia. Key features for diagnosis of dengue fever complicated by HLH include a history of prolonged fever exceeding seven days, splenomegaly, and worsening cytopenia. Early recognition and treatment are crucial for a successful outcome.
333	Janus Family kinase (JAK) inhibitors in HLH and severe COVID-19.	32918336	NaN
334	An Intriguing Presentation of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.	32905485	Hemophagocytic lymphohistiocytosis (HLH) is an immune related clinical syndrome with protean manifestations, varying presentation, clinically complex, with diverse causes, and is an under-recognized entity which carries high morbidity and mortality. It is precipitated by an immunological trigger in a susceptible host resulting in immune activation and dysregulation leading to disruption of immune homeostasis, cytokine storm and multi-organ failure. We describe a case of Epstein-Barr virus (EBV) associated HLH with its typical diagnostic challenges and associated high mortality rate. Certain diagnostic criteria and online tools may help to arrive at an earlier presumptive diagnosis which, in turn, may expedite treatment and lead to better clinical outcomes.
335	Unraveling subcutaneous panniculitis-like T-cell lymphoma: An association between subcutaneous panniculitis-like T-cell lymphoma, autoimmune lymphoproliferative syndrome, and familial hemophagocytic lymphohistiocytosis.	32894575	Germline HAVCR2 mutations, recently identified in a large subset of patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL), are associated with an increased risk of hemophagocytic lymphohistiocytosis (HLH). Discovery of this heritable HLH/SPTCL diathesis has expanded our understanding of a rare and molecularly heterogeneous lymphoma. Furthermore, patients with SPTCL have excellent survival unless they develop HLH. Therefore, through compiling data on SPTCL-related conditions that predispose patients to HLH, we are better able to predict which patients with SPTCL have the greatest risk of mortality. We present the first case of SPTCL with concomitant HLH and autoimmune lymphoproliferative syndrome (ALPS) in a patient who was subsequently diagnosed with familial HLH (F-HLH) attributable to a germline STXBP2 splice-site mutation. She had wild-type HAVCR2. Reports including ours show how SPTCL can evolve in the setting of an exaggerated host inflammatory response attributable to a variety of unusual underlying etiologies.
336	Challenges in HLH transplant: Tricks to prevent menace of mixed chimerism.	32893946	NaN
337	A Case of Hemophagocytic Lymphohistiocytosis Secondary to Disseminated Histoplasmosis.	32884850	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition characterized by a pathologic immune dysregulation resulting in extreme inflammation. Clinical manifestations are varied but can include severe multiorgan failure and death. HLH has been associated with malignancies, autoimmune diseases, and infections, such as histoplasmosis. Histoplasmosis commonly has subclinical manifestations but can also present in its disseminated form. We present the case of an immunocompromised patient with worsening liver function caused by hepatic histoplasmosis that later triggered HLH with severe multiorgan dysfunction.
338	Natural Killer Cell Dysfunction and Its Role in COVID-19.	32883007	When facing an acute viral infection, our immune systems need to function with finite precision to enable the elimination of the pathogen, whilst protecting our bodies from immune-related damage. In many instances however this "perfect balance" is not achieved, factors such as ageing, cancer, autoimmunity and cardiovascular disease all skew the immune response which is then further distorted by viral infection. In SARS-CoV-2, although the vast majority of COVID-19 cases are mild, as of 24 August 2020, over 800,000 people have died, many from the severe inflammatory cytokine release resulting in extreme clinical manifestations such as acute respiratory distress syndrome (ARDS) and hemophagocytic lymphohistiocytosis (HLH). Severe complications are more common in elderly patients and patients with cardiovascular diseases. Natural killer (NK) cells play a critical role in modulating the immune response and in both of these patient groups, NK cell effector functions are blunted. Preliminary studies in COVID-19 patients with severe disease suggests a reduction in NK cell number and function, resulting in decreased clearance of infected and activated cells, and unchecked elevation of tissue-damaging inflammation markers. SARS-CoV-2 infection skews the immune response towards an overwhelmingly inflammatory phenotype. Restoration of NK cell effector functions has the potential to correct the delicate immune balance required to effectively overcome SARS-CoV-2 infection.
339	Intravascular lymphoma with hypopituitarism: A case report.	32879852	Intravascular lymphoma (IVL) is a rare subtype of lymphoma involving the growth of lymphoma cells within the vessel lumina without lymphadenopathy. Because of various modes of presentation and its rarity, IVL is often diagnosed postmortem. Herein, we report a case of intravascular B-cell lymphoma with hypopituitarism, an extremely rare complication, that was successfully treated with chemotherapy.An 80-year-old Japanese woman presented with a 7-mo history of a tingling sensation in the lower limbs. She also presented with various other symptoms such as pancytopenia, high fever daily, and unconsciousness with hypoglycemia. Although the doctor who previously treated her diagnosed hypoglycemia as being due to hypopituitarism, the cause of the other symptoms remained uncertain despite a 7-mo evaluation period. We performed bone marrow aspiration to evaluate pancytopenia and found that she had hemophagocytic lymphohistiocytosis (HLH). On the basis of a random skin biopsy for assessing the cause of HLH, she was diagnosed with intravascular B-cell lymphoma. HLH and hypopituitarism were considered secondary to IVL. All her clinical findings matched the presentations of IVL. She was immediately treated with chemotherapy and achieved complete response. She was relapse free two years after treatment.IVL should be included in the differential diagnosis of hypopituitarism, which although life-threatening, is treatable through prompt diagnosis and appropriate chemotherapy.
340	Desperate times, desperate measures: successful use of chemotherapy in treatment of haemophagocytic lymphohistiocytosis (HLH) due to disseminated histoplasmosis.	32878853	We describe a case of haemophagocytic lymphohistiocytosis (HLH) secondary to disseminated histoplasmosis, which was treated with chemotherapy in addition to standard antifungal therapy. While HLH in the setting of infections is very well described, its treatment in this setting is controversial, with some physicians treating only the underlying infection, whereas others using immune suppression in addition to antimicrobials. To the best of our knowledge, this is the first report documenting the successful treatment of an adult patient with HLH due to disseminated histoplasmosis using etoposide chemotherapy after initial antifungal therapy failed to show improvement.
341	Cutaneous Manifestations in a Patient With Reactive Hemophagocytic Lymphohistiocytosis.	32874817	Hemophagocytic lymphohistiocytosis (HLH) is a rare hematologic disease caused by a disordered immune system. We present a case of reactive HLH (RHLH) with uncommon skin findings in a 35-year-old African American female with a history of hidradenitis suppurativa and morbid obesity. Skin findings on physical exam revealed bullous, ecchymotic, hypopigmented, and ulcerated lesions. The literature on the cutaneous lesions of RHLH is limited. Skin findings are an underrecognized feature of RHLH and can help alert to its presence or its recurrence after treatment.
342	Clinical profiles and risk factors of 7-day and 30-day mortality among 160 pediatric patients with hemophagocytic lymphohistiocytosis.	32867836	Hemophagocytic lymphohistiocytosis (HLH) is a relatively rare and life-threatening disorder. Early mortality remains significantly high among patients with HLH. Our aim was to investigate clinical features and risk factors associated with 7-day and 30-day mortality among pediatric HLH patients. We retrospectively collected medical records of patients with discharge diagnosis of HLH between August 2014 and October 2018 from a tertiary children's hospital in China. The main outcome measures were the 7-day and 30-day outcome after hospital admission. The associations between symptoms, concomitant diagnoses, laboratory test results, and the risk of 7-day and 30-day mortality were examined.Among 160 pediatric HLH patients, 18 (11.3%) patients were deceased within 7 days after admission, and 46 (28.8%) patients were deceased within 30 days. The identified strong risk factors (OR > 10 and p < 0.05) for 30-day mortality were myocardial damage, severe pneumonia, respiratory failure, coagulopathy, gastrointestinal disorder, and multiple organ dysfunction syndrome (MODS). Factors strongly associated with 7-day mortality were sepsis, myocardial damage, shock, and respiratory failure. All patients deceased within 7 days developed hepatic dysfunction, coagulopathy, and MODS.The identified risk factors could help to stratify patients with high risk of early death, and need to be considered in the development of treatment protocols. As early mortality of HLH remains high, studies are needed to investigate how to initiate adequate HLH-directed treatment strategies for patients at higher risk of early death.
343	Human Adenovirus 7-Associated Hemophagocytic Lymphohistiocytosis-Like Illness: Clinical and Virological Characteristics in a Cluster of Five Pediatric Cases.	32866230	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of immune dysregulation. Children often suffer from primary genetic forms of HLH, which can be triggered by infection. Others suffer from secondary HLH as a complication of infection, malignancy, or rheumatologic disease. Identifying the exact cause of HLH is crucial, as definitive treatment for primary disease is hematopoietic stem cell transplant. Adenoviruses have been associated with HLH but molecular epidemiology data are lacking.We describe the clinical and virologic characteristics of 5 children admitted with adenovirus infection during 2018-2019 who developed HLH or HLH-like illness. Detailed virologic studies, including virus isolation and comprehensive molecular typing were performed.All patients recovered; clinical management varied but included immunomodulating and antiviral therapies. A genetic predisposition for HLH was not identified in any patient. Adenovirus isolates were recovered from 4/5 cases; all were identified as genomic variant 7d. Adenovirus type 7 DNA was detected in the fifth case. Phylogenetic analysis of genome sequences identified two clusters - one related to strains implicated in 2016-2017 outbreaks in Pennsylvania and New Jersey, the other related to a 2009 Chinese strain.It can be challenging to determine whether HLH is the result of an infectious pathogen alone or genetic predisposition triggered by an infection. We describe 5 children from the same center presenting with an HLH-like illness after onset of adenovirus type 7 infection. None of the patients were found to have a genetic predisposition to HLH. These findings suggest that adenovirus 7 infection alone can result in HLH.
344	A founder RAB27A variant causes Griscelli syndrome type 2 with phenotypic heterogeneity in Qatari families.	32856792	Griscelli syndrome type 2 (GS2) is a rare autosomal recessive disorder caused by pathogenic variants in the RAB27A gene and characterized by partial albinism, immunodeficiency, and occasional hematological and neurological involvement. We reviewed and analyzed the medical records of 12 individuals with GS2 from six families belonging to a highly consanguineous Qatari tribe and with a recurrent pathogenic variant in the RAB27A gene (NM_004580.4: c.244C > T, p.Arg82Cys). Detailed demographic, clinical, and molecular data were collected. Cutaneous manifestations were the most common presentation (42%), followed by neurological abnormalities (33%) and immunodeficiency (25%). The most severe manifestation was HLH (33%). Among the 12 patients, three patients (25%) underwent HSCT, and four (33%) died. The cause of death in all four patients was deemed HLH, providing evidence for this complication's fatal nature. Interestingly, two affected patients (16%) were asymptomatic. This report highlights the broad spectrum of clinical presentations of GS2 associated with a founder variant in the RAB27A gene (c.244C > T, p.Arg82Cys). Early suspicion of GS2 among Qatari patients with cutaneous manifestations, neurological findings, immunodeficiency, and HLH would shorten the diagnostic odyssey, guide early and appropriate treatment, and prevent fatal outcomes.
345	A novel protein upstream stimulatory factor 2 identified in lamprey, Lethenteron reissneri.	32852621	Upstream stimulatory factors are kinds of multi-functional transcription factors, which are expressed in eukaryotes widely, including Upstream stimulatory factor 1 (USFl) and upstream stimulatory factor 2 (USF2). USF protein has a typical basic helix-loop-helix leucine zipper (b-HLH-LZ) structure, which is involved in cell cycle, cell proliferations, glucose and lipid metabolism, and other biochemical processes. Although the USF family is an important regulator of cellular processes, little is known about the USF genes of lampreys, especially their evolutionary relationships, expression profiles, and biological functions. Here, an upstream stimulatory factor 2 (USF2) homolog from lamprey (Lethenteron reissneri) was identified and characterized (designated as L-USF2) because it is closer to USF2 subfamily than to USF1 subfamily. The cDNA fragment of L-USF2 has an open reading frame (ORF) of 765-bp length, encodes 254 amino acids, and contains an HLH domain at the c-terminal of amino acids. Meanwhile, motifs and genetic structure analysis reveal that USF2 gene exons are conserved. Moreover, the 3D structure analysis indicates that L-USF2 adopts the general USF2 folding and has a high structural similarity with H-USF2. The synteny results showed that the L-USF2 adjacent gene changed greatly compared with the jaw vertebrates. By real-time quantitative experiment and Western blot analysis, we found that L-USF2 gene played a significant role in the immune responses. This study not only provides us with a further understanding of the evolution and function of the USF gene family but also provides a basis for exploring its immune responses and immune defenses in lampreys.
346	Hemophagocytic Lymphohistiocytosis: An Unusual Presentation of Scrub Typhus.	32850256	Scrub typhus is an important etiological cause for acute undifferentiated febrile illness in the Asia-Pacific region, including Sri Lanka. It is a mite-borne disease caused by Orientia tsutsugamushi. Hemophagocytic lymphohistiocytosis (HLH) is a rapidly progressive and potentially life-threatening hyperinflammatory syndrome rarely associated with scrub typhus. We herein describe a rare case of scrub typhus complicated by hemophagocytic lymphohistiocytosis in a 40-year-old previously healthy woman who presented with a history of an acute febrile illness. Following the observation of acute deterioration of hematological parameters despite the nature of the febrile illness, the rare association of hemophagocytic lymphohistiocytosis was considered, and this disease association was confirmed by fulfilling six out of eight of the diagnostic criteria of haemophagocytic lymphohistiocytosis. The patient made an uneventful recovery following treatment for the precipitating illness and with supportive care.
347	Hyperinflammation with COVID-19: The key to patient deterioration?	32838293	The potential risk of cytokine storm in patients with coronavirus disease 2019 (COVID-19) has been described [1]; we write to share our experience treating a 17-year-old male with haemophagocytic lymphohistiocytosis (HLH) secondary to COVID-19 infection.This patient presented with cough, sore throat, anorexia and pyrexia. On examination, he had gross cervical lymphadenopathy and palpable splenomegaly. Nose and throat swab for SARS-CoV-2 was positive and blood tests revealed pancytopaenia with very high ferritin, triglyceride and d-dimer levels. The patient's H-Score [2] was calculated at 220, suggesting probability of HLH of 93-96%. Considering Russell and colleagues' [3] comments about potential harm of corticosteroid use in patients with COVID-19 infection, the patient was commenced on treatment with the selective IL-1 receptor antagonist drug, Anakinra, and a two-day course of intravenous immunoglobulin.The patient responded rapidly to treatment, becoming apyrexial after 24 h. His lymph nodes and spleen began to normalise after the first 48 h, at which time point the ferritin also started to decrease. He was discharged after 11 days feeling fit and well.This case certainly illustrates the importance of hyperinflammation syndromes in COVID-19. It also raises the question - is the severe pneumonitis seen in patients with COVID-19 an immunological phenomenon? We know that the viral load of patients with COVID-19 seems to peak in the early stages of illness [4,5]; however, patients deteriorate later in the disease course, at around days 10-14. This patient, who had risk factors for deterioration (male, pancytopaenic), did not develop an oxygen requirement and clinically and biochemically improved rapidly on Anakinra with no adverse events. We might suggest Anakinra to the scientific community as a treatment option in COVID-19 infection.
348	COVID-19 associated Multisystem Inflammatory Syndrome in Children (MIS-C) guidelines; a Western New York approach.	32837142	NaN
349	Bioactive natural compounds against human coronaviruses: a review and perspective.	32834947	Coronaviruses (CoVs), a family of enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, unusually large RNA genome, and unique replication capability. CoVs are known to cause various potentially lethal human respiratory infectious diseases, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the very recent coronavirus disease 2019 (COVID-19) outbreak. Unfortunately, neither drug nor vaccine has yet been approved to date to prevent and treat these diseases caused by CoVs. Therefore, effective prevention and treatment medications against human coronavirus are in urgent need. In the past decades, many natural compounds have been reported to possess multiple biological activities, including antiviral properties. In this article, we provided a comprehensive review on the natural compounds that interfere with the life cycles of SARS and MERS, and discussed their potential use for the treatment of COVID-19.
350	SARS CoV 2 infection in chronic myelogenous leukemia: Severe hematological presentation.	32828694	Infection with SARS-CoV-2, the cause of coronavirus infectious disease-19 (COVID-19), has caused a pandemic. Few data are available about the risk of COVID-19 infection in persons with hematological cancer, but controversy whether these persons have the same clinical signs and outcomes. We describe a case of life-threatening COVID-19 infection complicated by severe anemia in patients affected also by chronic myelogenous leukemia. The screening for RBC antibodies and the direct antiglobulin test (DAT) turned positive. The identification of the antibodies, showed the presence of an alloantibody with anti-Lewis b specificity, which was reactive at room temperature, in the anti-human globulin phase (AGH) and with papain-treated red blood cells. At the same time hemophagocytic lymphohistiocytosis (HLH), on the basis of major laboratory findings including hyperferritnemia, increase of triglicerides levels and according to the HLH score was suspected. Patients received antiviral therapy, steroids and intravenous immunoglobulins. Hemolysis resolved and ferritin dramatically decreased after administration of Ig and a Afull recovery was achieved after viral infection resolution.This case highlights the novel and multifaceted hematological findings during sever COVID 19 infection. COVID 19-related pneumonia is mediated by hyper activation of effector T cells and excessive production of inflammatory cytokines, such as IL-6, IL-1, interferon-gamma, and TNF. This inflammatory process called "cytokine storm" is a life-threatening complication of COVID 19 infection. In this case severe immunohematological consequences are reported for the first time and recognition of this complications are probably underestimated.
351	Haemophagocytic lymphohistiocytosis (HLH) secondary to miliary tuberculosis.	32825870	HLH is a rare, life-threatening, hematologic disorder resulting from prolonged and excessive activation of antigen presenting cells (macrophages, histiocytes) and CD8+ T cells. It is characterized by fever, pancytopenia,splenomegaly and haemophagocytosis in bone marrow,liver or lymph node. This hyperinflammatory condition is often triggered by a variety of agents or events, mostly genetic or infectious. HLH secondary to TB, have 100 % mortality in absence of anti-tubercular treatment .Since it mimics other disorders, its timely diagnosis remains a challenge. We report a case of hemophagocytic syndrome associated with disseminated tuberculosis in an immunocompetent man managed with anti-tubercular treatment and corticosteroid as immune modulator.
352	A novel prognostic model for adult patients with Hemophagocytic Lymphohistiocytosis.	32819431	Hemophagocytic Lymphohistiocytosis (HLH) is a type of rare disease with low survival rate. We aimed to develop a model to evaluate the six-month prognosis in adult HLH patients. The data at discharge (will be called as post-treatment) for newly diagnosed adult HLH patients was collected and independent prognostic variables were selected for inclusion in the model.Three laboratory markers were confirmed to be the independent risk factors (ferritin: hazard ratio (HR) 0.101, 95% confidence interval (CI) 0.036-0.282, P<0.001; platelets: HR 4.799, 95% CI 1.884-12.223, P = 0.001; alanine aminotransferase (ALT): HR 0.423, 95% CI 0.180-0.997, P = 0.049). These were included in the final clinical prediction model. Receiver operating characteristic (ROC) curves disclosed that this model had a better discrimination (area under the curve (AUC) = 0.842, 95% CI 0.773-0.910, P < 0.001) than each of them alone and the calibration curves aligned completely with the model predictions and actual observations. Kaplan-Meier curves revealed a significant difference in the overall survival (OS) in patients stratified by the model with higher values associated with a better OS.These results point out that serum ferritin, platelets and ALT levels are independent elements of OS in adult patients with HLH, and that the proposed model have a better prognostic value than any of these markers alone.
353	Emapalumab treatment in an ADA-SCID patient with refractory hemophagocytic lymphohistiocytosis-related graft failure and disseminated bacillus Calmette-Guérin infection.	32817285	Emapalumab, a fully human anti-IFNγ monoclonal antibody, has been approved in the US as second-line treatment of primary hemophagocytic lymphohistiocytosis (HLH) patients and has shown promise in patients with graft failure (GF) requiring a second allogeneic hematopoietic stem cell transplantation (HSCT). The blockade of IFNγ activity may increase the risk of severe infections, including fatal mycobacteriosis. We report a case of secondary HLH-related GF in the context of HLA-haploidentical HSCT successfully treated with emapalumab in the presence of concomitant life-threatening infections, including disseminated tuberculosis (TB). A 4 years old girl with Adenosine Deaminase-Severe Combined Immunodeficiency complicated by disseminated TB came to our attention for ex-vivo hematopoietic stem cell-gene therapy. After engraftment failure of gene corrected cells, she received two HLA-haploidentical T-cell depleted HSCT from the father, both failed due to GF related to concomitant multiple infections and secondary HLH. Emapalumab administration allowed to control HLH, as well as to prevent GF after a third haplo-HSCT from the mother. Remarkably, all infections improved with antimicrobial medications and disseminated TB did not show any reactivation. This seminal case supports emapalumab use for treatment of secondary HLH and prevention of GF in patients undergoing haplo-HSCT even in the presence of multiple infections, including TB.
354	Hemophagocytic Lymphohistiocytosis and Relapsing Polychondritis with Acute Myelogenous Leukemia: Case Report and Review of the Literature.	32812531	BACKGROUND This case report describes rare disease entities with possible associations that include relapsing polychondritis, a rare disease with systemic manifestations characterized by bouts of inflammation in hyaline cartilage in multiple body sites, and hemophagocytic lymphohistiocytosis (HLH), another potentially life-threatening condition that occurs due to erratic activation of the immune system accompanied by pancytopenia. Both diseases constitute a real challenge to diagnose and treat. These entities, their associations, and treatment protocols and prognosis for them are highlighted. CASE REPORT A 16-year-old female presented with features and complications of both relapsing polychondritis (RP) and HLH including costochondritis, fever, splenomegaly, thrombocytopenia, and anemia. After admission to the intensive care unit, symptomatic management included paracetamol, intravenous fluids, prednisolone 60 mg orally, intravenous immune globlulin, and warfarin. Unfortunately, the patient developed acute myelogenous leukemia (FAB AML M5b) after a period of remission and died due to sepsis and multiorgan failure. CONCLUSIONS HLH and RP are two rare diseases that can present together. Whether this malignant process (AML) is a cause or a result of these diseases is unknown. In the case presented here, the patient developed features of AML after a period of remission from RP and HLH. This case report may provide perspective on diagnosis and treatment for clinicians faced with similar patients.
355	Drug reaction with eosinophilia and systemic symptoms (DRESS)-associated hemophagocytic lymphohistiocytosis (HLH), an important and underrecognized HLH mimic: A case report.	32808435	NaN
356	Spotlight on Tocilizumab in the Treatment of CAR-T-Cell-Induced Cytokine Release Syndrome: Clinical Evidence to Date.	32801727	Immune-based therapies such as chimeric antigen receptor (CAR)-T-cell therapy have revolutionized the landscape of cancer treatment in recent years. Although this class of therapy has demonstrated impressive clinical efficacy against cancers that were once thought to be incurable, its success is in part limited by unique toxicities which can be severe or even fatal. Cytokine release syndrome (CRS) is the most commonly observed toxicity and occurs as a result of non-antigen specific immune activation. Similar to macrophage activation syndrome (MAS)/hemophagocytic lymphohistiocytosis (HLH), CRS is associated with elevated levels of several cytokines including interleukin-6 (IL-6) that serve as a driver for host immune dysregulation. As a direct anti-cytokine drug, tocilizumab has been a cornerstone in the treatment of CAR-T-associated CRS through its ability to dampen CRS without compromising CAR-T-cell function. However, optimal timing of administration is yet unknown. Here, we review the use of tocilizumab in the management of CAR-T-associated CRS, emphasizing on the clinical efficacy across various CAR constructs and its role in current CRS management algorithms. We also discuss alternative therapies that may be considered for refractory CRS therapy and the use of tocilizumab in the current COVID-19 global pandemic.
357	[Analysis of Clinical Characteristics and Prognostic Risk Factors of HLH Children with Central Nervous System Involvement].	32798430	To investigate the clinical characteristics and prognostic risk factors of HLH children with central nervous system (CNS) involvement so as to provide more reference for further improving the prognosis of HLH children.The clinical data of 45 HLH children with CNS involvement treated in our hospital from January 2006 to October 2016 were collected and analyzed retrospectively. The clinical characteristics of HLH children with CNS involvement were recorded, moreover the possible factors influencing the prognosis of HLH children with CNS involvement were analyzed using univariate and multivariate analysis through the establishment of Cox risk ratio model.Among 45 HLH children with CNS involvement, male was 19 cases and female was 26 cases. The median age of 4.0 years old (1.0-15.1). The detection showed that EBV found in 38 cases (84.44%), CMV infection in 1 case (2.22%), bacterial infection in 3 cases (6.67%), connection tissue disease in 1 case (2.22%) and indefinite etiology infection in 2 cases (4.44%). After lumbar puncture of 27 HLH children with CNS involvement, 10 cases (37.04%) showed cerebrospinal fluid abnormality. In addition, 22 cases showed the craniography abnormality. The follow-up results showed that the OS rate of 1 year was 46.67% (21/45), the OS rate of 3 years was 44.44% (20/45); the median survival time was 5.0 months. The OS analysis indicated that 1 years OS rate of diseased children with cerebrospinal fluid abnormality was significantly lower than that of diseased children with cerebrospinal fluid normality (10/45 vs 17/45) (P＜0.05), and 1 years OS rate of diseased children who not received intrathecal injection was significantly lower that of diseased children who received intrathecal (10/45 vs 17/45) (P＜0.05). The univariate analysis showed that the symptoms of nervous system, abnormal cerebrospinal fluid, absence of intrathecal injection and treatment schedule all were the risk factors affecting the prognosis of HLH children with CNS involvement (P＜0.05). The multivariate analysis by Cox risk model showed that abnormal cerebrospinal fluid and absence of intrathecal injection were independent risk factors for of HLH children with CNS involvement (P＜0.05).The clinical prognosis of HLH children with CNS involvement is relatively poor, moreover some of HLH children with CNS involvement have neural sequelae. The cerebrospinal fluid abnormality and absence of intrathecal injection are independent risk factors leading to poor prognosis for HLH clildren with CNS involvement.HLH伴中枢神经系统累及的患儿临床特征及预后危险因素分析.探讨噬血细胞淋巴组织细胞增生症（HLH）伴中枢神经系统（CNS）累及患儿临床特征及预后危险因素，为进一步改善患儿预后提供更多参考.回顾性分析本院2006年1月-2016年10月收治HLH伴CNS累及的45例患儿临床资料，记录患儿临床特征，针对可能影响患儿预后危险因素建立Cox风险比例模型进行单因素和多因素分析.45例患儿中男19例，女26例，中位年龄为4.0（1-15.1）岁，其中EBV感染38例（84.44%），CMV感染1例（2.22%），细菌感染3例（6.67%），结缔组织疾病1例（2.22%），无明确病因2例（4.44%）。27例行腰椎穿刺检查后证实脑脊液异常10例（37.04%）；45例患儿中头颅影像学检查异常22例（48.89%）。45例患儿随访1年总生存率为46.67%（21/45），随访3年总生存率为44.44%（20/45），中位生存时间为5.0个月。脑脊液异常患儿随访1年总生存率显著低于脑脊液正常患儿（10 vs 17例）（P＜0.05）。未行鞘内注射患儿随访1年总生存显著低于行鞘内注射患儿（10 vs 17例）（P＜0.05）。单因素分析显示，神经系统症状、脑脊液异常、未行鞘内注射及治疗方式均是影响HLH伴CNS累及的患儿预后危险因素（P＜0.05）。以上变量引入Cox风险模型行多因素分析显示，脑脊液异常和未行鞘内注射是HLH伴CNS累及的患儿预后独立危险因素（P＜0.05）.HLH伴CNS累及的患儿临床预后相对较差，部分患儿后遗神经系统病变；而脑脊液异常和未行鞘注治疗是导致患儿临床预后不良的独立危险因素.
358	[Establishment of Secondary HLH Mouse Model and Effect of Ruxolitinib on Disease Manifestations of Model Mide].	32798429	To establish a secondary hemophagocytic lymphohistiocytosis(HLH) mouse model, and to investigate the effect of ruxolitinib on the disease manifestation of model mice.Wild type C57BL/6 mice were randomly divided into 4 groups: two groups of mice were intraperitoneally injected with CpG oligodeoxynucleotide 1826 (CpG-ODN1826) every other day to induce HLH, and other two groups were control groups. One group of the CpG-ODN1826 groups and one of the control groups were given ruxolitinib, and other two groups were given the same amount of PBS. Blood samples, serum ferritin and hepatic/spleen weights of experimental mice were detected and serum cytokine levels were measured by ELISA.Compared with the control groups, the levels of white blood cells, hemoglobin and platelets in the CpG-ODN1826 groups were significantly lower (P＜0.05); and liver/body weight, spleen/body weight, serum ferritin, sCD25, IL-10, IL-1β, IFN-Ƴ, IL-12p70, GM-CSF, TNF-α and IL-18 levels significantly increased (P＜0.05). There was no significant difference in the levels of IL-2, IL-4, IL-5, IL-6, IL-22, IL-13, IL-27 and IL-23 between the two groups (P＞0.05). The spleen in CpG group had disordered internal structure, expanding red pulp and hyperplastic nucleated cells. The liver had severe perivascular inflammations. The spleen/weight of the ruxolitinib-treated mice in the CpG-ODN1826 group was significantly smaller than that of the unapplied ruxolitinib (P＜0.05).The CpG-ODN1826 can induce secondary HLH symptoms in wild type C57BL/6 mice. Ruxolitinib can alleviate the symptoms of splenomegaly in HLH model mice.继发性HLH小鼠模型的建立及芦可替尼对模型小鼠疾病表现的影响.建立一种继发性噬血细胞性淋巴组织细胞增生症（HLH）小鼠模型，观察芦可替尼对模型小鼠疾病表现的影响。方法：。结果：。结论：。.将野生型C57BL/6小鼠随机分为4组，其中2组小鼠给予隔天腹腔注射CpG寡脱氧核苷酸1826（CpG-ODN1826）诱导产生HLH，另2组为对照组。CpG-ODN1826组和对照组中各有1组给予芦可替尼，其余2组给予等量PBS。检测实验小鼠血常规指标、血清铁蛋白和肝脾重量，用ELISA方法检测血清细胞因子水平.CpG-ODN1826组小鼠与正常对照组相比，白细胞数、血红蛋白水平、血小板数量明显降低（P＜0.05）；肝/体重、脾/体重、血清铁蛋白水平、sCD25、IL-10、IL-1 β、IFN-Ƴ、IL-12p70、GM-CSF、TNF-α和IL-18表达水平均明显升高（P＜0.05）；而IL-2、IL-4、IL-5、IL-6、IL-22、IL-13、IL-27、IL-23表达水平均无明显差异（P＞0.05）。CpG-ODN1826组小鼠脾组织内部结构排列紊乱，红髓部分明显扩大，有核细胞大量增生；肝组织血管周围有大量炎症细胞聚集。在CpG-ODN1826组中应用芦可替尼的小鼠脾/体重较未应用芦可替尼显著缩小（P＜0.05）.CpG-ODN1826可以诱导野生型C57BL/6小鼠产生继发HLH反应，建立继发HLH模型，而芦可替尼可以减轻HLH模型小鼠脾大症状.
359	Successful treatment of secondary macrophage activation syndrome with emapalumab in a patient with newly diagnosed adult-onset Still's disease: case report and review of the literature.	32793731	Here, we present a 22-year-old female patient with adult-onset Still's disease (AOSD) who was newly diagnosed in the setting of secondary macrophage activation syndrome (MAS), a rare, life-threatening inflammatory disease with 50% mortality due to multi-organ failure. She met the diagnostic criteria of AOSD and MAS, while genetic testing excluded primary causes of MAS. She had high fevers, anemia, thrombocytopenia, splenomegaly, hematophagocytosis, and elevated serum ferritin (37,950 ng/mL) and CD25 levels (11,870 pg/mL), which remained unresponsive to corticosteroids and anakinra. Her serum interferon gamma (IFN-γ) levels were elevated (7 pg/mL). She was markedly responsive to IFN-γ blockade with emapalumab that eliminated her fevers and all MAS-associated laboratory abnormalities. This report provides initial evidence for therapeutic efficacy for IFN-γ blockade in AOSD and secondary MAS.
360	Ferritin levels in patients with COVID-19: A poor predictor of mortality and hemophagocytic lymphohistiocytosis.	32790918	A hyperinflammatory environment has been a hallmark of COVID-19 infection and is thought to be a key mediator of morbidity. Elevated ferritin has been observed in many patients with COVID-19. Several retrospective studies have shown ferritin levels can be correlated and predictive of poor outcomes in COVID-19, though a rigorous analysis has been lacking.A retrospective analysis of 942 adult COVID-19 patients admitted in March 2020 at a large New York City health system with available ferritin levels.The primary outcome, all-cause mortality, was observed in 265 (28.1%) patients. Patients who died had a significantly higher median admission and maximum ferritin levels than those who did not. However, death was poorly predicted by admission and maximum ferritin levels on receiver operator curve (ROC) analysis, with AUCs of 0.677 and 0.638, respectively. AUCs increased when the cohort was limited to progressively younger patients. Ferritin levels were minimally better at predicting our secondary outcomes. These included mechanical ventilation, observed in 280 (29.7%) patients with an ROC yielding an area under the curve (AUC) of 0.769, and new renal replacement therapy, observed in 80 (8.5%) of patients with an ROC yielding an AUC of 0.787. We also performed a subset analysis on 22 patients with ferritins >20 000 ng/mL. None of the patients met HLH-2004 diagnostic criteria. Fifteen (68.2%) of these patients had suspected or confirmed bacterial infections.Though many patients with COVID-19 present with hyperferritinemia, elevated ferritin levels are not accurate predictors of outcomes and do not appear to be indicative of hemophagocytic lymphohistiocytosis.
361	Epstein-Barr Virus Versus Novel Coronavirus-Induced Hemophagocytic Lymphohistocytosis: The Uncharted Waters.	32787459	Hemophagocytic lymphohistocytosis (HLH) is a hyperinflammatory syndrome characterized by fever, hepatosplenomegaly, and pancytopenia. It may be associated with genetic mutations or viral/bacterial infections, most commonly Epstein-Barr virus (EBV) and cytomegalovirus. As for the novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), also known as COVID-19 (coronavirus disease-2019), the cytokine storm it triggers can theoretically lead to syndromes similar to HLH. In this article, we report a case of a 28-year-old female who presented with high-grade fevers, found to have both SARS-CoV-2 and EBV infections, and eventually began to show signs of early HLH. To our knowledge, this is the first case reported in literature that raises the possibility of SARS-CoV-2-related HLH development.
362	Hemophagocytic lymphohistiocytosis in advanced melanoma treated with dabrafenib and trametinib combination: two cases.	32784332	Hemophagocytic lymphohistiocytosis (HLH) has been only rarely reported in patients with BRAF-mutated advanced melanoma treated with targeted therapies and never with first-line dabrafenib/trametinib combination thus far. Two patients treated with first-line dabrafenib and trametinib combination therapy for metastatic melanoma presented with sudden occurrence of fever, cytopenia, rhabdomyolysis, hepatic cytolysis, hypertriglyceridemia and very high ferritin levels after few weeks of treatment, associated with concomitant epstein-barr virus (EBV) reactivation in one patient. In both cases, drug-induced HLH was primarily considered owing to a high H-score and the absence of other etiology. Patients rapidly improved after treatment discontinuation associated with oral steroids in one patient and did not relapse after subsequent treatment resumption with a concurrent anti-BRAF/anti-MEK combination. In metastatic melanoma HLH may occur either spontaneously in the absence of any treatment as a paraneoplastic condition, related to an intercurrent infection or drug-induced mainly with various immunotherapy or with dabrafenib and trametinib following immunotherapy. However, such observations are scarce and these are the first cases of HLH occurring during first-line treatment with dabrafenib and trametinib in advanced melanoma to our knowledge. Pathomechanisms remain to be elucidated since triggering factors may encompass the treatment itself but also other significant actors including viral reactivation along with the underlying disease. The liability of treatment should be considered in cases of HLH occurring in patients with advanced melanoma successfully treated with a combined targeted therapy. A rechallenge with a concurrent anti-BRAF/anti-MEK can be proposed in this setting.
363	Haemophagocytic lymphohistiocytosis in adult critical care.	32782466	Haemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune dysregulation, characterised by extreme inflammation, fever, cytopaenias and organ dysfunction. HLH can be triggered by conditions such as infection, autoimmune disease and malignancy, among others. Both a familial and a secondary form have been described, the latter being increasingly recognised in adult patients with critical illness. HLH is difficult to diagnose, often under-recognised and carries a high mortality. Patients can present in a very similar fashion to sepsis and the two syndromes can co-exist and overlap, yet HLH requires specific immunosuppressive therapy. HLH should be actively excluded in patients with presumed sepsis who either lack a clear focus of infection or who are not responding to energetic infection management. Elevated serum ferritin is a key biomarker that may indicate the need for further investigations for HLH and can guide treatment. Early diagnosis and a multidisciplinary approach to HLH management may save lives.
364	Stem cell transplantation for children with hemophagocytic lymphohistiocytosis: results from the HLH-2004 study.	3278084533351106	We report the largest prospective study thus far on hematopoietic stem cell transplantation (HSCT) in hemophagocytic lymphohistiocytosis (HLH), a life-threatening hyperinflammatory syndrome comprising familial/genetic HLH (FHL) and secondary HLH. Although all patients with HLH typically need intensive anti-inflammatory therapy, patients with FHL also need HSCT to be cured. In the international HLH-2004 study, 187 children aged <18 years fulfilling the study inclusion criteria (5 of 8 diagnostic criteria, affected sibling, or molecular diagnosis in FHL-causative genes) underwent 209 transplants (2004-2012), defined as indicated in patients with familial/genetic, relapsing, or severe/persistent disease. Five-year overall survival (OS) post-HSCT was 66% (95% confidence interval [CI], 59-72); event-free survival (EFS) was 60% (95% CI, 52-67). Five-year OS was 81% (95% CI, 65-90) for children with a complete response and 59% (95% CI, 48-69) for those with a partial response (hazard ratio [HR], 2.12; 95% CI, 1.06-4.27; P = .035). For children with verified FHL (family history/genetically verified, n = 134), 5-year OS was 71% (95% CI, 62-78) and EFS was 62% (95% CI, 54-70); 5-year OS for children without verified FHL (n = 53) was significantly lower (52%; 95% CI, 38-65) (P = .040; HR, 1.69; 95% CI, 1.03-2.77); they were also significantly older. Notably, 20 (38%) of 53 patients without verified FHL had natural killer cell activity reported as normal at diagnosis, after 2 months, or at HSCT, suggestive of secondary HLH; and in addition 14 (26%) of these 53 children had no evidence of biallelic mutations despite having 3 or 4 FHL genes analyzed (natural killer cell activity not analyzed after 2 months or at HSCT). We conclude that post-HSCT survival in FHL remains suboptimal, and that the FHL diagnosis should be carefully investigated before HSCT. Pretransplant complete remission is beneficial but not mandatory to achieve post-HSCT survival. This trial was registered at www.clinicaltrials.gov as #NCT00426101.
365	[Hemophagocytic lymphohistiocytosis on Epstein-Barr Virus reactivation].	32779901	We describe the case of a 43-years-old female patient admitted in the emergency department for impairment of general condition and dyspnea. This patient is on immunosuppressive medication for Behçet's disease and will develop a lymphoma and hemophagocytic lymphohistiocytosis following Epstein-Barr virus (EBV) reactivation. This clinical case introduces a brief literature review about hemophagocytic lymphohistiocytosis (HLH).Nous présentons le cas d’une patiente âgée de 43 ans se présentant aux urgences pour altération de l’état général et pour dyspnée. Cette patiente est sous immunosuppresseurs dans le cadre d’une maladie de Behçet et va développer un lymphome ainsi qu’une lymphohistiocytose hémophagocytaire suite à une réactivation de l’Epstein- Barr Virus (EBV). Ce cas clinique permet de présenter une brève revue de littérature sur la lymphohistiocytose hémophagocytaire (LHH).
366	Falciparum malaria-induced secondary hemophagocytic lymphohistiocytosis successfully treated with ruxolitinib.	32777582	JAK/STAT signaling plays a major role in the pathogenesis of secondary hemophagocytic lymphohistiocytosis. This case report on a critically ill patient with secondary hemophagocytic lymphohistiocytosis due to falciparum malaria treated successfully with ruxolitinib, demonstrates that JAK1/2 inhibition might be a promising treatment option for severe cases.
367	Pancytopenia induced by secondary hemophagocytic lymphohistiocytosis: A rare, overlooked dreadful complication of Plasmodium vivax.	32775294	Hemophagocytic lymphohistiocytosis (HLH) is an unusual multifaceted clinicopathological entity that often remains misdiagnosed and can be fatal if not timely detected or treated. It can be familial or associated with different types of infections, autoimmune disorders, and malignancies. Parasitic infection-associated HLH has been rarely documented in the literature with only a handful of them being reported due to Plasmodium vivax infection. We describe an extremely rare case of pancytopenia induced by HLH resulting from P. vivax infection in a 7-year-old girl, which posed as a diagnostic challenge and led to a therapeutic delay.
368	Treatment of pediatric primary hemophagocytic lymphohistiocytosis with the HLH-94/2004 regimens and hematopoietic stem cell transplantation in China.	32766934	We aimed to clarify the clinical characteristics, prognostic factors, and effectiveness of the HLH-94/2004 regimens and hematopoietic stem cell transplantation (HSCT) in pediatric patients with primary hemophagocytic lymphohistiocytosis (pHLH) in China. A retrospective analysis was performed on 38 patients with pHLH at Beijing Children's Hospital. PRF1 (34.2%) and UNC13D (31.6%) were the most common mutations in the pHLH. Thirty-eight patients were treated with the HLH-94/2004 regimens after diagnosis. Twenty-six patients (72.2%) responded to first-line treatment (complete response: 55.5%, partial response: 16.7%). The median survival time was 23 months. The overall survival (OS) rate at 3 years was 74.7%. There was no significant difference in the response rate (72% vs. 63.6%, P = 0.703) or 3-year OS (83.6% vs. 66.7%, P = 0.443) between the patients treated with the HLH-94 regimen and those treated with the HLH-2004 regimen. The incidences of all side effects in patients treated with the HLH-94 or HLH-2004 regimen were 32.0% and 18.2%, respectively (P = 0.394). Among 15 patients treated with HSCT, neither the preconditioning regimen nor the donor type affected patient prognosis (P = 0.205 and P = 0.161, respectively). The disease status (remission or nonremission) before preconditioning did not affect prognosis or the incidence of GVHD. Furthermore, a higher bilirubin level (≥ 30 μmol/L) was correlated with a poorer prognosis in pHLH patients (P = 0.026). The effectiveness rates of the HLH-94 and HLH-2004 regimens, chemotherapy, and HSCT were similar in pHLH patients. A bilirubin level ≥ 30 μmol/L might be an adverse prognostic factor in pHLH.
369	Hemophagocytic Lymphohistiocytosis (HLH) in a Patient with Disseminated Histoplasmosis.	32765915	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition characterized by an overwhelming inflammatory cascade activation which is often associated with rapid progression and high mortality. It may be familial with an underlying genetic mutation or triggered by infection, malignancy, and autoimmune disease. Disseminated histoplasmosis caused by histoplasma capsulatum is a granulomatous fungal disease seen typically in immunocompromised patients with varied clinical manifestations and requires long-term antifungal therapy. We present the case of a 61-year-old immunocompromised female with significant travel history who came with fever, pancytopenia, and liver failure raising suspicion for HLH that prompted a bone marrow biopsy procedure. Hemophagocytic figures consistent with HLH and numerous encapsulated fungi resembling histoplasma were visualized. She was treated with intravenous (IV) liposomal amphotericin B. Etoposide chemotherapy and interleukin-1 (IL-1) antagonist anakinra were deferred in order to limit her immunosuppression, and treatment was focused on antifungal therapy.
370	Ferritin in hemophagocytic lymphohistiocytosis (HLH): current concepts and controversies.	32745577	NaN
371	A pilot study of ruxolitinib as a front-line therapy for 12 children with secondary hemophagocytic lymphohistiocytosis.	32732367	Hemophagocytic lymphohistiocytosis (HLH) is an immune-regulatory disorder characterized by excessive production of inflammatory cytokines. The treatment recommendations of the HLH-1994 and HLH-2004 protocols have long been used in HLH therapy, but some patients still do not respond well to or have unacceptable side effects from conventional therapies. It is believed that cytokine-targeted strategies that directly target disease-driving pathways will be promising options for HLH. This prospective study aimed to investigate the efficacy and safety of ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, as a front-line therapy in children with secondary HLH. Twelve newly diagnosed patients without previous treatment were enrolled in this study with a median follow-up of 8.2 (7.1-12.0) months, including 8 cases of Epstein-Barr virus associated HLH (EBV-HLH), 2 cases of autoinflammatory disorder (AID)- associated HLH, and 2 cases of unknown etiology. Patients received oral ruxolitinib dosed on 2.5 mg, 5 mg or 10 mg twice daily depending on the body weight for 28 consecutive days. The overall response rate at the end of treatment (day 28) was 83.3% (10/12), with 66.7% (8/12) in complete response (CR), 8.3% (1/12) in partial response (PR), and 8.3% (1/12) in HLH improvement. Among the patients achieving CR, 87.5% (7/8) maintained CR condition for>6 months, and one patient with EBV-HLH relapsed following CR. For the EBV-HLH subgroup, all 8 patients responded to ruxolitinib, with a CR rate of 75% and a PR rate of 25%. Two patients with AID-associated HLH had quite different responses, with one showing reversal of the HLH abnormalities soon and the other showing no improvement, as did the two cases of unknown etiology. Patients who had no response or discontinued ruxolitinib all responded well to the subsequent HLH-1994 regimen. The expected 6-month event-free survival (EFS) rate was 58.3%±10.2%. No serious adverse effects were reported. Our study provides further support for the possibility of ruxolitinib targeted therapy for secondary HLH in children. This study was registered in the Chinese Clinical Trials Registry Platform (http://www.chictr.org.cn/) as ChiCTR2000029977.
372	Recalcitrant minocycline-induced DRESS mimicking HLH.	32730650	NaN
373	NK cells: energized yet exhausted in adult HLH.	3273057832356861	NaN
374	[Hemophagocytic syndrome, current diagnostic and therapeutic approach].	32730382	Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH) is a highly lethal hyper-inflammatory disorder that leads to a storm of cytokines, hemophagocytosis and multiple organ failure. It can be primary, which is inherited, or secondary. In the latter, virus infections are a frequent trigger, predominantly the family of herpes viruses, such as Epstein-Barr virus. An early treatment is recommended. Until recently there was no consensus about the management of secondary cases. The protocols for the treatment of primary HLH were used, which include cytotoxic agents and corticosteroids. We herein review the current diagnostic and therapeutic approach of HLH, based on a case associated with a reactivation of the Epstein-Barr virus in an immunocompetent adolescent. We highlight the importance of suspecting this disease in patients with a persistent inflammatory response state or with a fever of unknown origin, in order to carry out a timely treatment, with the least toxicity, and appropriate to the characteristics of each individual, which is the current therapeutic trend.
375	The use of anakinra in the treatment of secondary hemophagocytic lymphohistiocytosis.	32725881	Hemophagocytic lymphohistiocytosis (HLH) can be familial or secondary, which is often triggered by infection or malignancy. HLH therapy includes dexamethasone and etoposide. However, therapy is associated with significant morbidity and mortality. Anakinra, a recombinant interleukin-1 receptor antagonist, has been reported to treat macrophage activation syndrome (MAS), rheumatic sHLH. We report our experience with anakinra to treat patients with nonrheumatic secondary HLH (sHLH).Six children were diagnosed with HLH from December 2014 to August 2016 and were treated with subcutaneous anakinra (6-10 mg/kg/day divided over four doses) with or without dexamethasone (10 mg/m2 /day). Therapy was either escalated or weaned based on clinical and laboratory response.Five of six patients were treated with anakinra and dexamethasone, and one with anakinra alone due to active cytomegalovirus (CMV) pneumonitis. The median age of diagnosis was 1.8 years (range 0.8-14.9 years). No pathogenic mutations associated with HLH were identified, but three of six possessed genetic variants of unknown significance. Infectious triggers were identified for four patients and two patients had malignancies. The average treatment duration was 8 weeks with 3.5-5.5 years of follow up. No patient needed escalation of therapy to include etoposide. All patients achieved remission. Anakinra was well tolerated without significant adverse effects.Initial treatment with anakinra (with or without dexamethasone) is a feasible treatment alternative for patients with secondary HLH and may allow for avoidance of etoposide. We recommend early initiation of anakinra when HLH is suspected. A broader investigation of the use of anakinra as a first-line agent for HLH is ongoing.
376	Resolution of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis associated with rapid immune reconstruction after a single course of CHOP therapy.	32725606	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening disease characterized by the uncontrolled proliferation of EBV-infected T/NK cells with resultant immune system failure against EBV. While a CD5-HLA-DR+CD8+ T-cell population was previously shown to be EBV-infected cells and a useful marker for monitoring the response to treatment of EBV-HLH, changes in other lymphocyte subsets associated with EBV-HLH treatments have not been closely studied. We herein report a 25-year-old woman with EBV-HLH who presented with a fever, liver failure, and pancytopenia. CD8+ T cells harbored EBV. After failing steroid pulse therapy, one course of CHOP therapy immediately improved her fever and laboratory data and reduced the population of EBV-infected cells. Although the number of EBV-infected cells increased on day 20 of CHOP, a sharp increase in NK cells and normal activated T cells ensued, and the infected cells disappeared without an additional CHOP cycle. She has maintained remission without complications. This rapid immune reconstitution has not been observed in two other patients treated with HLH-2004 protocol-like regimens including prolonged immunosuppressants and etoposide. One cycle of CHOP was thought to have induced the resolution of EBV-HLH by eliminating infected cells as well as inducing the reconstruction of anti-EBV immunity.
377	Severe anaplasmosis represents a treatable cause of secondary hemophagocytic lymphohistiocytosis: Two cases and review of literature.	32723647	Anaplasmosis is an emerging infection in the United States and remains under-recognized in many areas including Pennsylvania. Presenting signs and symptoms are often nonspecific, but fulminant infection can occur in vulnerable populations. We present two cases of severe anaplasmosis that progressed to secondary hemophagocytic lymphohistiocytosis (HLH). This severe immune dysregulation syndrome has an extremely high mortality, but anaplasmosis represents one of the few treatable underlying etiologies. It is imperative for physicians to recognize this complication and start empiric doxycycline, as early treatment improves mortality. We also present a case of anaplasmosis-induced HLH successfully treated with a combination of doxycycline, steroids, and anakinra (an IL-1 receptor antagonist), highlighting that this primarily immune-mediated complication is amenable to treatment with both antibiotics and immune suppression.
378	Disseminated Juvenile Xanthogranuloma and Hemophagocytic Lymphohistiocytosis Developed During Treatment of Acute Lymphoblastic Leukemia: Case Report.	32719740	The association between acute lymphoblastic leukemia (ALL), non-Langerhans cell histiocytosis (non-LCH), and hemophagocytic lymphohistiocytosis (HLH), to the best of our knowledge, has not been published to date. Juvenile xanthogranuloma (JXG), as a type of non-LCH, is usually a benign disease limited to the skin. Systemic involvement is rarely reported. The present case report describes a 15-year-old boy diagnosed with disseminated JXG involving skin and bone marrow concurrent with severe symptoms of HLH during ALL therapy. Examination of immunoglobulin heavy chain genes in B-cell precursor leukemic blasts and histiocytes in the skin and bone marrow revealed identical rearrangements, confirming clonal relationship between both diseases. Implementation of corticosteroids, vinblastine, etoposide, cyclosporine, and tocilizumab resulted in partial skin lesion resolution with no improvement of bone marrow function; therefore, hematopoietic stem cell transplantation (HSCT) was eventually performed. The patient's hematological and general status has improved gradually; however, remarkable recovery of skin lesions was observed after empirical antitubercular therapy. Mycobacterium spp. infection should be considered as a possible secondary HLH trigger. Triple association of ALL, non-LCH, and HLH highlights heterogeneity of histiocytic disorders and possible common origin of dendritic and lymphoid cells.
379	Case Report: Systemic Inflammatory Response and Fast Recovery in a Pediatric Patient With COVID-19.	32719688	We report a case of an 8-year-old girl who underwent a SARS-CoV-2 infection manifesting with atypical symptoms spearheaded by abdominal discomfort and systemic inflammation and partially mimicking hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS), which however did not fulfill the HLH/MAS diagnostic criteria. In this case of what has since been described as Pediatric Inflammatory Multisystem Syndrome Temporally associated with SARS-COV-2 (PIMS-TS) we documented excellent clinical response to immunosuppression with systemic corticosteroids and intravenous immunoglobulins. We show a detailed longitudinal development of neutrophil immunophenotype which suggests activation and engagement of neutrophils during PIMS-TS with compensatory contraction of the response and contra-regulation of neutrophil phenotype during recovery.
380	The Oncogenic Role of miR-BART19-3p in Epstein-Barr Virus-Associated Diseases.	32714979	Accumulating evidence so far has shown that EBV's miRNAs have been found to be involved in cancer progression. However, the comprehensive EBV miRNA expression profiles and their biological significance in EBV-associated diseases are not well documented. A comprehensive profiling of EBV-encoded miRNAs expressed in CAEBV, EBV-HLH, and nasopharyngeal carcinoma (NPC) patients was constructed, and the results showed that miR-BART19-3p was upregulated in all these diseases. Ectopic expression of miR-BART19-3p induced EBV-negative cell proliferation and suppressed cell apoptosis. Molecularly, adenomatous polyposis coli (APC) was identified to be a direct target of miR-BART19-3p, and APC mRNA expression was inversely correlated with miR-BART19-3p in CAEBV samples. Our results demonstrated that miR-BART19-3p contributes to the tumorigenesis of EBV-associated diseases and may be a potential therapeutic target.
381	Hemophagocytic lymphohistiocytosis as a presentation of inflammatory bowel disease.	32713955	Hemophagocytic lymphohistiocytosis (HLH) is considered a medical emergency that should be recognized in patients with fever, splenomegaly, and progressive deterioration of the general condition. Laboratory findings include cytopenia, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. For HLH diagnosis, it is essential, although not mandatory, to perform a bone marrow biopsy. Given its nature, secondary causes of HLH should be sought, mainly infections, hemato-oncological disorders, autoimmune diseases, and auto-inflammatory conditions.We present the case of a female adolescent who presented with fever and lower gastrointestinal bleeding. Upon admission, acute liver failure and pancytopenia were documented. A bone marrow aspirate was performed, which revealed hemophagocytosis; other tests confirmed HLH diagnosis. During the diagnostic approach, inflammatory bowel disease was diagnosed. The patient received first-line treatment with an adequate response.Inflammatory bowel disease can be considered a cause of secondary HLH, particularly in patients with suggestive symptoms, such as digestive bleeding in the absence of other secondary causes of HLH.La linfohistiocitosis hemofagocítica (LHH) es considerada una urgencia médica que debe reconocerse en pacientes con deterioro progresivo del estado general, fiebre, pancitopenia y esplenomegalia. Los hallazgos de laboratorio incluyen citopenia, hipertrigliceridemia, hipofibrinogenemia e hiperferritinemia. Para su diagnóstico es importante, aunque no obligatoria, la realización de aspirado de médula ósea. Dada su naturaleza, se deben buscar causas secundarias de LHH, principalmente enfermedades infecciosas, hematooncológicas, autoinmunitarias y autoinflamatorias.Se presenta el caso de una adolescente que inició con fiebre y sangrado digestivo bajo. A su ingreso, se documentó falla hepática aguda y pancitopenia. Se realizó aspirado de médula ósea y se encontró hemofagocitocis; el resto de los exámenes concluyeron LHH. Durante su abordaje se diagnosticó enfermedad inflamatoria intestinal. La paciente recibió tratamiento de primera línea con adecuada respuesta.La enfermedad inflamatoria intestinal puede considerarse como una causa secundaria de LHH, en particular en pacientes con clínica sugestiva, como es el sangrado digestivo, en ausencia de otras causas secundarias de LHH.
382	Living-donor liver transplantation providing an adequate chemotherapy for a pediatric patient with anaplastic large cell lymphoma complicated with liver failure due to the aggravation of biliary hepatopathy by secondary hemophagocytic lymphohistiocytosis.	32710432	Anaplastic large cell lymphoma (ALCL) accounts for 10-15% of childhood non-Hodgkin lymphoma cases; it is generally chemo-sensitive and is one of the most curable pediatric cancers. We report here a case of pediatric ALCL complicated with acute liver failure due to the aggravation of pre-existing biliary hepatopathy by lymphoma-associated hemophagocytic lymphohistiocytosis (HLH). Although the initial treatment response against ALCL was very good, poor and irreversible liver function due to biliary cirrhosis worsening by lymphoma-associated HLH prevented the patient from receiving further consolidation chemotherapies. To make matters worse, his condition was accompanied with intrahepatic fungal pseudoaneurysm and invasive fungal infection. Thus, we decided to perform an urgent living-donor liver transplantation from his father to correct the patient's liver function and make it possible to proceed with further ALCL therapy. After the living-donor liver transplantation, the patient successfully received consolidation therapy with brentuximab vedotin. To our knowledge, this may be an early reported case of a pediatric patient undergoing liver transplantation during treatment for ALCL. In most patients with HLH-associated ALCL, liver function improves when ALCL is controlled. However, acute liver failure is occasionally observed in HLH cases with pre-existing liver dysfunction. In such cases, liver transplantation should be considered to correct liver dysfunctions if the disease control of HLH is satisfactory.
383	Emerging Roles of Inhibitor of Differentiation-1 in Alzheimer's Disease: Cell Cycle Reentry and Beyond.	32708313	Inhibitor of DNA-binding/differentiation (Id) proteins, a family of helix-loop-helix (HLH) proteins that includes four members of Id1 to Id4 in mammalian cells, are critical for regulating cell growth, differentiation, senescence, cell cycle progression, and increasing angiogenesis and vasculogenesis, as well as accelerating the ability of cell migration. Alzheimer's disease (AD), the most common neurodegenerative disease in the adult population, manifests the signs of cognitive decline, behavioral changes, and functional impairment. The underlying mechanisms for AD are not well-clarified yet, but the aggregation of amyloid-beta peptides (Aβs), the major components in the senile plaques observed in AD brains, contributes significantly to the disease progression. Emerging evidence reveals that aberrant cell cycle reentry may play a central role in Aβ-induced neuronal demise. Recently, we have shown that several signaling mediators, including Id1, hypoxia-inducible factor-1 (HIF-1), cyclin-dependent kinases-5 (CDK5), and sonic hedgehog (Shh), may contribute to Aβ-induced cell cycle reentry in postmitotic neurons; furthermore, Id1 and CDK5/p25 mutually antagonize the expression/activity of each other. Therefore, Id proteins may potentially have clinical applications in AD. In this review article, we introduce the underlying mechanisms for cell cycle dysregulation in AD and present some examples, including our own studies, to show different aspects of Id1 in terms of cell cycle reentry and other signaling that may be crucial to alter the neuronal fates in this devastating neurodegenerative disease. A thorough understanding of the underlying mechanisms may provide a rationale to make an earlier intervention before the occurrence of cell cycle reentry and subsequent apoptosis in the fully differentiated neurons during the progression of AD or other neurodegenerative diseases.
384	Incidence and treatment of hemophagocytic lymphohistiocytosis in hospitalized children with Ehrlichia infection.	32706439	We report a large cohort of pediatric patients with human monocytic ehrlichiosis (HME), enabling an estimated incidence of secondary hemophagocytic lymphohistiocytosis (HLH) in hospitalized children with HME. Among 49 children with PCR-confirmed Ehrlichia infection, 8 (16%) met current criteria for HLH. Those with HLH had more significant hematologic abnormalities and longer durations from symptom onset to admission and definitive anti-infective therapy. Among these eight, three received chemotherapy plus doxycycline, one of whom died; the other five were treated with doxycycline without chemotherapy, and all survived without HLH recurrence. Our findings demonstrate that antimicrobial therapy alone can successfully resolve Ehrlichia-associated HLH.
385	Cell-type-specific promoters for C. elegans glia.	32696701	Glia shape the development and function of the C. elegans nervous system, especially its sense organs and central neuropil (nerve ring). Cell-type-specific promoters allow investigators to label or manipulate individual glial cell types, and therefore provide a key tool for deciphering glial function. In this technical resource, we compare the specificity, brightness, and consistency of cell-type-specific promoters for C. elegans glia. We identify a set of promoters for the study of seven glial cell types (F16F9.3, amphid and phasmid sheath glia; F11C7.2, amphid sheath glia only; grl-2, amphid and phasmid socket glia; hlh-17, cephalic (CEP) sheath glia; and grl-18, inner labial (IL) socket glia) as well as a pan-glial promoter (mir-228). We compare these promoters to promoters that are expressed more variably in combinations of glial cell types (delm-1 and itx-1). We note that the expression of some promoters depends on external conditions or the internal state of the organism, such as developmental stage, suggesting glial plasticity. Finally, we demonstrate an approach for prospectively identifying cell-type-specific glial promoters using existing single-cell sequencing data, and we use this approach to identify two novel promoters specific to IL socket glia (col-53 and col-177).
386	Low dose ruxolitinib plus HLH-94 protocol: A potential choice for secondary HLH.	32690141	Hemophagocytic lymphohistiocytosis (HLH) is an immune-mediated syndrome resulting in cytokine storm. The uncontrolled cytokine storm leads to significant tissue damage and potentially life-threatening multiorgan failure. Conventional first-line treatment for HLH included HLH-94 protocol or HLH-2004 protocol. However, up to 30% of patients do not respond to first-line therapy. We reported 3 cases of secondary HLH/macrophage activation syndrome which were caused by lymphoma (2 cases) and adult-onset still's disease. They received low dose ruxolitinib plus HLH-94 protocol, and had rapid response to treatment without obvious adverse effects. Following the treatment, there was improvement seen in several disease markers, including fever, fibrinogen, serum ferritin, and liver function tests. Our report indicated that treatment with low dose ruxolitinib plus HLH-94 protocol might be a potential choice for secondary HLH, and clinical trials warrants to be further investigated in this treatment regimen.
387	Paediatric MAS/HLH caused by a novel monoallelic activating mutation in p110δ.	32681977	This study provides evidence for the first time for APDS-1 presenting as MAS/HLH, with evident clinical implications in patient's management and prognosis.
388	SARS-CoV-2 Infection-Associated Hemophagocytic Lymphohistiocytosis.	32681166	A subset of coronavirus disease 2019 (COVID-19) patients exhibit clinical features of cytokine storm. However, clinicopathologic features diagnostic of hemophagocytic lymphohistiocytosis (HLH) have not been reported. We studied the reticuloendothelial organs of 4 consecutive patients who died of COVID-19 and correlated with clinical and laboratory parameters to detect HLH.Autopsies were performed on 4 patients who died of COVID-19. Routine H&E staining and immunohistochemical staining for CD163 were performed to detect hemophagocytosis. Clinical and laboratory results from premortem blood samples were used to calculate H-scores.All 4 cases demonstrated diffuse alveolar damage within the lungs. Three of the 4 cases had histologic evidence of hemophagocytosis within pulmonary lymph nodes. One case showed hemophagocytosis in the spleen but none showed hemophagocytosis in liver or bone marrow. Lymphophagocytosis was the predominant form of hemophagocytosis observed. One patient showed diagnostic features of HLH with an H-score of 217, while a second patient likely had HLH with a partial H-score of 145 due to a missing triglyceride level. The remaining 2 patients had H-scores of 131 and 96.This is the first report of severe acute respiratory syndrome coronavirus 2-associated HLH. Identification of HLH in a subset of patients with severe COVID-19 will inform clinical trials of therapeutic strategies.
389	Ruxolitinib in Alleviating the Cytokine Storm of Hemophagocytic Lymphohistiocytosis.	32680878	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening syndrome classified into primary HLH and secondary HLH. Secondary HLH is always caused by autoimmune disease, infections, or cancer. The first-line therapy for secondary HLH is the HLH 2004 protocol, including dexamethasone, etoposide, and supportive therapy. However, up to 30% of patients, especially pediatric patients, remain unresponsive to first-line treatment, and the mortality rate reaches 50% in children with HLH. Furthermore, some children who have special conditions, such as an active virus infection, are not suitable for immunosuppressants treatment. Recently, several HLH-promoting cytokines have been identified, including interferon-γ, interleukin-2, and interleukin-6. Janus kinase 1 and 2 control the signaling of many cytokines, notably interferon-γ, interleukin-2, and interleukin-6. Janus kinase 1 and 2 inhibitors, such as ruxolitinib, have been successfully used to treat HLH in mice. Here, we report that a boy, diagnosed with HLH and high titer of hepatitis B virus-DNA copies, improved quickly, and the cytokine storm of HLH was alleviated after receiving ruxolitinib. Five days after ruxolitinib treatment, entecavir was introduced and serum titer results of hepatitis B virus-DNA returned negative. With 3 months of ruxolitinib treatment and following-up 1 year, the boy's situation maintained sustained remission. In this study, it is suggested that ruxolitinib might be a first-line drug, which could alleviate the cytokine storm of HLH. This treatment may be ushering in the age of glucocorticosteroid-free HLH treatment, which is particularly meaningful for children because it avoids the side effects of glucocorticosteroid.
390	Hemophagocytic lymphohistiocytosis in adults.	32675947	Hemophagocytic lymphohistiocytosis (HLH) is an underrecognized disorder due to the variability of its presentation and the fact that in adults, its diagnosis is based on cumbersome, pediatric-based criteria. Data regarding demographics, underlying causes, clinical features, laboratory results, complications, treatments received, and clinical outcomes were collected and analyzed in 41 patients who were diagnosed and treated at University of Arkansas for Medical Sciences between 2007 and 2019. In this group, 51% were male, the median age at diagnosis was 47 years, and 85% (35/41) met the HLH-2004 diagnostic criteria (5/8 variables). When evaluating seven extended variables easily obtained by routine laboratory test, 93% (38/41) of patients met 8 out of 15 criteria. The overall mortality in our patient population was 54% (22/41). The 30-day and 1-year overall survival estimates were 0.73 (95% confidence interval 0.56, 0.84) and 0.46 (95% confidence interval 0.29, 0.62), respectively. Thirty-five patients (85.4%) received HLH-directed therapy, and 19 patients (46.3%) achieved remission. The most common regimen for treating HLH was dexamethasone plus etoposide (53.7%). The patients with malignancy-related HLH had a worse prognosis than those without underlying malignancy, with a 73.33% (11/15) vs 34.62% (9/26) mortality (P = 0.02). In conclusion, despite increasing recognition, HLH remains an enigmatic disorder with increased mortality, even more so with malignancy-associated HLH.
391	Neonatal Dengue With HLH: Perks of Early Diagnosis and Management.	32675555	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, aggressive syndrome. It can be primary, which involves genetic mutation with an early presentation, or secondary to infections, malignancies, etc., due to absence of immune downregulation. It is a very rare condition in newborns. Dengue is a potential virus causing HLH, but, in newborns, there are only few case reports and limited clinical literature.Herein, in this report, we highlight a case of neonatal HLH, triggered by perinatal dengue. The neonate manifested clinically within the first week of life, the earliest reported timeline so far in the literature.HLH should be excluded in neonates especially when multisystem involvement cannot be explained by sepsis alone.
392	[Adenovirus pneumonia with hemophagocytic lymphohistiocytosis in children: a clinical analysis of 7 children].	32669173	To study the clinical features of children with adenovirus pneumonia and hemophagocytic lymphohistiocytosis (HLH).A retrospective analysis was performed on the mediacal data of 7 children with adenovirus pneumonia and HLH from March to September, 2019.The age of these children ranged from 11 months to 5 years, and among these children, 5 were aged <2 years and 5 were boys. None of these children had underlying diseases. All children were hospitalized due to persistent high fever and cough, and the peak temperature of fever was 39°C to 41°C. With disease progression, 7 children developed hepatomegaly and 6 developed splenomegaly. Routine blood test results showed reductions in two or three lineages of blood cells, with increases in serum ferritin (SF), C-reactive protein (CRP), procalcitonin (PCT), and lactate dehydrogenase (LDH). Phagocytosis of blood cells was observed in 6 children. Radiological examination of lungs showed pneumonia changes. All 7 children were diagnosed with human adenovirus type 7 infection based on pathogenic metagenome detection. No abnormality was found by HLH gene detection and the children were diagnosed with secondary HLH. All children received intravenous immunoglobulin. Among these children, 4 received dexamethasone and etoposide chemotherapy, 3 received dexamethasone alone, and 4 received plasma exchange. Of the 7 children, 2 died and 5 were recovered. Compared with those who survived, the children who died had significantly greater reductions in the three lineages of blood cells and significantly greater increases in serum levels of CRP, PCT, SF, and LDH.The children with adenovirus pneumonia and HLH have main clinical features of persistent high fever, progressive reductions in two or three lineages of peripheral blood cells, and involvement of other organ systems, including hepatosplenomegaly. Significant increases in serum levels of CRP, PCT, SF, and LDH may suggest a poor prognosis.
393	Short-term effectiveness of ruxolitinib in the treatment of recurrent or refractory hemophagocytic lymphohistiocytosis in children.	32666469	To investigate the efficacy and safety of the Janus kinase inhibitor ruxolitinib in the treatment of recurrent or refractory hemophagocytic lymphohistiocytosis (HLH) in children. We performed a retrospective analysis of ruxolitinib in children with recurrent or refractory HLH in Beijing Children's Hospital. All patients were treated firstly with HLH-94 protocol. Patients received oral ruxolitinib on a continuous 28-day cycle or until disease progression or unacceptable toxicity. The median age of enrolled patients was 1.7 years (range 9 months-5.0 years). The median time from the beginning of first-line treatment to the initiation of ruxolitinib was 3 (2-6) weeks. The median follow-up time was 14 months (1 week-24 months). Five children were diagnosed with EBV-related HLH, two with familial HLH, one with autoinflammatory disease-related HLH, and the other with unclear etiology. After the first-line treatment, seven patients had no remission (NR). The other two patients relapsed within 2-4 weeks after complete remission (CR). After oral administration of ruxolitinib, all patients' body temperatures decreased to the normal range within 48 h. At 1 week of receiving ruxolitinib, three patients (33.3%) achieved partial remission (PR). Five patients (55.6%) improved but did not achieve PR. One patient (11.1%) died. Compared with other causes of HLH, children with refractory or recurrent EBV-HLH had a poor response to ruxolitinib alone (Fisher's exact test, P = 0.048). Until the last follow-up, the three patients who achieved CR survived without recurrence. For children with recurrent or refractory HLH, ruxolitinib is a tolerable salvage therapy. Although some children could not achieve CR after one week of treatment, combination with chemotherapy could gain time for further treatment or bone marrow transplantation.
394	Could hemophagocytic lymphohistiocytosis be the core issue of severe COVID-19 cases?	32664932	COVID-19, a disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), commonly presents as fever, cough, dyspnea, and myalgia or fatigue. Although the majority of patients with COVID-19 have mild symptoms, some are more prone to serious outcomes, including pneumonia, acute respiratory distress syndrome (ARDS), and even death. Hemophagocytic lymphohistiocytosis (HLH) is a severe, life-threatening inflammatory syndrome associated with intense cytokine release (also known as a "cytokine storm"). Similar to COVID-19, HLH is characterized by aggressive course leading to multi-organ failure.The purpose of this review article is to draw attention to the possibility of the complication of HLH in patients with the severe course of COVID-19. Indeed, some of the clinical characteristics observed in the more severe cases of COVID-19 are reminiscent of secondary HLH (which can be triggered by infections, malignancies, rheumatological diseases, or autoimmune/immunodeficiency conditions). The pathogenesis of SARS-CoV-2 infection also suggests that HLH or a similar hyperinflammatory syndrome is the cause of the severe course of the infection.The pathogenesis and clinical symptoms of severe COVID-19 indicate that an increased inflammatory response corresponding to HLH is occurring. Therefore, patients with severe COVID-19 should be screened for hyperinflammation using standard laboratory tests to identify those for whom immunosuppressive therapy may improve outcomes.
395	Familial Hemophagocytic Lymphohistiocytosis Type 2 in a Chinese Infant with PRF1 Homozygous Mutation: a Case Report.	32658436	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and histiocytes. Familial HLH (fHLH) is an autosomal recessive disease.We report a case of fHLH in a 45-day-old Chinese female infant presenting with fever, hepatosplenomegaly, and pancytopenia. Typical laboratory findings were detected for the infant, and fHLH was diagnosed according to the HLH-2004 guidelines. The infant was initially treated with antibiotics and ganciclovir prior to diagnosis of HLH. No response to glucocorticoid and intravenous immunoglobulin treatment was observed, and the infant died of disseminated intravascular coagulation.A mutation in the perforin gene was identified in this patient. Direct sequencing analysis revealed a deleterious homozygous mutation of PRF1, c.65delC [p.P22Rfs*29], and her parents were heterozygous carriers of the mutant alleles.Therefore, familial HLH is still a potentially lethal childhood illness, and appropriate individualized therapies, including cell therapy and gene targeting therapy, need to be established.
396	Indian National Association for the Study of the Liver Consensus Statement on Acute Liver Failure (Part 1): Epidemiology, Pathogenesis, Presentation and Prognosis.	32655238	Acute liver failure (ALF) is an infrequent, unpredictable, potentially fatal complication of acute liver injury (ALI) consequent to varied etiologies. Etiologies of ALF as reported in the literature have regional differences, which affects the clinical presentation and natural course. In this part of the consensus article designed to reflect the clinical practices in India, disease burden, epidemiology, clinical presentation, monitoring, and prognostication have been discussed. In India, viral hepatitis is the most frequent cause of ALF, with drug-induced hepatitis due to antituberculosis drugs being the second most frequent cause. The clinical presentation of ALF is characterized by jaundice, coagulopathy, and encephalopathy. It is important to differentiate ALF from other causes of liver failure, including acute on chronic liver failure, subacute liver failure, as well as certain tropical infections which can mimic this presentation. The disease often has a fulminant clinical course with high short-term mortality. Death is usually attributable to cerebral complications, infections, and resultant multiorgan failure. Timely liver transplantation (LT) can change the outcome, and hence, it is vital to provide intensive care to patients until LT can be arranged. It is equally important to assess prognosis to select patients who are suitable for LT. Several prognostic scores have been proposed, and their comparisons show that indigenously developed dynamic scores have an edge over scores described from the Western world. Management of ALF will be described in part 2 of this document.
397	Hemophagocytic Lymphohistiocytosis: Lessons Learned from the Dark Side.	32654694	Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe form of immune dysregulation often presenting as unremitting fever, cytopenia, hepatosplenomegaly, coagulopathy, and elevation of typical HLH biomarkers. HLH is universally fatal, if left untreated. The HLH-2004 criteria are widely used to diagnose this condition, but there is growing concerns across different settings that its application may result in undertreatment of certain patients. There is an expanding spectrum of genetic conditions that can be complicated by HLH. This review summarizes the current concepts in HLH, the lessons learned from the past, and provide an overview of the latest diagnostic and treatment modalities.
398	[Clinical study of haploidentical hematopoietic stem cell transplantation on 15 cases of adult-onset primary hemophagocytic lymphohistiocytosis].	32654467	Objective: This study was designed to evaluate the efficacy of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) for adult-onset primary hemophagocytic lymphohistiocytosis (HLH) . Method: A retrospective study was carried out to analyze the clinical data of 15 adult patients with primary HLH who received haplo-HSCT from January 2013 to October 2019 in Beijing Friendship Hospital, Capital Medical University, Beijing, China. Results: Among the 15 patients included in the study, ten were males and five were females, with a median age of 21 years old (18-52) . Eight of the patients had familial hemophagocytic lymphohistiocytosis type 2 (FHL-2) , four had FHL-3, one had Griscelli syndrome type 2 (GS-2) , one had X-linked lymphoproliferative disease type 1 (XLP-1) , and the other had XLP-2. The median time from HLH diagnosis to transplantation was 7 months (2-46 months) . Seven patients were treated with Bu/Cy condition regimen prior to transplantation. Meanwhile, the other eight cases were treated with TBI/Cy. The median concentration of mononuclear cell (MNC) infusion was 12.6 (9.2-20.3) ×10(8)/kg and CD34(+) cells was 4.91 (2.51-8.37) ×10(6)/kg. The median time of leukocyte engraftment was on day 13 following transplantation (10-23 days) , and the platelet engraftment was on day 12 (9-36) . Graft failure (GF) finally occurred in two patients (one primary GF and one secondary GF) . The cumulative incidence of acute graft-versus-host-disease (GVHD) grades 2 to 4 was 71.4% (10/14) and chronic GVHD was 30.8% (4/13) , respectively. The five-year overall survival (OS) for all 15 cases of primary HLH was 65.5% (95% CI, 34.9%-73.3%) and the transplant-related mortality (TRM) was 26.7% (4/15) . The five-year OS was 87.5% (95% CI, 38.7%-66.3%) in eight patients who received haplo-HSCT subsequent to initial therapy and 42.9% (95% CI, 8.5%-65.2%) in patients seven patients who needed salvage therapy prior to haplo-HSCT (χ(2)=2.387, P=0.122) . The five-year OS was 85.7% (95% CI, 50.4%-89.8%) in eight patients who achieved complete response before haplo-HSCT and 42.9% (95% CI, 6.4%-53.0%) in seven patients with partial response (χ(2)=3.185, P=0.074) . Conclusion: The results indicated that haplo-HSCT is a promising method for the treatment of primary HLH in adults.目的： 探讨单倍型造血干细胞移植（haplo-HSCT）治疗成人原发性噬血细胞综合征（HLH）的疗效。 方法： 对2013年1月至2019年10月期间于首都医科大学附属北京友谊医院接受haplo-HSCT的15例成人原发性HLH患者进行回顾性分析。 结果： 全部15例患者中男10例、女5例，中位年龄21（18~52）岁。家族性噬血细胞综合征2型（FHL-2）8例，FHL-3 4例，Griscelli综合征2（GS-2）、X连锁淋巴组织增生综合征1型（XLP-1）、XLP-2各1例。确诊HLH至接受haplo-HSCT的中位时间为7（2~46）个月。所有患者移植前均接受HLH-94、HLH-2004或DEP方案诱导化疗，8例获得完全应答（CR），7例获得部分应答（PR）。移植预处理：Bu/Cy方案7例，TBI/Cy方案8例。输注单个核细胞12.6（9.2~20.3）×10(8)/kg，CD34(+)细胞4.91（2.51~8.37）×10(6)/kg。粒细胞植活中位时间为13（10~23）d，血小板植活中位时间为12（9~36）d。原发、继发性植入失败各1例。Ⅱ~Ⅳ度急性移植物抗宿主病（GVHD）发生率为71.4%（10/14），慢性GVHD发生率为30.8%（4/13）。移植后预期5年总生存（OS）率为65.5%（95%CI 34.9%~73.3%），移植相关死亡率为26.7%（4/15）。一线诱导治疗后立即接受移植（8例）、二线诱导治疗难治/复发患者（7例）的移植后预期5年OS率分别为87.5%（95%CI 38.7%~66.3%）、42.9%（95%CI 8.5%~65.2%）（χ(2)=2.387，P=0.122）。移植前CR（8例）、PR（7例）患者的移植后预期5年OS率分别为85.7%（95%CI 50.4%~89.8%）、42.9%（95%CI 6.4%~53.0%）（χ(2)=3.185，P=0.074）。 结论： haplo-HSCT是成人原发性HLH的有效治疗手段。.
399	[Clinical characteristics and prognostic analysis of advanced-stage extranodal NK/T cell lymphoma].	32654458	Objective: This study aimed to explore the clinical characteristics, survival rate, and prognostic factors of advanced-stage extranodal nasal type NK/T cell lymphoma (ENKTL) patients. Methods: The clinical data of 51 advanced-stage ENKTL patients in Peking Union Medical College Hospital from January 2012 to September 2019 were retrospectively analyzed. The clinical characteristics, treatment responses, survival rate, and prognostic factors were elucidated. The differences between nasal and non-nasal type and the significance of EBV-DNA in treatment response assessment and prognosis analysis were also evaluated. Results: The male-to-female ratio in the whole group was 2.9∶1 with a median age of 42 years old (range, 14-67 years) . The median follow-up time was 30 months (range, 1-78 months) . The one- and three-year progression-free survival (PFS) rates for the whole cohort were 34.1% and 24.6%, respectively, and the one- and three-year overall survival (OS) rates were 39.9% and 26.6%, respectively. The ratio of nasal to non-nasal type was 1.6∶1. The proportion of hemophagocytic lymphohistiocytosis (HLH) was significantly higher in non-nasal-type patients than nasal-type (P=0.039) , and the complete response (CR) rate of first-line chemotherapy is significantly lower in non-nasal type patients (P=0.008) . The median OS for nasal and non-nasal types were nine months and four months, respectively. The three-year PFS rates of nasal and non-nasal type patients were 36.0% and 10.0% (P=0.029) , respectively, and the three-year OS rates were 37.9% and 11.4% (P=0.050) , respectively. The correlation between the Epstein-Barr virus DNA (EBV-DNA) and treatment response were satisfactory. Survival curve between baseline EBV-DNA-negative and EBV-DNA-positive patients showed no significant difference. The three-year OS rates of EBV-DNA-negative and EBV-DNA-positive patients after one cycle of treatment were 77.9% and 8.1% (P=0.002) , respectively. In a multivariate analysis, EBV-DNA-positive following one cycle of treatment was an independent adverse prognostic factor for OS. Conclusions: The efficacy of pegaspargase-based chemotherapy and long-term survival of advanced-stage ENKTL patients were still poor. Clinical characteristics, treatment response, and long-term survival of non-nasal-type patients were worse than that of nasal-type patients. In a multivariate analysis, EBV-DNA-positive after one cycle of treatment was an independent adverse prognostic factor for OS. It can be used for early prediction of treatment response and prognosis.目的： 探讨非局限期结外鼻型NK/T细胞淋巴瘤（ENKTL）患者的临床特征及预后影响因素。 方法： 回顾性分析北京协和医院2012年1月至2019年12月收治的51例Ⅲ~Ⅳ期ENKTL患者的临床资料。分析其临床特征、疗效、生存情况及预后影响因素，依据起病时是否存在鼻咽部受累分为鼻型与非鼻型，对比分析二者的差异，探讨EB病毒DNA（EBV-DNA）在疗效监测及预后评价中的意义。 结果： 所有患者的中位发病年龄为42（14~67）岁，男女比例为2.9∶1。中位随访时间为30（1~78）个月，非局限期患者中位总生存（OS）时间为7个月，1年及3年无进展生存（PFS）率分别为34.1%和24.6%，1年及3年OS率分别为39.9%和26.6%。鼻型与非鼻型患者的比例为1.6∶1。非鼻型患者合并噬血细胞综合征的比例较鼻型患者高（65.0%对35.5%，P=0.039），非鼻型患者一线化疗的完全缓解（CR）率较鼻型患者低（10.0%对45.2%，P=0.008）。非局限期鼻型与非鼻型患者的中位OS时间分别为9个月和4个月，3年PFS率分别为36.0%和10.0%（P=0.029），3年OS率分别为37.9%和11.4%（P=0.050）。EBV-DNA的变化趋势与疗效有良好的相关性。基线EBV-DNA阴性与阳性患者生存曲线的差异无统计学意义。1个疗程化疗后EBV-DNA阴性与阳性患者的3年OS率分别为77.9%和8.1%，差异有统计学意义（P=0.002）。多因素分析结果显示，1个疗程化疗后EBV-DNA阳性为OS不良的独立预后因素。 结论： 应用以培门冬酶为基础化疗方案的非局限期ENKTL患者的治疗有效率及远期生存仍较差。非鼻型患者在临床特征、化疗疗效及远期预后方面均较鼻型患者差。1个疗程化疗后EBV-DNA阳性为OS不良的独立预后因素，可用于早期预测疗效及预后。.
400	Emapalumab for the treatment of hemophagocytic lymphohistiocytosis.	32648854	Emapalumab-Igsz (Gamifant) is a human monoclonal antibody directed against interferon-γ (IFN-γ), and the first Food and Drug Administration (FDA)-approved therapy for primary hemophagocytic lymphohistiocytosis (HLH). HLH is a disorder characterized by hypercytokinemia in the setting of unbridled immune activation, and emapalumab represents the first therapeutic developed to address the underlying pathophysiology of HLH. Emapalumab is approved for treatment of primary HLH that is refractory, recurrent, progressing or intolerant to current HLH treatments in both adult and pediatric patients. FDA approval was based on the results of a phase II/III clinical trial evaluating the safety and efficacy of emapalumab in 34 pediatric patients with primary HLH, 27 of whom were refractory to current therapies. Additional studies of emapalumab are currently ongoing in adults and other pediatric populations. Here, we will review the pharmacology, safety and efficacy of emapalumab for the treatment of HLH.
401	Epstein Virus Barr-Positive Diffuse Large B-Cell Lymphoma Associated with Hemophagocytic Lymphohistiocytosis.	32647871	Hemophagocytic lymphohistiocytosis (HLH) is a rare and often fatal disease if not diagnosed and treated promptly. HLH can be due to genetic factors or infections, malignancies and collagen-associated vascular diseases. Malignancy-associated HLH is not only more common in the setting of T/NK-cell lymphomas, but may also rarely be seen in the setting of B-cell lymphoma. Here, we describe a unique case of a patient who initially was diagnosed with HLH secondary to Epstein Barr virus (EBV) infection and subsequently developed EBV-positive diffuse large B-cell lymphoma affecting the brain. This case highlights the spectrum of findings associated with EBV infections and the challenges in diagnosing underlying diseases associated with HLH.
402	A Diagnostic Dilemma of Secondary Hemophagocytosis Lymphohistiocytosis in an Elderly Patient.	32642386	Hemophagocytic lymphohistiocytosis (HLH) is a rapidly progressive fatal condition. Although well described in the pediatric population, cases of secondary HLH are seen in adolescents and young adults. In the elderly, HLH has been shown to have a poor prognosis. Owing to its varied presentation and multisystemic involvement, diagnosis is often delayed. Due to its high mortality, prompt diagnosis and treatment are crucial. Here we present a case of secondary HLH in a 69-year-old male, who presented with fever for one week. Initial laboratory workup revealed a bicytopenia and elevated creatinine. He was initially treated with broad-spectrum antibiotics; however, a comprehensive infectious workup was negative. CT scan of the abdomen revealed splenomegaly. Further investigations revealed an elevated ferritin and triglycerides. Due to the constellation of findings, he was started on corticosteroids for concerns of HLH. Bone marrow biopsy was obtained, which revealed dysplastic changes and hemophagocytosis, consistent with HLH. This case highlights the diagnostic challenge and prognosis of HLH in the elderly population, suggesting that diagnosis and treatment should not be delayed for histological confirmation.
403	HEMOPHAGOCYTOSIS BY BLASTS IN A CHILD WITH ACUTE MONOCYTIC LEUKEMIA AFTER CHEMOTHERAPY.	32638943	To describe the case of a child who presented hemophagocytic lymphohistiocytosis (HLH) associated with acute monocytic leukemia after chemotherapy, with hemophagocytosis caused by leukemic cells.In a university hospital in Southern Brazil, a 3-year-old female was diagnosed with acute monocytic leukemia with normal karyotype. The chemotherapy regimen was initiated, and she achieved complete remission six months later, relapsing after four months with a complex karyotype involving chromosomes 8p and 16q. The bone marrow showed vacuolated blasts with a monocytic aspect and evidence of hemophagocytosis. The child presented progressive clinical deterioration and died two months after the relapse.HLH is a rare and aggressive inflammatory condition characterized by cytopenias, hepatosplenomegaly, fever, and hemophagocytosis in the bone marrow, lymph nodes, spleen, and liver. Although rare, malignancy-associated HLH (M-HLH) is fatal. The patient in this case report met five out of the eight established criteria for HLH. The evolution of the patient's karyotype, regardless of the diagnostic profile, seemed secondary to the treatment for acute monocytic leukemia. In this case, the cytogenetic instability might have influenced the abnormal behavior of leukemic cells. This is a rare case of HLH in a child with acute monocytic leukemia.
404	Neuroinflammatory Disease as an Isolated Manifestation of Hemophagocytic Lymphohistiocytosis.	32638196	Isolated neuroinflammatory disease has been described in case reports of familial hemophagocytic lymphohistiocytosis (FHL), but the clinical spectrum of disease manifestations, response to therapy and prognosis remain poorly defined. We combined an international survey with a literature search to identify FHL patients with (i) initial presentation with isolated neurological symptoms; (ii) absence of cytopenia and splenomegaly at presentation; and (iii) systemic HLH features no earlier than 3 months after neurological presentation. Thirty-eight (20 unreported) patients were identified with initial diagnoses including acute demyelinating encephalopathy, leukoencephalopathy, CNS vasculitis, multiple sclerosis, and encephalitis. Median age at presentation was 6.5 years, most commonly with ataxia/gait disturbance (75%) and seizures (53%). Diffuse multifocal white matter changes (79%) and cerebellar involvement (61%) were common MRI findings. CSF cell count and protein were increased in 22/29 and 15/29 patients, respectively. Fourteen patients progressed to systemic inflammatory disease fulfilling HLH-2004 criteria at a mean of 36.9 months after initial neurological presentation. Mutations were detected in PRF1 in 23 patients (61%), RAB27A in 10 (26%), UNC13D in 3 (8%), LYST in 1 (3%), and STXBP2 in 1 (3%) with a mean interval to diagnosis of 28.3 months. Among 19 patients who underwent HSCT, 11 neurologically improved, 4 were stable, one relapsed, and 3 died. Among 14 non-transplanted patients, only 3 improved or had stable disease, one relapsed, and 10 died. Isolated CNS-HLH is a rare and often overlooked cause of inflammatory brain disease. HLH-directed therapy followed by HSCT seems to improve survival and outcome.
405	Hemocyanins from Helix and Rapana Snails Exhibit in Vitro Antitumor Effects in Human Colorectal Adenocarcinoma.	32635655	Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of arthropods and mollusks that attract scientific interest with their diverse biological activities and potential applications in pharmacy and medicine. The aim of the present study was to assess the in vitro antitumor activity of hemocyanins isolated from marine snail Rapana venosa (RvH) and garden snails Helix lucorum (HlH) and Helix aspersa (HaH), as well the mucus of H. aspersa snails, in the HT-29 human colorectal carcinoma cell line. The effects of the hemocyanins on the cell viability and proliferation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the alterations in the tumor cell morphology were examined by fluorescent and transmission electron microscopy. The results of the MTT assay showed that the mucus and α-subunit of hemocyanin from the snail H. aspersa had the most significant antiproliferative activity of the tested samples. Cytomorphological analysis revealed that the observed antitumor effects were associated with induction of apoptosis in the tumor cells. The presented data indicate that hemocyanins and mucus from H. aspersa have an antineoplastic activity and potential for development of novel therapeutics for treatment of colorectal carcinoma.
406	Risk factors for mixed chimerism in children with hemophagocytic lymphohistiocytosis after reduced toxicity conditioning.	32618429	Reduced toxicity conditioning for hematopoietic stem cell transplantation of patients with hemophagocyticlymphohistiocytosis (HLH) results in favorable survival, however at the expense of relevant rates of mixed chimerism. Factors predisposing to mixed chimerism remain to be determined.Patients with primary HLH transplanted 2009-2016 after treosulfan- or melphalan-based conditioning regimens were analyzed in a retrospective multicenter study for survival, engraftment, chimerism, and adverse events. Mixed chimerism was considered substantial if < 25% donor chimerism occurred and/or if secondary cell therapy was administered. Donor type, graft source, type of alkylating agent, type of serotherapy, and remission status were analyzed as potential risk factors in a multivariable logistic regression model.Among 60 patients, engraftment was achieved in 95%, and the five-year estimated overall survival rate was 75%. Prevalence of any recipient chimerism was 48%. Substantial recipient chimerism was recorded in 32% of patients. Secondary post-HSCT cell therapy was administered in 30% of patients. A human leukocyte antigen (HLA)-mismatched donor (< 10/10) was the only significant risk factor for the occurrence of substantial recipient chimerism (P = 0.01; odds ratio, 5.8; CI 95%, 1.5-26.3).The use of an HLA-matched donor is the most important factor to avoid substantial recipient chimerism following treosulfan -or melphalan-based conditioning in primary HLH.
407	Ruxolitinib combined with doxorubicin, etoposide, and dexamethasone for the treatment of the lymphoma-associated hemophagocytic syndrome.	32617699	Case reports suggest that ruxolitinib-containing treatment could increase the clinical response rate of patients with hemophagocytic syndrome (HPS). This study aimed to explore the effect of ruxolitinib-containing treatment for patients with lymphoma-associated hemophagocytic syndrome (LAHS).This was a retrospective study of patients with LAHS hospitalized at the First Affiliated Hospital of Guangdong Pharmaceutical University between October 2017 and September 2019. Patients were treated with HLH-94 (etoposide and dexamethasone) or R-DED regimen (ruxolitinib, doxorubicin, etoposide, and dexamethasone). The clinical characteristics, treatment responses, and overall survival (OS) were compared. The patients were divided into the HLH-94 group (n = 34) and the R-DED group (n = 36).Compared with HLH-94, R-DED might effectively improve the clinical manifestations, including fever and splenomegaly in patients with LAHS, and control the systemic cytokine storm. The response rate at 2 weeks was 54.8% in the HLH-94 group, which was lower than in the R-DED group (83.3%) (p = 0.011). The OS was significantly prolonged in the R-DED group compared with the HLH-94 group (median, 5 vs. 1.5 months, p = 0.003). The multivariable analysis showed that lower IL-10 levels [hazard ratio (HR)] = 1.000, [95% confidence interval (CI)] 1.000-1.000, p = 0.012), R-DED regimen (HR = 0.196, 95% CI 0.084-0.457, p < 0.001), and non-NK/T-cell lymphoma (HR = 0.254, 95% CI 0.102-0.628, p = 0.003) were associated with better OS. The prognosis of patients with LAHS was generally poor.Ruxolitinib can be combined with chemotherapy in HPS. It is feasible, with no early signals of increased toxicity.
408	Haemophagocytic lymphohistiocytosis after heart transplantation: a case report.	32617508	Haemophagocytic lymphohistiocytosis (HLH) is an uncommon but serious systemic inflammatory response with high mortality rates. It can be triggered by malignancy or infectious agents, often in the context of immunosuppression. Literature covering HLH in heart transplantation (HTx) is scarce.A 25-year-old male with a history of celiac disease underwent HTx at Sahlgrenska Hospital in 2011 due to giant cell myocarditis and was treated with tacrolimus, mycophenolate mofetil (MMF), and prednisolone. He developed several episodes of acute cellular rejections (ACR) during the first 3 post-HTx years, which subsided after addition of everolimus. In May 2017, the patient was admitted to the hospital due to fever without focal symptoms. He had an extensive inflammatory reaction, but screening for infectious agents was negative. Haemophagocytic lymphohistiocytosis was discussed early, but first dismissed since two bone marrow biopsies revealed no signs of haemophagocytosis. Increasing levels of soluble IL-2 were considered confirmative of the diagnosis. Even with intense immunosuppressant treatment, the patient deteriorated and died in progressive multiorgan failure within 2 weeks of the symptom onset.A 25-year-old HTx recipient with an extensive inflammatory response, fulfilled criteria for HLH, but the diagnosis was delayed due to normal bone marrow biopsies. A background with autoimmune reactivity and immunosuppressive therapy may have contributed to HLH, but the actual trigger was not identified. Haemophagocytic lymphohistiocytosis can occur in HTx recipients in the absence of malignancy, identifiable infectious triggers and signs of haemophagocytosis. Early diagnosis and intervention are likely to be of importance for a favourable outcome.
409	HLA-mismatched GPBSC infusion therapy in refractory Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis: an observational study from a single center.	32611452	Hemophagocytic lymphohistiocytosis (HLH) is a severe or even fatal inflammatory state. Epstein-Barr virus (EBV) infection-associated HLH (EBV-HLH) is one of the most common secondary HLH and suffers a very poor prognosis. Allo-HSCT is often required for refractory EBV-HLH, but some patients still cannot proceed to the next allo-HSCT due to various factors. This study aimed to observe the efficacy of HLA-mismatched granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (GPBSCs) infusion for refractory EBV-HLH.A retrospective case-control study of refractory EBV-HLH patients with GPBSC infusion from HLA-mismatched donors after chemotherapy (as GPBSC group) and sole chemotherapy (as control group) was performed. Efficacy was evaluated 2 and 4 weeks and all patients were followed-up until March 1, 2018.There were 18 cases who accepted infusion between March 2016 and Sep 2017 and 19 were randomly selected from refractory EBV-HLH patients who underwent salvage therapy during the same period for the control group. In GPBSC group, WBC (p = 0.017), Fbg (p = 0.040), and ferritin (p = 0.039) improved significantly after treatment. The overall response rate was 66.7% (CR 22.2%, PR 44.4%). However, there are no significant differences in changes of WBC, HGB, PLT, TG, Fbg, Ferritin, AST, ALT, and T-bil between two groups. Only the Fbg level was recovered better in the GPBSC infusion group (p = 0.003). In the GPBSC group, EBV-DNA decreased significantly after 2 weeks (p = 0.001) and 4 weeks (p = 0.012) after treatment, and the effect of the decrease was significantly better than that of the chemotherapy alone group in 2 weeks but not 4 weeks (p2w = 0.011, p4w = 0.145). The median survival time in the infusion group was 20.4 weeks [95% CI 10.9, 29.9], and the median survival time in the control group was 10.8 weeks [95% CI 0-24.34]. In the short-term, the infusion group's survival rate was better (2-month 88.89% vs. 52.63%, p = 0.008; 3-month 83.33% vs. 47.09%, p = 0.012), but there was no difference in OS (p = 0.287).Infusing GPBSCs combined with chemotherapy is effective, especially in decreasing EBV-DNA, performs better than chemotherapy alone, and improves short-term survival rate. GPBSC infusion is suggested as a bridging treatment method to allo-HSCT.
410	Extended clinical and immunological phenotype and transplant outcome in CD27 and CD70 deficiency.	3260343133270851	Biallelic mutations in the genes encoding CD27 or its ligand CD70 underlie inborn errors of immunity (IEIs) characterized predominantly by Epstein-Barr virus (EBV)-associated immune dysregulation, such as chronic viremia, severe infectious mononucleosis, hemophagocytic lymphohistiocytosis (HLH), lymphoproliferation, and malignancy. A comprehensive understanding of the natural history, immune characteristics, and transplant outcomes has remained elusive. Here, in a multi-institutional global collaboration, we collected the clinical information of 49 patients from 29 families (CD27, n = 33; CD70, n = 16), including 24 previously unreported individuals and identified a total of 16 distinct mutations in CD27, and 8 in CD70, respectively. The majority of patients (90%) were EBV+ at diagnosis, but only ∼30% presented with infectious mononucleosis. Lymphoproliferation and lymphoma were the main clinical manifestations (70% and 43%, respectively), and 9 of the CD27-deficient patients developed HLH. Twenty-one patients (43%) developed autoinflammatory features including uveitis, arthritis, and periodic fever. Detailed immunological characterization revealed aberrant generation of memory B and T cells, including a paucity of EBV-specific T cells, and impaired effector function of CD8+ T cells, thereby providing mechanistic insight into cellular defects underpinning the clinical features of disrupted CD27/CD70 signaling. Nineteen patients underwent allogeneic hematopoietic stem cell transplantation (HSCT) prior to adulthood predominantly because of lymphoma, with 95% survival without disease recurrence. Our data highlight the marked predisposition to lymphoma of both CD27- and CD70-deficient patients. The excellent outcome after HSCT supports the timely implementation of this treatment modality particularly in patients presenting with malignant transformation to lymphoma.
411	Hemophagocytic Lymphohistiocytosis and Inflammatory Bowel Disease: Case Report and Systematic Review.	32596254	To report a case of a female patient with hemophagocytic lymphohistiocytosis (HLH) and to systematically review the available cases of the association between HLH and inflammatory bowel disease (IBD).In accordance to Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines, retrieval of studies was based on Medical Subject Headings and Health Sciences Descriptors, which were combined using Boolean operators. Searches were run on the electronic databases Scopus, Web of Science, MEDLINE (PubMed), Biblioteca Regional de Medicina, Latin American and Caribbean Health Sciences Literature, Cochrane Library for Systematic Reviews and Opengrey.eu. Languages were restricted to English, Spanish and Portuguese. There was no date of publication restrictions. The reference lists of the studies retrieved were searched manually.The search strategy retrieved 223 references. In the final analysis, 28 references were included, with the report of 35 cases. The most common clinical finding was fever, 57% of patients had a cytomegalovirus infection and 30 patients were on thiopurines previously to HLH diagnosis. Most patients were treated with steroids and antiviral therapy. All-cause mortality was 22%.These findings suggest that there might be a connection of HLH to IBD, opportunistic viral infections and the use of thiopurines. Due to the severity of such disease, the clinical suspicion is paramount to early diagnosis and therapy.
412	A Rare Case of Measles-Associated Hemophagocytic Lymphohistiocytosis in an Infant.	32596066	Measles continues to be a threat in most European countries due to suboptimum vaccination coverage. Although measles leads to several complications, measles-related hemophagocytic lymphohistiocytosis (HLH) has been rarely reported. Herein, we present a case of a four-month-old male infant, the first child of unrelated, healthy parents, with no significant medical history or unexplained infant death in the family, otherwise healthy, who was diagnosed with measles-associated HLH and was successfully treated with IV dexamethasone and IV immunoglobulin (IVIG). Additionally, we review previously reported cases of HLH secondary to measles and highlight the diagnostic and therapeutic challenges associated with its early recognition and treatment. High suspicion, early recognition, and appropriate treatment are essential for a favorable outcome of measles-associated HLH.
413	[The clinical significance of plasma PTX3 in patients with secondary hemophagocytic lymphohistiocytosis].	32594686	Objective: To investigate the significance of plasma pentraxin 3 (PTX3) in patients with secondary hemophagocytic lymphohistiocytosis (sHLH). Methods: Plasma PTX3 levels were tested by ELISA in 48 newly diagnosed sHLH patients, 18 healthy volunteers and 9 lymphoma controls in the First Affiliated Hospital of Nanjing Medical University from January 2017 to July 2019. Clinical parameters were collected, and the correlations with PTX3 levels were analyzed. Results: PTX3 level in newly diagnosed group was significantly higher than that of healthy control group [16.29(1.17-66.00) vs. 0.76(0.01-7.86) μg/L, P<0.01]. Patients with lymphoma-associated HLH(LHLH) had higher plasma level of PTX3 than Fhose with infection-associated HLH (IHLH) [24.29(3.36-66.00) vs. 9.56(1.17-36.50)μg/L, P<0.05]. Plasma PTX3 levels in 48 sHLH patients were positively correlated with serum ferritin (P<0.05). Receiver operating characteristic (ROC) curve for plasma PTX3 levels of sHLH and healthy controls produced a cutoff value at 3.9 μg/L, with its 86.7% sensitivity and 94.4% specificity. And ROC analysis showed that PTX3 17.5 μg/L was the critical value for diagnosis of LHLH from non-LHLH group, that the sensitivity and specificity were 63.0% and 76.2% respectively. The 1-year overall survival (OS) rate in patients with PTX3≥17.5 μg/L was significantly lower in those with PTX3<17.5 μg/L (18.5% vs. 75.8%, P<0.01). Conclusion: These results indicate the potential of PTX3 as a biomarker for diagnosis and prognosis in patients with sHLH.目的： 探讨血浆PTX3在继发性噬血细胞性淋巴组织细胞增多症（sHLH）的表达水平及其临床意义。 方法： 回顾性分析2017年1月至2019年7月南京医科大学第一附属医院48例sHLH患者外周血浆标本，以18名健康体检者、9例淋巴瘤患者为对照，ELISA法检测血浆PTX3水平。收集患者相关实验室检查数据，并根据血浆PTX3的表达水平进行诊断及预后分析等。 结果： 初诊sHLH组血浆PTX3表达水平明显高于健康对照组[16.29（1.17~66.00）比0.76（0.01~7.86）μg/L, P<0.01]；初诊淋巴瘤相关HLH组（LHLH）血浆PTX3水平明显高于感染相关HLH组（IHLH）[24.29（3.36~66.00）比9.56（1.17~36.50）μg/L, P<0.05]。48例sHLH患者血浆PTX3水平与血清铁蛋白正相关（P<0.05）。血浆PTX3以3.9 μg/L为最佳临界值时，其判断sHLH患者及健康对照组的敏感度及特异度分别为86.7%及94.4%；血浆PTX3以17.5 μg/L为最佳临界值时，其判断LHLH组与non-LHLH组的敏感度及特异度分别为63.0%及76.2%。对初诊sHLH组进行预后分析，多因素分析显示PTX3≥17.5 μg/L组1年总生存率（18.5%）与PTX3<17.5 μg/L组（75.8%）相比，差异有统计学意义（P<0.01）。 结论： 血浆PTX3在sHLH患者的诊断、LHLH鉴别诊断、预后分析等方面具有重要的临床意义。.
414	Interleukin-18 and cytotoxic impairment are independent and synergistic causes of murine virus-induced hyperinflammation.	3258970733152085	Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes typically associated with underlying hematologic and rheumatic diseases, respectively. Familial HLH is associated with genetic cytotoxic impairment and thereby to excessive antigen presentation. Extreme elevation of serum interleukin-18 (IL-18) has been observed specifically in patients with MAS, making it a promising therapeutic target, but how IL-18 promotes hyperinflammation remains unknown. In an adjuvant-induced MAS model, excess IL-18 promoted immunopathology, whereas perforin deficiency had no effect. To determine the effects of excess IL-18 on virus-induced immunopathology, we infected Il18-transgenic (Il18tg) mice with lymphocytic choriomeningitis virus (LCMV; strain Armstrong). LCMV infection is self-limited in wild-type mice, but Prf1-/- mice develop prolonged viremia and fatal HLH. LCMV-infected Il18-transgenic (Il18tg) mice developed cachexia and hyperinflammation comparable to Prf1-/- mice, albeit with minimal mortality. Like Prf1-/- mice, immunopathology was largely rescued by CD8 depletion or interferon-γ (IFNg) blockade. Unlike Prf1-/- mice, they showed normal target cell killing and normal clearance of viral RNA and antigens. Rather than impairing cytotoxicity, excess IL-18 acted on T lymphocytes to amplify their inflammatory responses. Surprisingly, combined perforin deficiency and transgenic IL-18 production caused spontaneous hyperinflammation specifically characterized by CD8 T-cell expansion and improved by IFNg blockade. Even Il18tg;Prf1-haplosufficient mice demonstrated hyperinflammatory features. Thus, excess IL-18 promotes hyperinflammation via an autoinflammatory mechanism distinct from, and synergistic with, cytotoxic impairment. These data establish IL-18 as a potent, independent, and modifiable driver of life-threatening innate and adaptive hyperinflammation and support the rationale for an IL-18-driven subclass of hyperinflammation.
415	Hemophagocytic lymphohistiocytosis: a review inspired by the COVID-19 pandemic.	32588191	Hemophagocytic syndrome (HPS) or hemophagocytic lymphohistiocytosis (HLH) is an acute and rapidly progressive systemic inflammatory disorder characterized by cytopenia, excessive cytokine production, and hyperferritinemia. Common clinical manifestations of HLH are acute unremitting fever, lymphadenopathy, hepatosplenomegaly, and multiorgan failure. Due to a massive cytokine release, this clinical condition is considered as a cytokine storm syndrome. HPS has primary and acquired (secondary, reactive) forms. Its primary form is mostly seen in childhood and caused by various mutations with genetic inheritance and, therefore, is called familial HLH. Secondary HLH may be caused in the presence of an underlying disorder, that is, secondary to a malignant, infectious, or autoimmune/autoinflammatory stimulus. This paper aims to review the pathogenesis and the clinical picture of HLH, and its severe complication, the cytokine storm, with a special emphasis on the developed classification criteria sets for rheumatologists, since COVID-19 infection has clinical symptoms resembling those of the common rheumatologic conditions and possibly triggers HLH. MED-LINE/Pubmed was searched from inception to April 2020, and the following terms were used for data searching: "hemophagocytic syndrome" OR "macrophage activation syndrome" OR "hemophagocytic lymphohistiocytosis", OR "cytokine storm". Finally, AND "COVID-19" was included in this algorithm. The selection is restricted to the past 5 years and limited numbers of earlier key references were manually selected. Only full-text manuscripts, published in an English language peer-reviewed journal were included. Manuscript selection procedure and numbers are given in Fig. 2. Briefly, the database search with the following terms of "Hemophagocytic syndrome" OR "Macrophage activation syndrome" OR "Hemophagocytic lymphohistiocytosis" OR "Cytokine storm" yielded 6744 results from inception to April 2020. The selection is restricted to the past 5 years and only limited numbers of earlier key references were selected, and this algorithm resulted in 3080 manuscripts. The addition of (AND "COVID-19") resulted in 115 publications of which 47 studies, together with four sections of an online book were used in the final review. No statistical method was used. HLH is triggered by genetic conditions, infections, malignancies, autoimmune-autoinflammatory diseases, and some drugs. In COVID-19 patients, secondary HLH and cytokine storm may be responsible for unexplained progressive fever, cytopenia, ARDS, neurological and renal impairment. Differentiation between the primary and secondary forms of HLH is utterly important, since primary form of HLH requires complicated treatments such as hematopoietic stem cell transplantation. Further studies addressing the performance of HScore and other recommendations in the classification of these patients is necessary.
416	Molecular characterization, inducible expression and functional analysis of an IKKβ from the tropical sea cucumber Holothuria leucospilota.	32585358	The inhibitory kappa B kinase (IKK) is a critical regulator for the nuclear factor-κB (NF-κB) pathway. In this study, an IKKβ named as HLIKKβ was identified from the sea cucumber Holothuria leucospilota. The full-length cDNA of HLIKKβ is 4246 bp in size, containing a 132 bp 5'-untranslated region (UTR), a 1783 bp 3'-UTR and a 2331 bp open reading frame (ORF) encoding a protein of 776 amino acids with a deduced molecular weight of 89.66 kDa. HLIKKβ contains a kinase domain (KD) at its N-terminal, a leucine zipper (LZ) and a helix-loop-helix (HLH) motif at its C-terminal. In the KD, a conserved active loop (SXXXS) were identified. The results of luciferase reporter assay and ELISA assay showed that over-expressed HLIKKβ in HEK293T cells could activate the nuclear factor-κB (NF-κB) and induce the secretion of proinflammatory cytokines TNF-α and IL-1β. When HLIKKβ was silenced by siRNA, the apoptosis rate of sea cucumber coelomocytes was increased significantly, indicating the anti-apoptotic function of HLIKKβ. Moreover, the up-regulation of HLIKKβ mRNA was observed in the sea cucumber coelomocytes after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge, suggesting that the HLIKKβ might play important roles in the innate immune defense of sea cucumber against the viral and bacterial infections.
417	COVID-19 Associated Hemophagocytic Lymphohistiocytosis and Coagulopathy: Targeting the Duumvirate.	32583809	Preliminary data on coexistence of secondary hemophagocytic lymphohistiocytosis syndrome (HLH) and disseminated intravascular coagulation (DIC) in critically ill children with novel coronavirus disease (COVID-19) are emerging. Herein, we summarize the available literature and fill-in the gaps in this regard.We have performed a literature search for articles in PubMed, EMBASE and Google Scholar databases till May 12, 2020, with following keywords: "COVID-19", "SARS-CoV-2", "HLH", "HScore", "coagulopathy", "D-dimer", "cytokine storm", "children" and "pediatrics" with interposition of Boolean operator "AND".Children presenting with moderate-severe COVID-19 and Kawasaki disease shock-like syndrome exhibit peripheral blood picture analogous to HLH. HScore, a validated tool to diagnose HLH, has been suggested to screen severe COVID-19 patients for cytokine storm. However, HScore faces certain limitations in this scenario. It may be more pragmatic to use 'high D-dimer' (> 3 µg/mL) instead of 'low fibrinogen' to facilitate early detection of cytokine storm. COVID-19 associated coagulopathy resembles hypercoagulable form of DIC with bleeding being rarely reported. Although the International Society on Thrombosis and Haemostasis (ISTH) interim guidance recommends low molecular weight heparin in all hospitalized patients, data is lacking in population below 14 years of age. However, in the presence of life-threatening thromboembolic event or symptomatic acro-ischemia, unfractionated heparin (UFH) should be used with caution.HScore can be used as a complement to clinical decision for initiating immunosuppression. Children with moderate-to-severe COVID-19, especially those with documented thrombocytopenia or chilblains, should be regularly monitored for coagulopathy.
418	Hemophagocytic lymphohistiocytosis secondary to visceral leishmaniasis in an endemic area in the north of Minas Gerais, Brazil.	32578702	Visceral leishmaniasis (VL) is an ill-studied disease that is endemic to several regions of Brazil. It is often complicated by hemophagocytic lymphohistiocytosis (HLH), a potentially fatal disorder resulting from excessive non-malignant activation/proliferation of T lymphocytes and macrophages. Considering the overlapping clinical and laboratory characteristics of these diseases, diagnosing HLH is a challenge. Therefore, tracking the association between VL and HLH is necessary in endemic areas. Although HLH can be inapparent and resolve with antileishmanicides, this may not always occur. HLH causes high lethality; therefore, immunosuppressive therapy should be instituted immediately in order to avoid a fatal outcome.We described the epidemiological, clinical, laboratory, and therapeutic profile of this association in a region of Brazil endemic for VL.We presented 39 patients with this association in a retrospective cohort of 258 children who were admitted from January 2012 to June 2017. Of the 39 patients, 31 were from urban areas (79.5%), and 21 (53%) were males. The mean age and weight were 2.86 (2.08) years and 14.03 (5.96) kg, respectively. The main symptoms were fever (100%), hepatosplenomegaly (100%), pallor of the skin and mucosa (82.5%), edema (38.5%), bleeding (25%), and jaundice (7.5%). Hemophagocytosis was identified in 16/37 (43.24%) patients, and direct examination revealed that 26/37 (70.27%) patients were positive for VL. The patients were treated as recommended by the Ministry of Health.It was observed that HLH is a common complication in endemic areas, and its diagnosis must consider the overlapping of clinical characteristics and pancytopenia.
419	[A genetic analysis of children with Epstein-Barr virus-positive hemophagocytic lymphohistiocytosis and its association with T-helper type 1/T-helper type 2 cytokines].	32571462	To study the effect of genetic variation on the prognosis of children with Epstein-Barr virus (EBV)-positive hemophagocytic lymphohistiocytosis (HLH) and its association with cytokines.A total of 81 EBV-positive HLH children who received the sequencing of related genes were enrolled. According to the results of gene detection, they were divided into a non-mutation group and a mutation group. According to the pattern of gene mutation, the mutation group was further divided into three subgroups: single heterozygous mutation (SHM), double heterozygous mutation (DHM), and homozygous or compound heterozygous mutation (H-CHM). The serum levels of cytokines were measured and their association with HLH gene mutations was analyzed.UNC13D gene mutation had the highest frequency (13/46, 28%). The STXBP2 c.575G>A(p.R192H) and UNC13D c.604C>A(p.L202M) mutations (likely pathogenic) were reported for the first time. The mutation group had a significantly higher level of tumor necrosis factor alpha (TNF-α) than the non-mutation group, while it had a significantly lower level of interferon gamma (IFN-γ) than the non-mutation group (P<0.05). The IL-4 level of the DHM subgroup was higher than that of the non-mutation group, while the IL-4 level of the H-CHM subgroup was lower than that of the DHM group (P<0.0083). The H-CHM subgroup had a significantly lower 1-year overall survival rate than the non-mutation group, the SHM subgroup, and the DHM subgroup (39%±15% vs 85%±6%/86%±7%/91%±9%, P=0.001).There is a significant reduction in IFN-γ level in the mutation group. Children with homozygous or compound heterozygous mutation tend to have poorer prognosis, while other mutations do not have a significant impact on prognosis.
420	Emerging trends in COVID-19 treatment: learning from inflammatory conditions associated with cellular therapies.	32565132	Coronavirus disease 2019 (SARS-CoV2) is an active global health threat for which treatments are desperately being sought. Even though most people infected experience mild to moderate respiratory symptoms and recover with supportive care, certain vulnerable hosts develop severe clinical deterioration. While several drugs are currently being investigated in clinical trials, there are currently no approved treatments or vaccines for COVID-19 and hence there is an unmet need to explore additional therapeutic options. At least three inflammatory disorders or syndromes associated with immune dysfunction have been described in the context of cellular therapy. Specifically, Cytokine Release Syndrome (CRS), Immune Reconstitution Inflammatory Syndrome (IRIS), and Secondary Hemophagocytic Lymphohistiocytosis (sHLH) all have clinical and laboratory characteristics in common with COVID19 and associated therapies that could be worth testing in the context of clinical trials. Here we discuss these diseases, their management, and potential applications of these treatment in the context of COVID-19. We also discuss current cellular therapies that are being evaluated for the treatment of COVID-19 and/or its associated symptoms.
421	Multilevel regulation of muscle-specific transcription factor hlh-1 during Caenorhabditis elegans embryogenesis.	32556563	hlh-1 is a myogenic transcription factor required for body-wall muscle specification during embryogenesis in Caenorhabditis elegans. Despite its well-known role in muscle specification, comprehensive regulatory control upstream of hlh-1 remains poorly defined. Here, we first established a statistical reference for the spatiotemporal expression of hlh-1 at single-cell resolution up to the second last round of divisions for most of the cell lineages (from 4- to 350-cell stage) using 13 wild-type embryos. We next generated lineal expression of hlh-1 after RNA interference (RNAi) perturbation of 65 genes, which were selected based on their degree of conservation, mutant phenotypes, and known roles in development. We then compared the expression profiles between wild-type and RNAi embryos by clustering according to their lineal expression patterns using mean-shift and density-based clustering algorithms, which not only confirmed the roles of existing genes but also uncovered the potential functions of novel genes in muscle specification at multiple levels, including cellular, lineal, and embryonic levels. By combining the public data on protein-protein interactions, protein-DNA interactions, and genetic interactions with our RNAi data, we inferred regulatory pathways upstream of hlh-1 that function globally or locally. This work not only revealed diverse and multilevel regulatory mechanisms coordinating muscle differentiation during C. elegans embryogenesis but also laid a foundation for further characterizing the regulatory pathways controlling muscle specification at the cellular, lineal (local), or embryonic (global) level.
422	[Expression and Clinical Significance of Soluble B7-H3 in Patient with Secondary Hemophagocytic Lymphohistiocytosis].	32552973	To investigate the expression and clinical significance of soluble B7-H3 (sB7-H3) in patients with secondary hemophagocytic lymphohistiocytosis (sHLH).The plasma samples of 85 newly diagnosed sHLH patients from December 2012 to April 2018 were collected. The patients were divided into lymphoma-related HLH（LHLH）group and infection-related HLH（IHLH）group. The expression of sB7-H3 in plasma was detected by ELISA, and the clinical data were collected for analysis. Fifteen healthy people were chosen as control group.The expression level of sB7-H3 in lymphoma-related HLH and infection-related HLH group significant increased as compared with the control group, (P＜0.05), and the expression level of sB7-H3 in lymphoma-related HLH group was significant higher than that in infection-related HLH group [(35.75± 9.90) vs (28.70±8.95) ng/ml)] (P＜0.001). There were no significant statistical difference in the expression of some clinical factors (including age, fever, splenomegaly, ANC, Plt, FIB, calcium ion, serum albumin, LDH, serum ferritin, sCD25) in lymphoma-related HLH and infection-related HLH group (P＞0.05). The evaluation of expression and significance of sB7-H3 in sHLH by using ROC curve, showed that the area under ROC curve comparison of patients in lymphoma-related HLH group and infection-related HLH group was 0.718 (95% CI 0.610-0.810) (P=0.0002), and predicting the sensitivity and specificity of the lymphoma-related HLH patients were 77.36% and 59.38%, respectively. The best cut-off value of patients in sB7-H3 was 29.81 ng/ml, the overall survival time of sB7-H3 high-expression group (≥29.81 ng/ml) was significant shorter than that in low-expression group (＜29.81 ng/ml) (24 vs 440 d) (P＜0.001). The clinical factors affecting the survival status of sHLH patients were neutrophils, albumin, serum ferritin, serum calcium ions and sB7-H3 levels.sB7-H3 associates with poor prognosis of sHLH patients, and may be involved in disease progression.可溶性B7-H3在继发性噬血细胞性淋巴组织细胞增多症患者中的表达及临床意义.探讨可溶性B7-H3（sB7-H3）在继发性噬血细胞性淋巴组织细胞增多症（sHLH）患者中的诊断及预后意义.收集2012年12月至2018年4月收治的85例初诊sHLH患者初次就诊时的血浆标本，并按照病因将患者分为淋巴瘤相关HLH（LHLH）组和感染相关HLH（IHLH）组。应用ELISA实验检测患者血浆中sB7-H3的表达，同时收集患者的临床资料进行分析并以15例健康体检者为对照组.与对照组相比较，淋巴瘤相关HLH组和感染相关HLH组sB7-H3的表达均明显升高（P＜0.05），淋巴瘤相关HLH组sB7-H3的表达明显高于感染相关HLH组（(35.75±9.90) vs (28.70±8.95) ng/ml）（P＜0.001）。淋巴瘤相关HLH组与感染相关HLH组患者部分临床指标(包括年龄、发热、脾肿大、ANC、Plt、FIB、钙离子、血清白蛋白、LDH、血清铁蛋白、sCD25)的表达均无明显统计学差异（P＞0.05）。应用ROC曲线评价sB7-H3在sHLH的表达及意义结果显示，在与感染相关HLH患者的对比中，ROC（AUC）曲线下面积为0.718（95% CI 0.610-0.810）（P=0.0002)，预测淋巴瘤相关HLH患者的敏感性和特异性分别为77.36%和59.38%。ROC曲线分析sB7-H3的最佳cut-off值为29.81 ng/ml，sB7-H3高表达组（≥29.81 ng/ml）总生存中位时间较低表达组（＜29.81 ng/ml）明显缩短（24 vs 440 d）（P＜0.001）。影响sHLH患者生存状态的临床指标分别是中性粒细胞、白蛋白、血清铁蛋白、血清钙离子和sB7-H3水平.sB7-H3与sHLH影响不良预后的相关因素并可能参与疾病进展.
423	A Case of Cytomegalovirus-Induced Hemophagocytic Lymphohistiocytosis in a Patient with an Underlying Rheumatic Disease.	32550050	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition caused by overproduction of inflammatory cytokines and overactivation of macrophages that can progress to multiorgan dysfunction and failure. Although there are guidelines that attempt to recognize the condition in its early stage, diagnosis can be very challenging due to heterogeneous presentations of HLH. Symptoms and clinical findings include fever, neurologic complaints, respiratory issues, liver dysfunction, cytopenias, amongst others most of which are not specific to HLH. In addition, response to treatment can be highly variable, necessitating an individualized treatment plan based on the presentation. We present a case of a 21-year-old female with a history of biopsy-proven inflammatory myositis on azathioprine and prednisone who presented with fever, hypotension, and pancytopenia. Additional imaging studies showed multiorgan involvement, including pneumonia, pyelonephritis, and splenomegaly. A bone marrow biopsy of her iliac crest showed hemophagocytosis and the infectious workup confirmed cytomegalovirus (CMV) infection, which led to the diagnosis of CMV-induced HLH. She was treated initially with anakinra for macrophage activation syndrome (MAS) in addition to dexamethasone and ganciclovir. Unfortunately, she did not respond to anakinra and was subsequently switched to etoposide with dexamethasone and valganciclovir, which subsequently helped our patient to recover clinically. Our case highlights the challenging nature of HLH and the importance of early detection and a personalized treatment plan in achieving optimal outcomes in patients with HLH.
424	Frequency and spectrum of disease-causing variants in 1892 patients with suspected genetic HLH disorders.	32542393	This article explores the distribution and mutation spectrum of potential disease-causing genetic variants in hemophagocytic lymphohistiocytosis (HLH)-associated genes observed in a large tertiary clinical referral laboratory. Samples from 1892 patients submitted for HLH genetic analysis were studied between September 2013 and June 2018 using a targeted next-generation sequencing panel approach. Patients ranged in age from 1 day to 78 years. Analysis included 15 genes associated with HLH. A potentially causal genetic finding was observed in 227 (12.0%) samples in this cohort. A total of 197 patients (10.4%) had a definite genetic diagnosis. Patients with pathogenic variants in familial HLH genes tended to be diagnosed significantly younger compared with other genes. Pathogenic or likely pathogenic variants in the PRF1 gene were the most frequent. However, mutations in genes associated with degranulation defects (STXBP2, UNC13D, RAB27A, LYST, and STX11) were more common than previously appreciated and collectively represented >50% of cases. X-linked conditions (XIAP, SH2D1A, and MAGT1) accounted for 17.8% of the 197 cases. Pathogenic variants in the SLC7A7 gene were the least encountered. These results describe the largest cohort of genetic variation associated with suspected HLH in North America. Merely 10.4% of patients were identified with a clearly genetic cause by this diagnostic approach; other possible etiologies of HLH should be investigated. These results suggest that careful thought should be given regarding whether patients have a clinical phenotype most consistent with HLH vs other clinical and disease phenotypes. The gene panel identified known pathogenic and novel variants in 10 HLH-associated genes.
425	Three novel prevention, diagnostic, and treatment options for COVID-19 urgently necessitating controlled randomized trials.	32534175	Asymptomatic or minimally symptomatic infection with COVID-19 can result in silent transmission to large numbers of individuals, resulting in expansion of the pandemic with a global increase in morbidity and mortality. New ways of screening the general population for COVID-19 are urgently needed along with novel effective prevention and treatment strategies.A hypothetical three-part prevention, diagnostic, and treatment approach based on an up-to-date scientific literature review for COVID-19 is proposed. Regarding diagnosis, a validated screening questionnaire and digital app for COVID-19 could help identify individuals who are at risk of transmitting the disease, as well as those at highest risk for poor clinical outcomes. Global implementation and online tracking of vital signs and scored questionnaires that are statistically validated would help health authorities properly allocate essential health care resources to test and isolate those at highest risk for transmission and poor outcomes. Second, regarding prevention, no validated protocols except for physical distancing, hand washing, and isolation exist, and recently ivermectin has been published to have anti-viral properties against COVID-19. A randomized trial of ivermectin, and/or nutraceuticals that have been published to support immune function including glutathione, vitamin C, zinc, and immunomodulatory supplements (3,6 Beta glucan) could be beneficial in preventing transmission or lessening symptomatology but requires statistical validation. Third, concerning treatment, COVID-19 induced inflammation and "cytokine storm syndrome" with hemophagocytic lymphohistiocytosis (HLH)/Macrophage Activation Syndrome (MAS) have resulted in extreme morbidity and mortality in those with certain comorbidities, secondary to "acute respiratory distress syndrome" (ARDS) and multiorgan dysfunction with disseminated intravascular coagulation (DIC). Deficiency in red blood cell, serum and alveolar glutathione has been published in the medical literature for ARDS, as well as viral and bacterial pneumonias, resulting from increased levels of free radical/oxidative stress. A randomized controlled trial of blocking NF-κB and cytokine formation using glutathione precursors (N-acetyl-cysteine [NAC] and alpha lipoic acid) and PO/IV glutathione with associated anti-viral effects should be performed, along with an evaluation of Nrf2 activators (curcumin, sulforaphane glucosinolate) which have been scientifically proven to lower inflammation. Since high mortality rates from sepsis induced DIC due to COVID-19 infection has also been associated with thrombotic events and elevated levels of D-dimer, randomized controlled trials of using anticoagulant therapy with heparin is urgently required. This is especially important in patients on ventilators who have met certain sepsis induced coagulopathy (SIC) criteria. The use of acetazolamide with or without sildenafil also needs to be explored with or without heparin, since increased oxygen delivery to vital organs through prevention of thrombosis/pulmonary emboli along with carbonic anhydrase inhibition may help increase oxygenation and prevent adverse clinical outcomes.A three-part prevention, diagnostic, and treatment plan is proposed for addressing the severe complications of COVID-19. Digital monitoring of symptoms to clinically diagnose early exposure and response to treatment; prevention with ivermectin as well as nutritional therapies that support a healthy immune response; treatment with anti-inflammatory therapies that block NF-κB and activate Nrf2 pathways, as well as novel therapies that address COVID-19 pneumonia and ARDS with DIC including anticoagulation and/or novel respiratory therapies with or without acetazolamide and sildenafil. These three broad-based interventions urgently need to be subjected to randomized, controlled trials.
426	JAK/STAT pathway inhibition sensitizes CD8 T cells to dexamethasone-induced apoptosis in hyperinflammation.	3253003932761218	Cytokine storm syndromes (CSS) are severe hyperinflammatory conditions characterized by excessive immune system activation leading to organ damage and death. Hemophagocytic lymphohistiocytosis (HLH), a disease often associated with inherited defects in cell-mediated cytotoxicity, serves as a prototypical CSS for which the 5-year survival is only 60%. Frontline therapy for HLH consists of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide. Many patients, however, are refractory to this treatment or relapse after an initial response. Notably, many cytokines that are elevated in HLH activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine HLH models and humans with refractory disease. We recently reported that cytokine-induced JAK/STAT signaling mediates DEX resistance in T cell acute lymphoblastic leukemia (T-ALL) cells, and that this could be effectively reversed by RUX. On the basis of these findings, we hypothesized that cytokine-mediated JAK/STAT signaling might similarly contribute to DEX resistance in HLH, and that RUX treatment would overcome this phenomenon. Using ex vivo assays, a murine model of HLH, and primary patient samples, we demonstrate that the hypercytokinemia of HLH reduces the apoptotic potential of CD8 T cells leading to relative DEX resistance. Upon exposure to RUX, this apoptotic potential is restored, thereby sensitizing CD8 T cells to DEX-induced apoptosis in vitro and significantly reducing tissue immunopathology and HLH disease manifestations in vivo. Our findings provide rationale for combining DEX and RUX to enhance the lymphotoxic effects of DEX and thus improve the outcomes for patients with HLH and related CSS.
427	Acute human immunodeficiency virus infection associated hemophagocytic lymphohistiocytosis.	32528850	Although acute HIV-induced HLH is rare in literature, HIV is an important differential diagnosis in patients with HLH. In our study, a 33-year-old previously healthy male patient was admitted with fever of unknown origin, lymphadenopathy, generalized edema, transaminitis, acute renal failure, oliguria, myalgias, night sweats, unintentional weight loss, and leukopenia. Disease course was indicative of a viral-like prodrome of roughly 2-month duration. At an outside hospital, full viral work-up (including EBV, CMV, HIV antibodies, hepatitis panel) was negative. HIV p24 antigen assay was not available at the outside facility. Outside liver chemistry and lymph node biopsy were suggestive of HLH. HLH was confirmed via serum ferritin, white cell receptor, and cytokine studies. Repeat viral and rheumatologic studies revealed a positive p24 antigen with indeterminant HIV antibody. We demonstrate efficacy of a specific treatment plan as well as importance of p24 antigen studies in patients with HLH and/or the HIV window-period, adding to available literature/documentation of a rare disease process.
428	Secondary HLH is uncommon in severe COVID-19.	32526046	NaN
429	L-DEP regimen salvage therapy for paediatric patients with refractory Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis.	32525580	Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis (EBV-HLH) is one of the most common subtypes of secondary HLH. However, more than 30% of patients do not respond to traditional treatment. Here, we investigated the efficacy and safety of the L-DEP regimen as a salvage therapy for paediatric refractory EBV-HLH. We retrospectively analysed 26 paediatric patients with refractory EBV-HLH who received the L-DEP regimen at Beijing Children's Hospital from 1 January 2016 to 31 March 2019. Five of the patients achieved complete response (CR) and 11 achieved partial response (PR), indicating an overall response rate of 61·5% (CR + PR). Ten of the 16 patients who achieved CR or PR received allogenic haematopoietic stem cell transplantation (allo-HSCT), and seven were still alive at the last follow-up on 30 April 2020. Two of the 10 patients who did not respond were alive after allo-HSCT. Major side effects included increased amylase, bone marrow suppression and coagulation disorders, though these could be controlled by supportive therapy in most cases. Thus, we conclude that the L-DEP regimen is an effective and relatively safe salvage therapy for children with refractory EBV-HLH. This regimen also provides a bridge to allo-HSCT.
430	[Clinical features and outcomes of cancer-related versus non-cancer-related sepsis in pediatric intensive care unit].	32521960	Objective: To compare the clinical features and outcomes of cancer-related and non-cancer-related sepsis in children who were admitted pediatric intensive care unit (PICU). Methods: The clinical history of patients with sepsis, who were admitted to PICU in Shanghai Children's Hospital, Shanghai Jiao Tong University from August 2016 to July 2019, were retrospectively reviewed. A total of 768 patients were divided into the cancer-related sepsis group (135 cases) and the non-cancer-related sepsis group (633 cases). The patients in the cancer-related group were further categorized into three subgroups including hematological malignancy (80 cases), solid tumor (43 cases) and hemophagocytic lymphohistiocytosis (HLH) (12 cases). The variables of clinical features, laboratory tests, pathogens, management strategies and in-hospital mortality were compared between the two groups by student t test, Mann-Whitney U test or Chi-square test. Results: The patients with cancer-related sepsis accounted for 17.6% of all patients (135/768). Regarding the site of initial infection, the incidence of gastrointestinal infection (43.0% (58/135) vs. 28.6% (181/633), χ(2)=10.718, P=0.001), blood stream infection (29.6% (40/135) vs. 17.1% (108/633), χ(2)=11.297, P=0.001) and skin and soft tissue infection (22.2% (30/135) vs. 4.1% (26/633), χ(2)=54.013, P<0.01) were higher in the patients with cancer-related sepsis than in those with non-cancer-related sepsis. On first PICU admission, the levels of hemoglobin (71 (61, 83) vs. 106 (92, 116) g/L, Z=13.594, P<0.01), white blood cell (1.4 (0.3, 5.2) vs. 9.8 (5.8, 15.1)×10(9)/L, Z=11.213, P<0.01), platelet count (51 (15, 121) vs. 286 (192, 384)×10(9)/L, Z=13.336, P<0.01), CD19(+)cells (0.106 (0.008, 0.274) vs. 0.325 (0.224, 0.454), Z=6.555, P<0.01), and neutrophil (0.449 (0.170, 0.730) vs. 0.683 (0.537, 0.800), Z=5.974, P<0.01) were significantly lower in patients with cancer-related sepsis; however, the levels of C-reactive protein (82 (25, 155) vs. 36 (11, 86) mg/L, Z=-5.257, P<0.01), procalcitonin (1.5 (0.3, 12.0) vs. 0.8 (0.2, 4.0) μg/L, Z=-2.767, P=0.006), CD8(+)cells (0.329 (0.253, 0.514) vs. 0.209 (0.156, 0.275), Z=-5.699, P<0.01), interleukin (IL) -6 (0.1 (0.1, 522.4) vs. 0.1 (0.1, 0.1) ng/L, Z=-2.747, P=0.006), IL-8 (0.1 (0.1, 177.0) vs. 0.1 (0.1, 4.5) ng/L, Z=-2.087, P=0.037), and IL-10 (0.1 (0.1, 42.7) vs. 0.1 (0.1, 6.6) ng/L, Z=-2.148, P=0.032) were significantly higher in patients with cancer-related sepsis. Similarly, the rate of continuous renal replacement therapy (CRRT) (34.8% (47/135) vs. 16.9% (107/633), χ(2)=26.267, P<0.01) and the use of intravenous immunoglobulin (IVIG) (83.0% (112/135) vs. 66.2% (419/633), χ(2)=14.667, P<0.01) were significantly higher in cancer-related sepsis group. Moreover, the incidence of co-infection with fungi was also higher in cancer-related sepsis group (14.1% (19/135) vs. 0.5%(3/633), χ(2)=73.965, P<0.01), and so was the number of multiple organ dysfunction (3 (2, 5) vs. 2 (1, 3), Z=-6.988, P<0.01). Finally, the in-hospital mortality rate of cancer-related sepsis and non-cancer-related sepsis were 36.3% (49/135) and 9.3% (59/633), respectively, also significantly different (χ(2)=67.000, P<0.01). There was no difference in the in-hospital mortality among children with hematologic tumors, solid tumors and HLH (35.0% (28/80) vs. 32.6% (14/43) vs. 7/12, χ(2)=2.838, P=0.242). Conclusions: The site of initial infection, inflammatory markers on PICU admission, and co-infection pathogen during hospitalization are different between patients with cancer-related sepsis and non-cancer-related sepsis. Besides, the in-hospital mortality of cancer-related sepsis is about 4-fold that of non-cancer-related sepsis. The monitoring of clinical features and organ dysfunction, and timely treatment are crucial for cancer-related sepsis.目的： 对比分析儿童肿瘤相关与非肿瘤相关脓毒症的临床特征与预后。 方法： 回顾性总结2016年8月至2019年7月上海交通大学附属儿童医院儿童重症监护病房（PICU）收治的768例脓毒症患儿资料，根据是否存在肿瘤性疾病，分为肿瘤相关脓毒症组（135例）与非肿瘤相关脓毒症组（633例）；前者又根据肿瘤类型分为血液系统肿瘤组（80例）、实体瘤组（43例）和噬血细胞综合征组（12例）。采用t检验、非参数检验（Mann-WhitneyU检验）或χ(2)检验比较组间临床特征、主要治疗措施和住院病死率差别。 结果： 768例脓毒症患儿中，肿瘤相关脓毒症患儿135例，占17.6%。肿瘤相关脓毒症组消化道感染比例[43.0%（58/135）比28.6%（181/633）,χ(2)=10.718，P=0.001]、血液感染[29.6% （40/135）比17.1%（108/633）, χ(2)=11.297，P=0.001]、皮肤软组织感染[22.2%（30/135）比4.1%（26/633）, χ(2)=54.013，P<0.01]明显高于非肿瘤相关脓毒症组；肿瘤相关脓毒症组患儿入PICU后首次血红蛋白[71（61,83）比106 (92,116) g/L, Z=13.594, P<0.01]、白细胞计数[1.4（0.3,5.2）比9.8(5.8,15.1)×10(9)/L,Z=11.213,P<0.01]、血小板计数[51（15,121）比286（192，384）×10(9)/L,Z=13.336, P<0.01]、中性粒细胞[0.449（0.170,0.730）比0.683（0.537,0.800）,Z=5.974,P<0.01]和CD19(+)细胞[0.106（0.008,0.274）比0.325（0.224,0.454）, Z=6.555, P<0.01]明显低于非肿瘤相关脓毒症组，差异均有统计学意义；C反应蛋白[82（25,155）比36（11,86） mg/L，Z=-5.257, P<0.01]、降钙素原[1.5（0.3,12.0）比0.8（0.2,4.0） μg/L, Z=-2.767，P=0.006]、CD8(+)细胞[0.329（0.253,0.514）比0.209（0.156,0.275）, Z=-5.699, P<0.01]、白细胞介素（IL）-6[0.1（0.1,522.4）比0.1（0.1,0.1）ng/L，Z=-2.747，P=0.006]、IL-8[0.1（0.1,177.0）比0.1（0.1,4.5）ng/L,Z=-2.087, P=0.037]、IL-10[0.1（0.1,42.7）比0.1（0.1,6.6）ng/L, Z=-2.148, P=0.032]明显高于非肿瘤相关脓毒症患儿组，差异均有统计学意义。肿瘤相关脓毒症组需要连续性肾替代治疗（CRRT)[34.8%（47/135）比16.9%（107/633）, χ(2)=26.267,P<0.01]和静脉注射人免疫球蛋白[83.0%（112/135）比66.2%（419/633）, χ(2)=14.667,P<0.01]支持的比例均高于非肿瘤相关脓毒症组。住PICU期间，肿瘤相关脓毒症患儿发生真菌感染比例高于非肿瘤相关脓毒症[14.1%（19/135）比0.5%（3/633）,χ(2)=73.965，P<0.01]；器官功能障碍个数多于非肿瘤相关脓毒症组[3（2,5）比2（1,3）个,Z=-6.988，P<0.01]。肿瘤相关与非肿瘤相关脓毒症患儿的住院病死率分别为36.3%（49/135）和9.3% （59/633），组间比较差异有统计学意义（χ(2)=67.000，P<0.01）；血液系统肿瘤、实体肿瘤与噬血细胞综合征相关脓毒症病死率分别为35.0% （28/80）、32.6% （14/43）和7/12，组间比较差异无统计学意义（χ(2)=2.838，P=0.242）。 结论： 肿瘤相关脓毒症患儿的感染部位、入PICU时炎症指标和合并感染病原菌与非肿瘤相关脓毒症存在差异。肿瘤相关脓毒症患儿病死率为非肿瘤相关脓毒症的4倍，应特别重视肿瘤性疾病合并感染的监测、评估及治疗策略。.
431	Hemophagocytic Lymphohistiocytosis Secondary to PD-1 and IDO Inhibition in a Patient with Refractory Glioblastoma.	32518546	Immune checkpoint inhibition (ICI)-based approaches have transformed the treatment landscape of numerous solid tumors. Glioblastoma (GBM) is an aggressive and almost universally fatal disease which is in need of novel treatment options, and combinations of immune checkpoint inhibitors, including dual agent therapy, are starting to be explored in refractory GBM. Growing adoption of ICI-based approaches in solid tumors has been met with improved understanding of immune-related adverse events (IRAEs), including primary hematologic adverse events. Although management guidelines for multiple hematologic IRAEs have been established, the emergence of hemophagocytic lymphohistiocytosis (HLH) secondary to ICI therapy has only rarely been described, and its pathogenesis and optimal management are incompletely understood. We present the case of a 74-year-old male with a history of refractory GBM treated with PD-1 and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition who experienced acute liver injury, followed by progressive fevers, altered mental status, and cytopenias. Serum studies and examination of spleen and bone marrow pathology were consistent with HLH, which was refractory to steroids and ultimately resulted in his rapid clinical decline. Here, we review prior cases of HLH secondary to ICI therapy across solid tumors, and explore potential mechanisms contributing to the rapid onset and refractory nature of our patient's HLH syndrome. We hope to further highlight HLH as an emerging hematologic IRAE secondary to ICI therapy, and suggest that new practice guidelines begin to recognize HLH as a characteristic hematologic IRAE in patients treated with PD-1 and other immune checkpoint inhibitors.
432	Treatment outcomes and prognostic factors for non- malignancy associated secondary hemophagocytic lymphohistiocytosis in children.	32517812	Secondary hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome that requires prompt diagnosis and appropriate treatment. A risk-stratification model that could be used to identify high-risk pediatric patients with HLH who should be considered for second-line therapies, including salvage regimens and allogeneic hematopoietic cell transplantation (HCT), was developed.The medical records of 88 pediatric patients (median age 1.4 years, range 0.2-15 years) with non-malignancy associated secondary HLH were retrospectively reviewed. Treatment strategies included dexamethasone, etoposide, and cyclosporine.Survival analysis showed HLH patients with infections other than Epstein-Barr virus (EBV) and unknown causes experienced better 5-year overall survival (OS) than patients with HLH due to autoimmune disease, EBV or immunodeficiency (76% vs. 65, 33.3, 11%, p < 0.001). On multivariate analysis, among all patients, non-response at 8 weeks was the most powerful predictor of poor OS. When treatment response was excluded, hemoglobin < 60 g/L and albumin < 25 g/L at diagnosis were associated with poor OS. In patients with EBV-HLH, hemoglobin < 60 g/L at diagnosis was associated with poor OS. A prognostic risk score was established and weighted based on hazard ratios calculated for three parameters measured at diagnosis: hemoglobin < 60 g/L (2 points), platelets < 30 × 109/L (1 point), albumin < 25 g/L (2 points). Five-year OS of low-risk (score 0-1), intermediate-risk (score 2), and poor-risk (score ≥ 3) patients were 88, 38, and 22%, respectively (p < 0.001).These findings indicate that clinicians should be aware of predictive factors at diagnosis and consider 8-week treatment response to identify patients with high-risk of disease progression and the need for second-line therapy and allogeneic HCT.
433	Hemophagocytic lymphohistiocytosis is associated with Bartonella henselae infection in a patient with multiple susceptibility genes.	32517705	Adult-onset hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening condition, which is often triggered by certain types of infection, cancer and numerous autoimmune diseases; however, of the numerous infectious triggers associated with HLH, the consequences of Bartonella henselae infection have been rarely reported.A 48-year-old female presented with a 20-day history of intermittent fever accompanied by systemic rash, fatigue, anorexia and weight loss later she developed shock and unconsciousness. Blood tests showed a reduction of leukocyte, anemia and thrombocytopenia, and pathological results of a bone marrow biopsy confirmed hemophagocytic activity. Metagenomic next-generation sequencing (mNGS) analysis of the lymph node detected the presence of B. henselae. Whole exome sequencing revealed two gene variants, STXBP2 and IRF5, in this adult patient with secondary HLH. Then, she received minocycline and rifampin combination anti-infective therapy. Intravenous immunoglobulin for 5 days followed by a high dose of methylprednisolone were also administered. The patient was successfully discharged from the intensive care unit and remained in good condition after 2 months of follow-up.mNGS served crucial roles in obtaining an etiological diagnosis, which suggested that screening for B. henselae should be considered in patients with HLH, especially those with a cat at home. In addition, the genetic defects were discovered to not only be present in primary HLH, but also in secondary HLH, even in the elderly.
434	Novel XIAP mutation causing enhanced spontaneous apoptosis and disturbed NOD2 signalling in a patient with atypical adult-onset Crohn's disease.	32514016	X-linked inhibitor of apoptosis (XIAP) is the most potent human inhibitor of apoptosis, and is also involved in NOD2-dependent NFκB and MAPK signalling cascade activation. The absence or defective function of XIAP leads to the development of a rare and severe primary immunodeficiency known as X-linked lymphoproliferative syndrome type 2 (XLP-2), which is characterized by a triad of clinical manifestations, including a high incidence of haemophagocytic lymphohistiocytosis (HLH), lymphoproliferation and inflammatory bowel disease (IBD), usually with very early onset. Here, we present a novel XIAP mutation identified in a patient with atypical adult-onset IBD complicated by relapsing HLH, splenomegaly and sarcoid-like disease. The c.266delA mutation in the XIAP gene creates a premature stop codon, and causes a severe reduction in XIAP protein expression. The mutation is also associated with impaired spontaneous and staurosporine- and PMA-induced apoptosis accompanied by significantly increased expression of pro-apoptotic genes. We also confirmed the negative impact of this particular XIAP mutation on NOD2-dependent NFκB and MAPK activation, while NOD2-independent activation was found to be unaffected. Moreover, we assume that the mutation has an impact on the overproduction of IL-12 and IFNγ, the shift towards the Th1 immune response and increased numbers of central memory and effector memory CD4+ and CD8+ T cells. All these changes contribute to immune dysregulation and the clinical manifestation of XLP-2.
435	Rising visceral leishmaniasis in Holy Himalayas (Uttarakhand, India) - A cross-sectional hospital-based study.	32509616	Apart from the rarity of the visceral leishmaniasis (VL) cases in high altitude (>2000 ft), the combination triad of VL, hemophagocytic lymphohistiocytosis (HLH) syndrome, and Himalayas is rarely being reported. Here, we studied the triad in the Himalayan region, attending a single tertiary care hospital over a period of 2 years.The study was a cross-sectional analysis of case records of seven confirmed VL patients. A systematic master chart review analyzed the demographic, clinical, laboratory, treatment, and outcome details of these patients.These cases were diagnosed as VL by clinical findings and confirmed by rk-39 anti-body and demonstration of LD bodies in bone marrow smears. All cases without any travel history to endemic regions presented with prolonged fever (>1 months duration), anorexia, weight loss, and having hepatosplenomegaly and bi-or pan-cytopenia. All cases were having HLH, confirmed based on the HScore system (online calculation), and liver injury having transaminitis. Kidney involvement was seen in 27% cases. All cases improved with liposomal amphotericin-B, but one had cardiac arrest after blood transfusion reaction.Clinician of the non-endemic zone should suspect VL in patients with fever of unknown origin and have a high suspicion in cases of HLH and liver involvement and vice versa. Kidney involvement is seen in one-third of the VL cases. Liposomal amphotericin-B is recommended in this region. The leishmaniasis prevalent in these areas should further be subject to comparison with endemic parts, and a large-scale study is needed to find the reason of the rising vector from the holy Himalayas.
436	A rare case of diffuse large B-cell lymphoma-associated hemophagocytic lymphohistiocytosis.	32507098	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome of excessive immune activation. Herein, we aimed to report a diffuse large B-cell lymphoma (DLBCL) case that was presented as HLH.A 32-year-old man presented to a hospital with complaints of vomiting, nausea and diarrhea in October 2019. Fever and hepatosplenomegaly was detected in physical investigation. Bone marrow aspiration investigation revealed the hemaphagocytosis. HLH-2004 protocol was started for hemophagocytosis. Whole body magnetic resonance imaging (MR) revealed no lymphadenopathy. Bone marrow biopsy revealed high-grade B-cell lymphoma, favoring DLBCL. There were no pathologic cells in lumber puncture investigation.He was diagnosed with secondary hemaphagocytic syndrome due to DLBCL, and chemotherapy was switched to rituximab, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin (R-EPOCH) regimen. After three cycles of R-EPOCH chemotherapy regimen, complete remission was confirmed with positron emission tomography-computerised tomography (PET-CT) scan.Our patients' findings are suitable for six out of eight criteria of hemaphagocytic syndrome. The H-score of our patient was more than 250, reflecting the >99% probability of HLH syndrome. Compatible with literature knowledge, our patient had responded very well to etoposide-containing regimens. In our patient, no lymphadenopathy was detected by physical examination or MR scan, and the diagnosis of DLBCL was only made by the result of bone marrow investigation. In conclusion, herein, we have reported a DLBCL case that had presented with HLH, and clinicians should be aware that B-cell lymphomas may be the underlying cause of HLH.
437	Dynamics of hematopoiesis is disrupted by impaired hematopoietic microenvironment in a mouse model of hemophagocytic lymphohistiocytosis.	32506245	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory disorder. We found recently that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated mice (SAMP1/TA-1) but not in senescence-resistant control mice (SAMR1). In this study, we analyzed the dynamics of hematopoiesis in this mouse model of HLH. When treated repeatedly with LPS, the numbers of myeloid progenitor cells (CFU-GM) and B-lymphoid progenitor cells (CFU-preB) in the bone marrow (BM) rapidly decreased after each treatment in both strains. The number of CFU-GM in SAMP1/TA-1 and SAMR1, and of CFU-preB in SAMR1, returned to pretreatment levels by 7 days after each treatment. However, the recovery in the number of CFU-preB in SAMP1/TA-1 was limited. In both strains, the BM expression of genes encoding positive regulators of myelopoiesis (granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6), and negative regulators of B lymphopoiesis (tumor necrosis factor (TNF)-α) was increased. The expression of genes encoding positive regulators of B lymphopoiesis (stromal-cell derived factor (SDF)-1, IL-7, and stem cell factor (SCF)) was persistently decreased in SAMP1/TA-1 but not in SAMR1. Expression of the gene encoding p16INK4a and the proportion of β-galactosidase-positive cells were increased in cultured stromal cells obtained from LPS-treated SAMP1/TA-1 but not in those from LPS-treated SAMR1. LPS treatment induced qualitative changes in stromal cells, which comprise the microenvironment supporting appropriate hematopoiesis, in SAMP1/TA-1; these stromal cell changes are inferred to disrupt the dynamics of hematopoiesis. Thus, hematopoietic tissue is one of the organs that suffer life-threatening damage in HLH.
438	Hodgkin lymphoma-associated hemophagocytic lymphohistiocytosis-a dangerous disease.	32500223	This study investigated the clinical characteristics of Hodgkin lymphoma-associated hemophagocytic lymphohistiocytosis (HLH-HL). Clinical data of 8 patients with HLH-HL and 20 non-HLH-HL patients were included. All eight HLH-HL patients tested positive for plasma Epstein-Barr virus (EBV)-DNA and EBV-encoded small RNA (EBER), and six patients were positive for EBV-DNA in the peripheral blood mononuclear cells (PBMCs). Two out of the 20 non-HLH-HL patients were confirmed positive for EBER, and the remaining 18 patients were negative. Among the HLH-HL patients, five patients received ABVD (doxorubicin/bleomycin/vinblastine/dacarbazine) chemotherapy regimens in other hospitals, and their conditions were considered to be worse, for which reason they were transferred to our center, and three patients were treated with DEP (doxorubicin-etoposide-methylprednisolone) regimens to target HLH and were alive as of the writing of this article. Two patients were critically ill upon admission and were not able to undergo chemotherapy. Significant differences in survival time were observed between the HLH-HL and non-HLH-HL patients (P = 0.005). HL patients found positive for EBV (plasma/PBMCs EBV-DNA(+)/EBER(+)) may be more likely to develop HLH-HL. It may be beneficial to target HLH during the acute phase of HLH, followed by treating HL once the HLH condition has stabilized. HLH-HL patients have worse prognosis and higher mortality than non-HLH-HL patients.
439	Prognostic Value of Blood-Based Inflammatory Biomarkers in Secondary Hemophagocytic Lymphohistiocytosis.	32495220	Hemophagocytic lymphohistiocytosis (HLH) is a rare systematic immune disease manifested with excessive activation of lymphocytes and macrophages. This study was designed to explore the feasible prognostic factors of secondary HLH (sHLH).We retrospectively analyzed 179 patients with newly diagnosed sHLH from January 2016 to May 2019 according to the HLH-2004 protocol. Baseline characteristics and laboratory results were reviewed.The median age of all patients was 53 years, with a male/female ratio of 1.45. The commonest cause of HLH was malignancy. Of the 179 patients, 48.6% presented with Epstein-Barr virus (EBV) infection, 92.8% with hemocytopenia (at least 2 lineages), 60.3% with hypofibrinogenemia, 43.0% with hypertriglyceridemia (≥ 3 mmol/L), 99.4% with high ferritin, 97.8% with fever, 72.1% with splenomegaly, and 72.6% with hemophagocytosis. As to their prognosis, 122 patients died; the median survival was 88 days, with a 2-year survival rate of 26.72%. Univariate analysis confirmed neutrophil-to-lymphocyte ratio ˃ 2.53, lymphocyte-to-monocyte ratio (LMR) ≤ 4.43, platelet-to-lymphocyte ratio ˃ 227.27, red blood cell distribution width ˃ 14.6, red blood cell distribution width-to-platelet ratio (RPR) > 0.33, EBV infection, platelet ≤ 34 × 109 /L, fibrinogen ≤ 1.34 g/L, alkaline phosphatase ˃ 182.4 U/L, adenosine deaminase ˃ 69.2 U/L, and ferritin ˃ 2318 ng/mL were associated with an inferior survival. In a multivariate model, LMR, RPR, and ferritin were considered as three independent factors.Some blood-based inflammatory markers, which can be easily and cheaply detected, are significantly associated with the OS of HLH patients. LMR and RPR, superior to NLR, PLR, RDW, can be taken to predict the OS of patients with HLH.
440	Visceral leishmaniasis with hemophagocytic lymphohistiocytosis (HLH).	32489870	NaN
441	A case of pembrolizumab-induced hemophagocytic lymphohistiocytosis successfully treated with pulse glucocorticoid therapy.	32489850	Treatments using immune checkpoint inhibitors such as pembrolizumab lead to immune mediated adverse effects including hemophagocytic lymphohistiocytosis (HLH). Herein, we present a case where HLH developed after pembrolizumab administration, which was treated using high dose prednisolone. He developed high-grade fever complicated with liver dysfunction and diarrhea 7 days after pembrolizumab administration. Although treatment with oral prednisolone alleviated the symptoms, other adverse effects arose owing to a tapered prednisolone dose. Hyperferritinemia suggested the diagnosis of HLH and met the criteria for HLH diagnosis. He was thus administered intravenous pulses of methylprednisolone followed by high-dose oral prednisolone, which resolved these symptoms.
442	Hemophagocytic Lymphohistiocytosis Complicating Methotrexate Toxicity.	32477983	Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal disease caused by dysregulated activation of macrophages against one's blood cells. Major pathologic feature of HLH is hemophagocytosis. We present a case of HLH complicating methotrexate toxicity in a 65-year-old psoriatic patient with history of renal disease. Diagnosis of HLH was established as he fulfilled five out of eight HLH diagnostic criteria. This case report is presented to enlighten clinicians about the clinical entity of HLH and to suspect and recognize this rare and generally fatal disease at the earliest.
443	Hemophagocytic lymphohistiocytosis associated with Leishmania: A hidden passenger in endemic areas.	32473845	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome characterized by excessive immune activation. We analyzed the presentation, diagnosis and prognosis of our cohort of HLH-Leishmania cases.We studied HLH cases in patients over 14 years of age in the province of Granada (Spain), from January 2008 to November 2019.In this study, Leishmania was the predominant trigger of adult HLH in our region. There were no differences in the clinical-analytical presentation between HLH triggered by Leishmania and those initiated by a different cause. RT-PCR was the best tool to identify Leishmania as the trigger of HLH, given that the other microbiological tests showed low sensitivity to detect the parasite in our HLH-Leishmania cases.A comprehensive search for Leishmania is mandatory in HLH cases. Based on our findings, we propose that RT-PCR for Leishmania in bone marrow samples must be included in HLH differential diagnostic protocols.
444	The amount of cytokine-release defines different shades of Sars-Cov2 infection.	32460624	The recent outbreak of coronavirus disease (COVID 19), spreading from China all around the world in early 2020, has led scientists to investigate the immuno-mediated mechanisms underlying the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) infection. Depending on the amount of cytokines released as the result of the immunological activation induced by SARS-CoV2, three major clinical phenotypes can be identified: "mild",symbolized as a "drizzle" of cytokines, severe as a "storm", and critical as a "hurricane". In patients with mild symptoms, the release of pro-inflammatory cytokines is balanced to obtain a defense response against the virus which is often self-limiting and overcomes without tissue damage. In severe phenotype, resembling a "cytokine-release syndrome", SARS-CoV2 causes the lysis of the immune-mediators leading to a cytokine storm able to induce lung epithelium damage and acute respiratory distress syndrome. In critical patients, the immune response may become uncontrolled, thus the cytokine burst resembles a form of secondary hemophagocytic lymphohistiocytosis which may result in a multi organ failure. In addition to the standard of care, an immune-modulatory therapy tailored to each one of the different phenotypes should be used in order to prevent or reduce the release of cytokines responsible for organ damage and disease progression.
445	Considering immunologic and genetic evaluation for HLH in neuroinflammation: A case of Griscelli syndrome type 2 with neurological symptoms and a lack of albinism.	32459386	NaN
446	Cell Versus Cytokine - Directed Therapies for Hemophagocytic Lymphohistiocytosis (HLH) in Inborn Errors of Immunity.	32457750	Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous hyperinflammatory syndrome with different pathways of pathogenesis resulting in similar clinical presentations. It is best defined and understood if presenting in the context of genetic immunodeficiencies associated with defects of lymphocyte cytotoxicity. In these "primary" forms of HLH, cellular and soluble immune effectors are relatively well characterized. While etoposide-based broad cell-directed therapies remain standard of care, more specific therapies targeting these effectors individually are increasingly available. Anti-CD52 as a cell-directed therapy and anti-IFN-gamma, IL-18BP, and JAK-inhibition as cytokine-directed therapies are expected to broaden the therapeutic options, but the precise role of these drugs in first-line and rescue treatment indications remains to be defined. A number of additional inborn errors of immunity are associated with episodes of immune activation fulfilling the clinical criteria of HLH. Impaired pathogen control is a key driver of hyperinflammation in some conditions, while others are characterized by a strong autoinflammatory component. This heterogeneity of disease-driving factors and the variable severity in disease progression in these conditions do not allow a simple adaptation of protocols established for "primary" HLH to HLH in the context of other inborn errors of immunity. Cytokine-directed therapies hold significant promise in these increasingly recognized disorders.
447	Ehrlichiosis Presenting as Hemophagocytic Lymphohistiocytosis in an Immunocompetent Adult.	32455115	Hemophagocytic Lymphohistiocytosis (HLH) is a fatal, immunologic syndrome characterized by dysregulated tissue inflammation. HLH can be either primary or secondary; with the latter typically resulting from an infection. Diagnosis requires five or more of the following: fever, splenomegaly, cytopenia, hypertriglyceridemia, hemophagocytosis via biopsy, low natural killer (NK) cell activity, elevated ferritin and soluble CD25 level (sCD25). We present a case of HLH related to ehrlichiosis. In order to mount an effective immune response against microbes such as Ehrlichia chaffeensis, the host must have preserved NK cell function. Being that HLH Is characterized as a state of depleted NK cell function, It is crucial to investigate the role NK cell function has in the setting of HLH on the infectivity of Ehrlichia species.
448	Hemophagocytic lymphohistiocytosis in critically ill patients: diagnostic reliability of HLH-2004 criteria and HScore.	32448380	Hemophagocytic lymphohistiocytosis (HLH) is a rare though often fatal hyperinflammatory syndrome mimicking sepsis in the critically ill. Diagnosis relies on the HLH-2004 criteria and HScore, both of which have been developed in pediatric or adult non-critically ill patients, respectively. Therefore, we aimed to determine the sensitivity and specificity of HLH-2004 criteria and HScore in a cohort of adult critically ill patients.In this further analysis of a retrospective observational study, patients ≥ 18 years admitted to at least one adult ICU at Charité - Universitätsmedizin Berlin between January 2006 and August 2018 with hyperferritinemia of ≥ 500 μg/L were included. Patients' charts were reviewed for clinically diagnosed or suspected HLH. Receiver operating characteristics (ROC) analysis was performed to determine prediction accuracy.In total, 2623 patients with hyperferritinemia were included, of whom 40 patients had HLH. We found the best prediction accuracy of HLH diagnosis for a cutoff of 4 fulfilled HLH-2004 criteria (95.0% sensitivity and 93.6% specificity) and HScore cutoff of 168 (100% sensitivity and 94.1% specificity). By adjusting HLH-2004 criteria cutoffs of both hyperferritinemia to 3000 μg/L and fever to 38.2 °C, sensitivity and specificity increased to 97.5% and 96.1%, respectively. Both a higher number of fulfilled HLH-2004 criteria [OR 1.513 (95% CI 1.372-1.667); p <  0.001] and a higher HScore [OR 1.011 (95% CI 1.009-1.013); p <  0.001] were significantly associated with in-hospital mortality.An HScore cutoff of 168 revealed a sensitivity of 100% and a specificity of 94.1%, thereby providing slightly superior diagnostic accuracy compared to HLH-2004 criteria. Both HLH-2004 criteria and HScore proved to be of good diagnostic accuracy and consequently might be used for HLH diagnosis in critically ill patients.The study was registered with www.ClinicalTrials.gov (NCT02854943) on August 1, 2016.
449	Thinking Beyond HLH: Clinical Features of Patients with Concurrent Presentation of Hemophagocytic Lymphohistiocytosis and Thrombotic Microangiopathy.	32447592	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive immune system activation driven mainly by high levels of interferon gamma. The clinical presentation of HLH can have considerable overlap with other inflammatory conditions. We present a cohort of patients with therapy refractory HLH referred to our center who were found to have a simultaneous presentation of complement-mediated thrombotic microangiopathy (TMA). Twenty-three patients had therapy refractory HLH (13 primary, 4 EVB-HLH, 6 HLH without known trigger). Sixteen (69.6%) met high-risk TMA criteria. Renal failure requiring renal replacement therapy, severe hypertension, serositis, and gastrointestinal bleeding were documented only in patients with HLH who had concomitant complement-mediated TMA. Patients with HLH and without TMA required ventilator support mainly due to CNS symptoms, while those with HLH and TMA had respiratory failure predominantly associated with pulmonary hypertension, a known presentation of pulmonary TMA. Ten patients received eculizumab for complement-mediated TMA management while being treated for HLH. All patients who received the complement blocker eculizumab in addition to the interferon gamma blocker emapalumab had complete resolution of their TMA and survived. Our observations suggest co-activation of both interferon and complement pathways as a potential culprit in the evolution of thrombotic microangiopathy in patients with inflammatory disorders like refractory HLH and may offer novel therapeutic approaches for these critically ill patients. TMA should be considered in children with HLH and multi-organ failure, as an early institution of a brief course of complement blocking therapy in addition to HLH-targeted therapy may improve clinical outcomes in these patients.
450	Hemophagocytic lymphohistiocytosis (HLH) caused by disseminated histoplasmosis by H. capsulatum var. duboisii in HIV patient: A case report.	32446648	NaN
451	A comprehensive analysis of adult patients with secondary hemophagocytic lymphohistiocytosis: a prospective cohort study.	32440790	Secondary hemophagocytic lymphohistiocytosis (HLH) is a rare but fatal condition with various underlying disorders in adult patients and is diagnosed based on the HLH-2004 criteria, which were established based on experience in pediatric patients. However, few studies have prospectively evaluated the treatment outcomes and diagnostic performance of HLH criteria in adult patients with secondary HLH. Thus, we performed a single-center, prospective cohort study of adult patients with suspected HLH, and we analyzed treatment outcomes of patients enrolled between 2017 and 2019 as an interim analysis ( ClinicalTrials.gov Identifier: NCT03117010). Of the 73 patients with suspected HLH, 70 patients completed the evaluation for ≥ 7 of the HLH-2004 criteria, and 55 patients were diagnosed with HLH (55/73, 75%). Although serum ferritin and fever had a sensitivity of more than 90%, both had exceptionally low specificity, whereas soluble CD25 had a sensitivity of more than 90% and specificity of 80%. Forty patients with malignancy-associated HLH had B cell (n = 19) or T- or NK-cell (n = 21) lymphoid malignancy, whereas 15 patients had non-malignant disorders. Non-malignancy-associated HLH had greater than 90% 1-year overall survival (OS) after diagnosis of HLH, whereas that for malignancy-associated HLH was less than 40%. In conclusion, our study showed promising treatment outcomes for patients enrolled in our prospective cohort study, and prospectively demonstrated the diagnostic performance of the HLH-2004 criteria in adult patients with suspected HLH. Given that lymphoma was the most common cause of HLH in adults, thorough evaluation for lymphoma should be performed in adults with suspected HLH.
452	New variant (Val597Ile) in transmembrane region of the TSH receptor with human chorionic gonadotropin hypersensitivity in familial gestational hyperthyroidism.	32437589	Only two mutations at the lysine 183 amino acid in the extracellular N-terminal domain of human TSH receptor (hTSHR) have been associated with hypersensitivity to hCG and familial gestational hyperthyroidism.Describe a new variant of the TSHR gene with hCG hypersensitivity found in two women of the same family diagnosed with gestational hyperthyroidism.A 38-year-old woman was seen during the first trimester of her second pregnancy for thyrotoxicosis with increased fT3 and fT4 concentrations and low TSH levels without anti-TSH receptor antibody. Thyrotoxicosis improved spontaneously during the 2nd trimester and persisted at the 3rd trimester. Similar clinical symptoms (weight loss, nausea, vomiting) were also reported during the first trimester of her first pregnancy and the first pregnancy of her mother.DNA sequencing of the hTSHR gene of this woman and her mother identifies a heterozygous variant changing valine to isoleucine residue at codon 597 in the transmembrane domain (TMD) of this receptor. In vitro functional studies of this variant showed increased constitutive activity in regard to the basal level of cAMP and IP3 production and to the low cell-surface expression, while response to TSH was reduced compared to that of the wild-type receptor. The Val597Ile variant presented a dose-dependent increase in cAMP response to hGC and human luteinizing hormone (hLH). Simulation of the protein dynamics showed a high structural impact of the Val597Ile variant on helices 3 (TMH3) and 5 (TMH5) of the transmembrane domain participating to constitutive activity and hCG sensitivity.We describe a new variant in the transmembrane region of the hTSHR gene with increased constitutive activity and hCG hypersensitivity in familial gestational hyperthyroidism.
453	Mitochondrial translation, dynamics, and lysosomes combine to extend lifespan.	3243422032259199	In this issue, Liu et al. (2019. J. Cell. Biol.https://doi.org/10.1083/jcb.201907067) find that the inhibition of mitochondrial ribosomes in combination with impaired mitochondrial fission or fusion increases C. elegans lifespan by activating the transcription factor HLH-30, which promotes lysosomal biogenesis.
454	Early Use of Blood Purification in Severe Epstein-Barr Virus-Associated Hemophagocytic Syndrome.	32430444	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a common type of hemophagocytic lymphohistiocytosis (HLH) that exhibits high rates of morbidity and fatalities. Multiorgan failure caused by Epstein-Barr virus (EBV)-induced hypercytokinemia is one of the main reasons for early deaths. Blood purification techniques have been successfully applied in previously treated hypercytokinemia. However, there were insufficient studies to support the combination of plasma exchange (PE) and continuous renal replacement therapy (CRRT) in treating patients with severe EBV-HLH. In this article, we have summarized the effects of early incorporation of PE and CRRT, together with HLH-2004 chemoimmunotherapy, in 8 pediatric patients with severe EBV-HLH. Early use of PE and CRRT appeared to be well tolerated, and no serious side effects and early deaths were observed. After PE and CRRT procedures, cytokine levels were reduced to normal values, except for soluble interleukin 2 receptor, and significant reductions in EBV DNA, serum ferritin, aspartate transaminase, total bilirubin, total bile acid, lactate dehydrogenase, and body temperature values and increases in the neutrophil count in addition to hemoglobin, albumin, and cholinesterase values were observed. Furthermore, through continuous HLH-2004 treatment regimens, lower limits of detection were exhibited for EBV DNA levels, and all other observational indicator levels were restored to normal. Finally, 7 patients achieved and maintained complete remission for 15 to 24 months, culminating in August 2019. Therefore, it is our suggestion that early incorporation of PE and CRRT with chemoimmunotherapy might be a safe and effective treatment for patients with severe EBV-HLH.
455	A case of pediatric visceral leishmaniasis-related hemophagocytic lymphohistiocytosis diagnosed by mNGS.	32425641	Visceral leishmaniasis-related hemophagocytic lymphohistiocytosis (VL-HLH) is a secondary hemophagocytic syndrome, which can be life-threatening, caused by leishmania and transmitted by infected sandflies. Rapid and accurate identification of leishmania is crucial for clinical strategies.Here, we report an infantile infection in a non-epidemic area of China. The infant was a 9.5-month-old girl with fever, pancytopenia and hepatosplenomegaly, which meet the HLH-2004 standard, and the negative gene results exclude congenital HLH. However, chemotherapy is ineffective and is accompanied by severe infection. Fortunately, she is diagnosed with VL-HLH (visceral leishmaniasis-related hemophagocytic lymphohistiocytosis), as leishmania is detected by next-generation meta-genome sequencing (mNGS) and quickly relieved after treatment with libosomal amphotericin B (L-AMB).mNGS can detect leishmania in pediatric HLH, and should be performed as a new detection for VL-HLH, particularly for infants, who may not respond to HLH-2004 regimen.
456	Neurologic complications of COVID-19.	32425321	Much of the focus regarding the global pandemic of coronavirus disease of 2019 (COVID-19) has been on the cardiovascular, pulmonary, and hematologic complications. However, neurologic complications have arisen as an increasingly recognized area of morbidity and mortality.This brief report summarizes the neurologic complications associated with COVID-19 with an emphasis on the emergency medicine clinician.COVID-19 has infected over 3.5 million people and killed over 240,000 people worldwide. While pulmonary complications are profound, the neurologic system is also significantly impacted, with complications including acute cerebrovascular events, encephalitis, Guillain-Barré syndrome, acute necrotizing hemorrhagic encephalopathy, and hemophagocytic lymphohistiocytosis. Additionally, patients on immunosuppressive medications for pre-existing neurologic issues are at an increased risk for complications with COVID-19 infection, and many of the currently proposed COVID-19 therapies can interact with these medications.When caring for COVID-19 patients, emergency medicine clinicians should be aware of the neurologic complications from COVID-19.
457	Inherited chromosomally integrated human herpesvirus-6 in a patient with XIAP deficiency.	32424944	Human herpesvirus-6 (HHV-6) is a common pathogen affecting the human population. Primary HHV-6 infection generally occurs during infancy and causes exanthema subitum. Moreover, HHV-6 may exhibit inherited chromosomally integrated HHV-6 (iciHHV-6) in certain individuals. Although iciHHV-6 is generally known to be nonpathogenic, it may cause reactivation in patients with primary immunodeficiency disease (PID). XIAP deficiency is a rare PID characterized by recurrent hemophagocytic lymphohistiocytosis (HLH). It has been reported that the Epstein-Barr virus primarily causes HLH; however, the other pathogens, including HHV-6, can also cause this complication. We encountered a case of XIAP deficiency accompanied by iciHHV-6. He suffered from recurrent HLH, for which allogeneic bone marrow transplantation (BMT) was performed as a curative therapy. During the course of BMT, the patient experienced HLH three times, but there was no reactivation of endogenous HHV-6 from iciHHV-6. Finally, the patient achieved complete donor chimerism and a decline in HHV-6 DNA copy number in whole blood. This case report demonstrates no evidence of reactivation of iciHHV-6 during BMT in a patient with XIAP deficiency.
458	Children with lymphoma presenting with hemophagocytic lymphohistiocytosis.	32419422	< 7b > Hemophagocytic lymphohistiocytosis (HLH) may precede malignancy, in particular lymphomas and leukemias. However, the causative factors, appropriate treatment and the prognosis of this association is not established.Herein, we present two patients, one with nodular sclerosing Hodgkin lymphoma (HL) and concomitant Epstein-Barr virus (EBV) infection, and the other with anaplastic large cell lymphoma (ALCL), presented as malignancy associated HLH.In our patients, malignancy directed therapy was sufficient to treat HLH symptoms both at presentation and at recurrence in the second patient.
459	Trigger-dependent differences determine therapeutic outcome in murine primary hemophagocytic lymphohistiocytosis.	32419134	Familial hemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory syndrome affecting patients with genetic cytotoxicity defects. Perforin-deficient (PKO) mice recapitulate the full clinical picture of FHL after infection with lymphocytic choriomeningitis virus (LCMV). Hyperactivated CD8+ T cells and IFN-γ have been identified as the key drivers of FHL and represent targets for therapeutic interventions. However, the response of patients is variable. This could be due to trigger-dependent differences in pathogenesis, which is difficult to address in FHL patients, since the trigger frequently escapes detection. We established an alternative FHL model using intravenous infection of PKO mice with murine CMV (MCMV)Smith . PKO mice developed acute FHL after both infections and fulfilled HLH diagnostic criteria accompanied by excessive IFN-γ production by disease-inducing T cells, that enrich in the BM. However, direct comparison of the two infection models disclosed trigger-dependence of FHL progression and revealed a higher contribution of CD4 T cells and NK cells to IFN-γ production after MCMV infection. Importantly, therapeutic intervention by IFN-γ neutralization or CD8+ T-cell depletion had less benefit in MCMV-triggered FHL compared to LCMV-triggered FHL, likely due to MCMV-induced cytopathology. Thus, the context of the specific triggering viral infection can impact the success of targeted immunotherapeutic HLH control.
460	Macrophage activation syndrome in children with Kawasaki disease: diagnostic and therapeutic approaches.	32418074	Macrophage activation syndrome (MAS) is a rare, life-threatening complication of Kawasaki disease (KD). Early recognition and treatment of MAS are very important, but sometimes it is difficult to distinguish MAS from a severe form of KD.A PubMed search was performed in Clinical Queries using the key terms "macrophage activation syndrome or secondary hemophagocytic lymphohistiocytosis (HLH)" and "Kawasaki disease".KD patients with MAS show high intravenous immunoglobulin (IVIG) resistance and coronary complications. Mortality is also as high as MAS in other diseases. Persistent fever greater than 10 days is highly associated with development of MAS in KD. Splenomegaly is observed in more than two-thirds of KD patients with MAS. Thrombocytopenia is often the earliest laboratory finding of MAS. Hyperferritinemia is highly specific and sensitive for detecting MAS in KD; so, ferritin levels should be checked if there are unexplained clinical exacerbations in KD patients. Given the under-recognition of MAS in KD, it is prudent to consider resistant KD as occult/subclinical MAS. Many KD patients with MAS have good outcomes on immune modulators. However, if KD patients fulfill the HLH-2004 diagnostic criteria, they may undergo longer and more intensive treatment than needed.The possible existence of MAS should be taken into account when a KD patient shows persistent fever, splenomegaly, thrombocytopenia, hyperferritinemia, or IVIG resistance. The under-diagnosis of MAS in patients with KD is an important issue to be addressed. Therapeutically, however, there is a possibility of over-treatment of MAS in patients with KD.
461	Etoposide-based therapy for severe forms of COVID-19.	32416415	The new coronavirus infection COVID-19 has quickly become a global health emergency. Mortality is principally due to severe Acute Respiratory Distress Syndrome (ARDS) which relays only on supportive treatment. Numerous pathological, clinical and laboratory findings rise the similarity between moderate to severe COVID-19 and haemophagocytic lymphohistiocytosis (HLH). Etoposide-based protocol including dexametasone is the standard of care for secondary HLH. The protocol has been successfully used in HLHs that are secondary to EBV and H1N1 infections by inducing complete response and prolonged survival. These observations prompt to consider this cytotoxic therapy in HLH associated to moderately severe to severe forms of COVID-19.
462	Hemophagocytic Lymphohistiocytosis in Renal Transplant Recipients: A 2-Case Report.	32389490	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by an excessive immune activation. HLH can be triggered by a variety of events that disrupt immune homeostasis, such as infections and immunosuppression. HLH presents with heterogeneous clinical symptoms and laboratory findings such as pancytopenia, elevated liver enzymes, impaired renal function, and hyperferritinemia.Case 1. A 58-year-old man was admitted because of a high fever and diarrhea. Laboratory findings showed acute renal impairment, pancytopenia, Epstein-Barr virus (EBV) DNA seropositivity with a replication index of 2 million copies/mL, and hyperferritinemia. A diagnosis of HLH was confirmed by bone marrow aspiration. He was treated with etoposide, steroids, and rituximab with initial good response and good kidney function restoration. He was discharged after 31 days but eventually died after 44 days after a disease relapse. Case 2. A 51-year-old kidney transplant recipient was admitted because of a fever of unknown origin. A worsening renal function, pancytopenia, EBV DNA of 4,356,222 copies/mL on blood, D-Dimer 7505 ng/mL, ferritinemia 9180.9 ug/L, and triglycerides 1273 mg/dL were found. Bone marrow aspiration was negative for HLH; a few days later, a diagnosis of HLH was made after a positive bone marrow biopsy. Continuous renal replacement therapy was started in the intensive care unit because of severe lactic acidosis due to sepsis. She died few days later.EBV infection could be a trigger for HLH in a renal transplant patient. Hyperferritinemia is useful for differential diagnosis in a septic patient. The outcome is very poor even with prompt treatment.
463	Retrospective single-centre analysis of diagnostic approach to adult-onset haemophagocytic lymphohistiocytosis.	32388900	Haemophagocytic lymphohistiocytosis (HLH) is a rare disorder with a poor prognosis characterised by substantial immune activation leading to end-organ failure. In childhood, genetic defects that impair cytotoxic function of natural killer cells and T cells. (HLH) are often identified. In adults, clinical manifestations are similar to those observed in children but the aetiology is often unclear.To evaluate whether poor prognosis for adult HLH is in part due to lack of awareness of the disorder, which results in incomplete investigation and failure to implement timely treatment.We performed a retrospective case series of adult-onset HLH in a tertiary hospital in Australia. We evaluated clinical characteristics, treatment and outcome, and related these to application of standard diagnostic criteria for HLH.In our centre, incomplete assessment of HLH criteria was common. Serum ferritin was the criterion most commonly assessed. Hyperferritinaemia ≥10 000 μg/L was highly sensitive in detecting patients with adult-onset HLH; however, the majority of patients who had hyperferritinaemia ≥10 000 μg/L did not have adult-onset HLH.The present study highlights the importance of comprehensive application of diagnostic criteria to improve accuracy and timelines of the diagnosis of adult onset HLH.
464	Hemophagocytic lymphohistiocytosis: An update on pathogenesis, diagnosis, and therapy.	32387063	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening state of immune hyperactivation that arises in the setting of genetic mutations and infectious, inflammatory, or neoplastic triggers. Sustained, aberrant activation of cytotoxic CD8+ T cells and resultant inflammatory cytokine release are core pathogenic mechanisms. Key clinical features include high persistent fever, hepatosplenomegaly, blood cytopenia, elevated aminotransferase and ferritin levels, and coagulopathy. HLH is likely under-recognized, and mortality remains high, especially in adults; thus, prompt diagnosis and treatment are essential. Familial forms of HLH are currently treated with chemotherapy as a bridge to hematopoietic stem cell transplantation. HLH occurring in rheumatic disease (macrophage activation syndrome) is treated with glucocorticoids, IL-1 blockade, or cyclosporine A. In other forms of HLH, addressing the underlying trigger is essential. There remains a pressing need for more sensitive, context-specific diagnostic tools. Safer, more effective therapies will arise with improved understanding of the cellular and molecular mechanisms of HLH.
465	Targeted busulfan-based reduced-intensity conditioning and HLA-matched HSCT cure hemophagocytic lymphohistiocytosis.	3238454232818225	Reduced-intensity/reduced-toxicity conditioning and allogeneic T-cell replete hematopoietic stem cell transplantation are curative in patients with hemophagocytic lymphohistiocytosis (HLH). Unstable donor chimerism (DC) and relapses are clinical challenges . We examined the effect of a reduced-intensity conditioning regimen based on targeted busulfan to enhance myeloid DC in HLH. The European Society for Bone and Marrow Transplantation-approved reduced-intensity conditioning protocol comprised targeted submyeloablative IV busulfan, IV fludarabine, and serotherapy comprising IV alemtuzumab (0.5-0.8 mg/kg) for unrelated-donor and IV rabbit anti-T-cell globulin for related-donor transplants. We assessed toxicity, engraftment, graft-versus-host disease (GHVD), DC in blood cell subtypes, and overall survival/event-free survival. Twenty-five patients from 7 centers were treated (median age, 0.68 year). The median total dose and cumulative area under the curve of busulfan was 13.1 mg/kg (6.4-26.4) and 63.1 mg/L × h (48-77), respectively. Bone marrow, peripheral blood stem cell, or cord blood transplants from HLA-matched related (n = 7) or unrelated (n = 18) donors were administered. Donor cells engrafted in all patients (median: neutrophils d+20/platelets d+28). At last follow-up (median, 36 months; range, 8-111 months), the median DC of CD15+ neutrophils, CD3+ T cells, and CD16+56+ natural killer cells was 99.5% (10-100), 97% (30-100), and 97.5% (30-100), respectively. Eight patients (32%) developed sinusoidal obstruction syndrome, resolving after defibrotide treatment. The 3-year overall survival and event-free survival rates were both 100%. None of the patients developed acute grade III to IV GHVD. Limited chronic GVHD was encountered in 4%. This regimen achieves excellent results with stable DC in patients with HLH.
466	Epstein-Barr Virus Infection-associated Hemophagocytic Lymphohistiocytosis.	32382465	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening syndrome characterized by uncontrolled immune activation. There is an aberrant activation of lym-phocytes and macrophages that results in hypercytokinemia. We aim to describe a case of secondary HLH due to primary Epstein-Barr virus (EBV) infection. A Hispanic 28-year-old man presented with sore throat and fatigue for one week. He was diagnosed with mononucleosis and discharged and was treated according to the currently available treatment. HLH is treated by diminishing the inflammation by myelosuppressive and immunosuppressive therapy. EBV infection-associated HLH is a rare disease with high mortality. It is crucial to think about it when facing a patient with fever, cytopenia, hepatosplenomegaly, and high levels of ferritin. Despite medical treatment, the patient died from multiorgan failure.
467	Chemotherapy-sparing treatment of haemophagocytic lymphohistiocytosis with intravenous immunoglobulins and corticosteroids.	32376661	Haemophagocytic lymphohistiocytosis (HLH) can be a rapidly fatal disease. Current treatment in adults is extrapolated from the HLH-2004 protocol that specifies a regimen of etoposide, dexamethasone and cyclosporine. However, HLH presents as a spectrum of disease severity. A therapeutic challenge arises for milder cases where the harms of potent chemotherapy such as etoposide may outweigh its benefit. We present a case of an adult with HLH who developed significant pancytopenia but was otherwise not critically ill and who responded to treatment with a chemotherapy-sparing approach consisting of intravenous immunoglobulins and corticosteroids alone. The case illustrates that tailored therapy may allow effective treatment of the disorder while minimising therapy-related toxicities.
468	Fatal COVID-19 infections: Is NK cell dysfunction a link with autoimmune HLH?	3237640132268212	NaN
469	Genotype characteristics and immunological indicator evaluation of 311 hemophagocytic lymphohistiocytosis cases in China.	32375849	Primary hemophagocytic lymphohistiocytosis (pHLH) is a genetic disorder that is classically diagnosed by genetic testing. Secondary HLH (sHLH) is usually caused by infections, malignancies, or autoimmune disorders, but may display some mutations or polymorphisms. Rapid immunological assays examining natural killer (NK) cell activity, degranulation function (CD107a), and protein expression related to genetic deficiencies have been recommended for early pHLH identification.A retrospective analysis of 311 HLH patients from a Chinese population was performed to evaluate the potential correlations between genetic testing and rapid immunological assays; genotyping characteristics, age of onset, and etiology were examined.Among the 128 (128/311) patients who were positive in the genetic screening, the most frequently detected mutant gene was UNC13D (29%), followed by LYST (21%), PRF1 (17%), and STXBP2 (10%). Among pHLH patients (n = 39), the majority (67%) had PRF1 and UNC13D defects. FHL-2 was predominant (12/27, 44%) in patients aged under 18, while FHL-3 was the most common (6/12, 50%) in adults. Differences in genetic variant types and etiological components were noted in HLH patients based on the age of onset. NK cell activity and CD107a were observed to show a consistent trend (Ptrend < 0.001) when grouping patients according to the severity of the genetic variant type. Moreover, NK cell activity was generally consistent within a certain range of ΔCD107a values (Ptrend < 0.001). The PPV for bi-allelic degranulation gene mutations in patients with CD107a < 5% was 38.9% (7/18), while the PPV in patients with CD107a ≤10% was 16.7% (13/78). The PPV for pHLH was 41.4% (29/70) with NK cell activity ≤13%. To further evaluate the diagnostic efficacy of NK cell activity assay in pHLH, a receiver operating characteristic (ROC) curve was generated and showed an area under the curve (AUC) of 0.872, and the optimal cutoff value was determined to be 13.425% with a sensitivity of 84.21% and specificity of 80.67% when the corresponding Youden index was maximized. Flow cytometry screening for deficient proteins, including perforin, SAP, and XIAP, showed a relatively high sensitivity (83.33-93.33%). The positive predictive values (PPVs) of perforin and XIAP were relatively low (20.83-26.92%), but the negative predictive values (NPVs) for all three were excellent (all > 98%).Various immunological indicators have different clinical prediction and application values for the diagnosis of pHLH. The degree of reduction of immunological indicators also needs attention, and choosing appropriate cutoff value may be of important significance in guiding clinical judgment for pHLH.
470	Successful treatment of mycobacterial infection associated hemophagocytic lymphohistiocytosis with etoposide and anti-tuberculous therapy: a case report.	32370734	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially life-threatening disorder characterized by an exacerbated but ineffective inflammatory response, which can be classified as primary and secondary HLH. HLH associated with Mycobacterium tuberculosis is uncommon. This case report accounted an immunocompetent patient who was confirmed to be Mycobacterium infection, or rather, highly suspected tuberculosis (TB) associated HLH, with a favorable outcome.A 36-year-old man presented with persistent fever, pancytopenia, and hyperferritinemia. A bone marrow smear demonstrated hemophagocytosis, and pathological examination of lung biopsy was positive for acid-fast bacilli, which established the diagnosis of Mycobacterium infection and HLH. Then the patient treated successfully with anti-TB therapy, along with 8 weeks of etoposide.This case emphasizes that HLH should be kept in mind when clinicians encounter a patient with severe infection presenting with pancytopenia and hyperferritinemia. Given the high mortality, early diagnosis and appropriate therapy can provide patients with a favorable prognosis.
471	Functional and genetic testing in adults with HLH reveals an inflammatory profile rather than a cytotoxicity defect.	3235686132730578	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life as a result of monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infection, lymphoma, or rheumatic disease. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of natural killer (NK) cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtype, NK cell phenotype, perforin expression and degranulation, and natural or antibody-dependent cell cytotoxicity, in comparison with patients affected by the same underlying disease without HLH (disease controls [DCs]) and with healthy controls (HCs). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group and 34 each in the DC and HC groups. In HLH patients, severe and transient lymphopenia, activated NK cell phenotype (eg, increased CD69, ICAM-1, HLADR, and CCR5 expression), and decreased capacity of interferon γ production were observed; mean perforin expression was normal; and degranulation tests and NK cell cytotoxicity were not different from those in DCs. A monoallelic variant of uncertain significance affecting a lymphocyte cytotoxicity gene or the perforin variant A91V was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH patients compared with DCs and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon γ production seems similar to those of other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis.
472	Clinical and imaging features in adult patients with bone marrow haemophagocytosis with and without haemophagocytic lymphohistiocytosis: a single-institution experience.	32354424	To evaluate clinical, laboratory, imaging findings, and outcomes of adult patients with bone marrow haemophagocytosis (BMH) who meet the diagnostic criteria for haemophagocytic lymphohistiocytosis (HLH) with those who do not meet the criteria.A pathology database search was performed from 2009 to 2019 to identify adult patients with BMH. Electronic medical records of 41 patients were reviewed to distinguish those who fulfil the HLH-2004 diagnostic guidelines, which identified 22 patients (11 men; mean age, 53.5 years) who met the criteria (HLH+) and 19 patients (13 men; mean age, 54.7 years) who did not meet the criteria (HLH-). Multi-modality imaging was reviewed to record imaging features. Clinical, laboratory, imaging findings, and outcomes were compared between the two groups using Fisher's exact test and Wilcoxon test.Malignancy (non-Hodgkin's lymphoma) was the major trigger for both groups. 86% of HLH+ and 31% of HLH- patients presented with fever. Compared to the HLH- group, the HLH+ group exhibited higher serum ferritin, triglycerides, and lower fibrinogen levels (p<0.05). Alveolar opacities and hepatosplenomegaly were the most common imaging findings identified in both groups. Median overall survival of HLH+ and HLH- were 123.5 (interquartile range [IQR]: 40.7-681.7 days) and 189 days (IQR: 52-1680 days), respectively. Distribution of imaging features and overall survival did not differ between the groups.Malignancy is the major trigger for BMH in both HLH+ and HLH- groups. HLH+ and HLH- groups have similar imaging manifestations or clinical outcomes. Therefore, presence of BMH alone is correlated with high morbidity and mortality.
473	Soluble interleukin-2 receptor (sIL-2r) level is a limited test for the diagnosis of adult secondary hemophagocytic lymphohistiocytosis.	32353917	Soluble interleukin-2 receptor (sIL-2r) level is used as a diagnostic tool in hemophagocytic lymphohistiocytosis (HLH). However, evidence supporting its use among adults is inadequate.We conducted a retrospective study to assess the performance characteristics of sIL-2r for the diagnosis of adult HLH.One hundred thirty-two adults with sIL-2r levels sent for evaluation of HLH over a ten-year period were included. Sixty-five (49%) met criteria for HLH. Mean sIL-2r was significantly higher among patients with HLH relative to all patients without HLH (12942U/ml vs. 6308 U/mL, P = .00311). However, when comparing mean sIL-2r in the HLH group to those in the non-HLH group with primary diagnoses of hematologic malignancy (8911 U/mL), sepsis (7127 U/mL), and rheumatologic disease (4624 U/mL), no significant differences were found (P = .241, P = .178, and P = .0607, respectively). There was only weak correlation between sIL-2r and diagnosis of HLH (r = .253). The standard cutoff sIL-2r > 2400 U/ml yielded a sensitivity of 89.2% and specificity of 38.8%. The area under the curve for the corresponding receiver-operator curve was 0.691, consistent with a poor discriminating ability for the diagnosis of HLH.sIL-2r is a limited test for the diagnosis of adult secondary HLH, and its role in this setting should be reevaluated.
474	HLH Associated with Disseminated Tuberculosis.	32348646	NaN
475	Drs. Cron and Chatham reply.	32335514	We appreciate our Italian colleagues' interest in our editorial denoting the rheumatologist's role in helping to diagnose and treat cytokine storm syndrome (CSS) in the setting of the coronavirus disease 2019 (COVID-19) pandemic1 It is encouraging that none of the 123 pediatric rheumatology patients (primarily juvenile idiopathic arthritis) treated with background biological disease-modifying antirheumatic drug (bDMARD) therapies in Milan, Italy, surveyed over a 7-week period from February 25 through April 14, 2020 (during which time COVID-19 was hyperendemic there), had either confirmed or suspected COVID-192.
476	Case series of three adult patients with exceptional clinical presentations of haemophagocytic lymphohistiocytosis.	32332189	Macrophage activation syndrome (MAS) is a secondary form of haemophagocytic lymphohistiocytosis (HLH). MAS-HLH is an underrecognised and life-threatening condition associated with a heterogeneous group of diseases including connective tissue disease and inflammatory disorders. Here, we report three cases of adult patients with MAS-HLH triggered by different entities, including systemic lupus erythematosus, Griscelli syndrome type 2, and Adult onset Still's disease.
477	Hemophagocytic Lymphohistiocytosis in Patients With Primary Immunodeficiency.	32324696	Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled and excessive immune responses with high mortality. We aimed to define mortality-related parameters in HLH secondary to primary immunodeficiency (PID). A total of 28 patients with HLH between the years 2013 and 2017 were enrolled in the study. The patients were evaluated in 2 groups including PID with hypopigmentation (n=7) (Chédiak-Higashi syndrome [CHS] and Griscelli syndrome type 2 [GS2]) and other PIDs (n=21). The median age of the study population was 23 (4.3 to 117.0) months at the time of the diagnosis of HLH. Central nervous system involvement was recorded in 7 (GS2/CHS patients [n=4], other PIDs [n=3], P=0.026), and death was observed in 9 patients (GS2/CHS patients [n=1], other PIDs [n=8], P=0.371). Five patients (3 GS2/CHS and 2 other PID patients) underwent hematopoietic stem cell transplantation. Low serum albumin level was the only variable associated with the mortality and albumin levels less than the cut-off value of 3.07 g/dL increased mortality 5.8 times in patients with HLH secondary to PID. We presented a single-center experience consisting of patients with HLH secondary to PID with a mortality rate of 32.1%. Hypoalbuminemia was the only risk factor to increase the overall mortality rate of HLH.
478	IL6-R blocking with tocilizumab in critically ill patients with hemophagocytic syndrome.	3232156330992265	NaN
479	[Clinical Characteristics and Prognostic Analysis of 59 Adult Patients with Epstein-Barr Virus-Associated Hemophagocytic Syndrome].	32319412	To analyze the clinical characteristics and the prognostic risk factors of adult patients with Epestein-Barr virus-associated hemophagocytic syndrome (EBV-HLH) so as to enhance the understanding of EBV-HLH and diagnosis and treatment level.The clinical manifestation and survival data of 59 adult patients with EBV-HLH admitted in our hospital from January 2013 to August 2018 were analyzed retrospectively.The most common clinical manifestations of 59 patients were high fever (100%), liver dysfunction (91.5%), however the skin rashes (1.7%), and neurologic abnormality (3.4%) were rare. 96.6% of the patients showed the elevation of serum ferritin and LDH level, and hypoproteinemia and sCD25≥2 400 U/ml were found in 93.2% and 92.3% of the patients, respectively. The median survival time of 59 patients was 2.5±0.7 months; overall survival rate of 1, 3, 6 and 12-month was 69.5%±6.0%, 44.7%±6.6%, 23.9%±5.8%, 19.7%±5.5%, respectively. Univariate survival analysis showed that the patients with EBV-DNA copies≥5×105/ml (P＜0.05), LDH level≥600 U/L (P＜0.05) and Plt count＜20×109/L (P＜0.05) had poor prognosis, and there was statistically difference in the overall survival rate (P＜0.01) between HLH-94/2004 group and the group treated without etoposide (not HLH-94/2004). Multivariate analysis revealed that LDH level≥600 U/L (P＜0.05), Plt count＜20×109/L (P＜0.05) and treatment protocol (not HLH-94/2004) (P＜0.01) were independent prognostic risk factors in 59 patients with EBV-HLH.EBV-HLH assocites with severe clinical features, high mortality rate and poor prognosis of patients. EBV-DNA copies≥5×105/ml (P＜0.05), LDH level≥600 U/L (P＜0.05) and Plt count＜20×109/L (P＜0.05) are the poor prognostic factors, and the treatment with HLH-94/2004 protocol can effectively improve the survival of EBV-HLH patients, should be applied as early as possible.59例成人EB病毒相关噬血细胞综合征临床特点和预后分析.分析成人EB病毒相关噬血细胞综合征（EBV-HLH）患者的临床特点和预后危险因素，提高对该疾病的认识和诊治水平.对2013年1月至2018年8月间南昌大学第一附属医院诊治的EBV-HLH 59例成人患者的临床特点和生存资料进行回顾性综合分析.59例患者中以发热（100%）、肝功能异常（91.5%）最常见，皮疹（1.7%）、神经系统症状（3.4%）少见；血清铁蛋白升高（96.6%）、乳酸脱氢酶（LDH）升高（96.6%）、白蛋白（Alb）下降（93.2%）及可溶性CD25（sCD25）水平≥2400 U/ml（92.3%）均比较常见。本组患者中位生存时间为2.5±0.7个月，1、3、6、12个月总体生存率分别是69.5%±6.0%、44.7%±6.6%、23.9%±5.8%和19.7%±5.5%。生存资料单因素分析显示，初诊时EBV-DNA拷贝数≥5×105拷贝数/ml（P=0.028）、LDH水平≥600 U/L（P=0.012）、Plt计数＜20×109/L（P=0.022）的患者预后更差，系统使用HLH-94/2004方案治疗能有效改善EBV-HLH患者预后（P＜0.001）。多因素分析显示，LDH水平≥600 U/L（P=0.01）、Plt计数＜20×109/L（P=0.013）、未系统使用HLH-94/2004方案治疗（P＜0.001）均是影响患者生存时间的独立预后不良因素.EBV-HLH患者病情凶险，早期死亡率高，初诊时EBV-DNA拷贝数≥5×105/ml、LDH水平≥600 U/L、Plt计数＜20×109/L的患者预后不良，HLH-94/2004方案治疗能有效改善患者生存情况，应尽早系统治疗.
480	[Relationship between Single Nucleotide Polymorphisms of IL2RA, IL-10 Gene and Epstein-Barr Virus Associated Hemophagocytic Lymphohistiocytosisin in children].	32319410	To investigate the correlation of single nucleotide polymorphisms (SNPs) of IL2RA and IL-10 gene with the pathogenesis of Epstein Barr Virus associated hemophagocytic lymphohistiocytosis (EBV-HLH) in children and the effect of correlated SNPs on the prognosis of children with EBV-HLH.For EBV-HLH group (51 cases), EBV-associated infectious mononucleosis (EBV-IM) group (48 cases) and EBV-positive healthy children group (52 cases), the genotypes at rs2104286, rs12722489, rs11594656 of IL2RA gene and rs1800896, rs1800871 and rs1800872 of IL-10 gene were detected with the SNaPshot technique. The distribution differences of genotype frequency and allele frequency of each SNP in each group were analyzed, and the correlated SNPs were taken as the research object for survival analysis.the frequency of AA genotype at rs1800896 of IL-10 gene in EBV-HLH group was higher than that in IM group (58.8% vs 25.0%) and healthy control group (58.8% vs 26.9%). The frequency of A allele in EBV-HLH group was higher than that in IM group (74.5% vs 54.2%) and healthy control group (74.5% vs 57.7%). Similarly, the frequency of AA genotype at rs2104286 of IL2RA gene in EBV-HLH group was higher than that in both IM (54.9% vs 27.1%) and healthy control group (54.9% vs 25.0%). The frequency of A allele in EBV-HLH group was higher than that in both IM (70.6% vs 51.0%) and healthy control group (70.6% vs 46.2%). Kaplan-Meier survival curves of EBV-HLH children with different genotypes were not statistically significant.The polymorphism of rs1800896 in IL-10 gene and rs2104286 in IL2RA gene may be related with the incidence of EBV-HLH in children, and the AA genotype and A allele of the both sites may be the susceptible risk factors for EBV-HLH.IL2RA，IL-10基因单核苷酸多态性与儿童EBV-HLH相关性的研究.探讨IL2RA，IL-10基因单核苷酸多态性（SNPs）与儿童EBV-HLH发病的关系及关联SNPs对患儿预后的影响.对EBV-HLH 组（51例），EBV相关传染性单核细胞增多症（EBV-IM）组（48例）和EBV血清阳性的健康儿童组（52例），用SNaPshot基因分型检测技术检测IL2RA基因的rs2104286、rs12722489、rs11594656位点和IL-10基因的rs1800896、rs1800871、rs1800872位点的基因型，分析各SNP的基因型频率、等位基因频率在每组的分布差异；以关联SNPs进行生存分析.IL-10基因rs1800896位点AA基因型在EBV-HLH组的出现频率高于IM组（58.8% vs 25.0%）和健康对照组（58.8% vs 26.9%）；A等位基因在EBV-HLH组的出现频率高于IM组（74.5% vs 54.2%）和健康对照组（74.5% vs 57.7%）。IL-2RA基因 rs2104286位点AA基因型在EBV-HLH组的出现频率高于IM组(54.9% vs 27.1%)及健康对照组（54.9% vs 25.0%）；A等位基因在EBV-HLH组的出现频率高于IM组（70.6% vs 51.0%）及健康对照组（70.6% vs 46.2%）。不同基因型的EBV-HLH患儿的Kaplan-Meier生存曲线，差异无统计学意义.IL-10基因rs1800896位点及IL-2RA基因rs2104286位点多态性可能与儿童EBV-HLH发病相关，两位点的AA基因型、A等位基因可能是儿童EBV-HLH的易感危险因素.
481	[Clinical Characteristics and Prognostic Factors of Children with Non-Hodgkin's Lymphoma of Different Pathological Subtypes].	32319386	To investigate the clinical characteristics and prognostic factors of children with non-Hodgkin's lymphoma of different pathological subtypes.Ninety-three patients with newly-diagnosed childhood NHL in Fujian Medical University Union Hospital from March 2011 to September 2017 were salected. The diagnosis of patients was performed according to the World Health Organization classification of tumors 2008 ys. The chemotherapy regimens were based on immune phenotype, pathological type and clinical stages. The 5-years event-free survival rate (EFS) were calculated and analyzed by Kaplan-Meier method, and difference of survival rate between groups were compared. The possible factors influencing 5-years EFS was analyzed using Cox proportional hazards model.Among the 93 patients, male to female ratio was 2.88:1, the median age at diagnosis was 6 (0.9 to 13) years old. According to pathological types, Burkitt's lymphoma was the most common, follow by ALK+ anaplastic large-cell lymphoma (ALCL) and lymphoblastic lymphoma (LBL). Clinically, the most common initial symptoms observed at diagnosis were swelling of superficial lymph node, and abdominal pain and abdominal mass in mature B-cell neoplasms, and the swelling of mediastinal lymph nodes in LBL, and hemophagocytic syndrome (HPS) in mature T-cell and natural killer cell NHL. Seventy-nine cases completed 2 courses of induction chemotherapy, and 64 cases (81.01%) reached complete remission (CR). In a median follow up for 32.5(1.0-88.5) months, ten patients (11.90%) relapsed, the median relapsed time was 5.7(3.4-15.7) months. 5-year EFS rate in 84 patients received standardized treatments were (77.1±4.9)%. As compared with lymphoblastic lymphoma and extranodal NK/T cell lymphoma, there was a trend towards better outcomes in B-LBL, and mature B-cell neoplasms and ALK+ ALCL showing 5-year EFS was (86.2±5.2)% and (93.8±6.1)% vs (53.3%±16.1)% and (28.6±17.1)%. Univariate analysis showed that B symptoms, LDH level, secondary HLH, immunophenotype, pathological subtypes, clinical stage and whether reached CR after induction chemotherapy significantly correlated with prognosis. Cox regression analysis showed that no CR after 2 courses was an independent unfavorable prognostic factor (HR0.001, 95%CI: 0.000-0.122).The clinical characteristics and prognosis of patients with NHL of different pathological types are different. Whether reached CR after induction chemotherapy is the imdependent risk factor affecting the prognosis.儿童不同病理亚型非霍奇金淋巴瘤的临床特征及预后因素.研究儿童不同病理亚型非霍奇金淋巴瘤（NHL）的临床特征及预后影响因素.收集2011年3月到2017年9月期间福建医科大学附属协和医院收治的初发儿童NHL 93例，按照WHO-2008淋巴系恶性肿瘤分类标准诊断，根据不同免疫标记、病理类型及肿瘤分期选择化疗方案；采用Kaplan-Meier方法计算5年无事件生存率（EFS），用Cox比例风险模型对预后影响因素进行分析.93例NHL患儿中，男女比例为2.88∶1，中位年龄6（0.9-13）岁，病理亚型伯基特淋巴瘤（BL）、ALK+间变大细胞淋巴瘤（ALCL）、淋巴母细胞淋巴瘤（LBL）、弥漫大B细胞淋巴瘤（DLBCL）和结外NK/T细胞淋巴瘤（ENKTL）分别为35、17、17、11和7例。在临床上，浅表淋巴结肿大为最常见的首诊表现，而腹腔瘤块（尤其是回盲部）伴LDH明显升高最常见于成熟B-NHL（B-NHL），纵膈肿物最常见于LBL，噬血细胞综合征（HPS）最常见于成熟T/NK细胞型NHL。79例完成2个疗程诱导化疗并评估，总体完全缓解（CR）率为81.01%；中位随访时间为32.5（1.0-88.5）个月，10例复发，复发率为11.90%，中位复发时间为5.9（3.4-15.7）个月；总体5年EFS为（77.1±4.9）%，T-LBL、B-LBL、成熟B淋巴细胞淋巴瘤、ALK+ALCL及ENKTL的5年EFS分别为（53.5±16.1）%、100%、（86.2±5.2）%、（87.5±8.3）%和（28.6±17.1）%。单因素分析显示，B症状、LDH水平、继发HLH、免疫分型、病理亚型、临床分期、诱导化疗是否CR等因素影响患儿的预后。多因素分析显示，诱导化疗2个疗程能否CR为独立的预后危险因素（HR0.001, 95%CI:0.000-0.122）.不同病理亚型的儿童NHL临床特征及预后不同，诱导化疗后能否达CR是儿童NHL独立的预后因素.
482	Association of soluble interleukin-2 receptor alpha with laboratory parameters and clinical findings of hemophagocytic lymphohistiocytosis patients: The first report from South of Iran.	32318616	Hemophagocytic lymphohistiocytosis (HLH) is caused by overactivation of immune system. Gene mutations, infections, malignant, and autoimmune trigger the development of the disease.Clinical data and peripheral blood samples of 21 patients suspected of HLH were collected in Shiraz Medical Centers 2017-2018. Peripheral blood samples were analyzed for soluble interleukin-2 receptor alpha (sIL2Rα) marker (sCD25), and the results were compared with 36 normal controls as well as comparison with clinical findings and other laboratory parameters.Twenty-one patients (11 males and 10 females) with an average age of 5.2 were investigated. In this study, peripheral blood samples were taken from 16 newly diagnosed patients before treatment, and five were posttreatment blood samples. The mean sIL2Rα level before treatment in 16 patients was 9023 pg/ml. The mean peripheral blood sample of the 36 controls was 3025 pg/ml. The mean of the five posttreatment samples was 4198 pg/ml. Significant difference between pretreatment and the control group was observed. However, no significant difference was detected between after treatment samples and the control group. By comparing the sIL2Rα levels between patients with increased aspartate aminotransaminase (AST) and patients with normal AST level, there was a significant difference in the amount of IL2Rα level.This study highlights the importance of IL2Rα marker in the diagnosis and follow-up, during treatment and suppression. Furthermore, a significant difference with respect to AST level requires further investigation.
483	A Rare and Fatal Case of Hemophagocytic Lymphohistiocytosis Associated with Sarcoidosis.	32315294	BACKGROUND Sarcoidosis is a systemic inflammatory disorder characterized by a classic pathologic feature of non-caseating granulomas involving any organ system. Hemophagocytic lymphohistiocytosis (HLH) is a catastrophic cytokine surge characterized by dysregulation of the macrophage response, which can be rapidly fatal. Recognition of HLH has been increasing over the past decade. HLH can present with features of sepsis that can make the diagnosis challenging and requires high clinical suspicion. CASE REPORT We report a case of a 48-year-old African American male with a past medical history of sarcoidosis infiltrating the lymph nodes, liver, and bone marrow with initial presentation of abdominal pain, nausea, vomiting, and weight loss of 100 pounds over 8 months. Sepsis was suspected, but the patient clinically deteriorated with vancomycin and cefepime. Fevers, bone marrow biopsy, anemia, thrombocytopenia, elevated ferritin, and elevated soluble receptor interleukin 2 confirmed HLH. The patient was treated with etoposide and dexamethasone with poor response and died from cardiac arrest. CONCLUSIONS Sarcoidosis associated with HLH is an extremely rare phenomenon with only 10 cases reported in the literature. Early clinical suspicion can be challenging as this condition is a sepsis-mimicker. To reduce mortality, prompt initiation of therapy is a key determinant in patients who are clinically deteriorating despite treatment for sepsis.
484	Outcome analysis of pediatric hemophagocytic lymphohistiocytosis.	32307323	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease associated with rapid clinical deterioration and the need for intensive care; therefore, it is essential to identify clinical parameters related to mortality and establish prognostic factors correlated with unfavorable outcome in high risk patients whose treatment may fail.Between January 2004 and December 2018, a total of 51 pediatric patients (less than 18 years old) who fulfilled the diagnostic criteria of HLH-2004 with documented results of bone marrow investigations at Kaohsiung Chang Gung Memorial Hospital were enrolled. The treatment protocol was based on hemophagocytic lymphohistiocytosis-94 (HLH-94) and HLH-2004. We retrospectively reviewed electronic medical records (EMR) including clinical features, length of intensive care unit (ICU) stay, serological tests, microscopic reports of bone marrow examination, and ultrasound examination reports at diagnosis to identify prognostic factors. The patients were divided into four groups based on etiology; these included infection associated hemophagocytic syndrome (IAHS), macrophage activation syndrome (MAS), malignancy associated hemophagocytic lymphohistiocytosis (MA-HLH), and idiopathic hemophagocytic lymphohistiocytosis (IHLH) to identify differences among the groups.Out of 51 patients enrolled, 27 patients had IAHS, 12 MAS, 8 MA-HLH, and 4 IHLH. The median age at diagnosis was 7 years. The overall mortality rate was 15.7% (there was no mortality in the MA-HLH group); the mean length of ICU stay was 6 ± 20.8 days. Longer activated partial thromboplastin time (aPTT) (p = 0.007), lower sodium concentration (p = 0.0007), and higher creatinine (p = 0.032) and aspartate aminotransferase (AST) (p = 0.017) were significantly related to mortality. Multivariate Cox regression analysis demonstrated that aPTT (p = 0.045, HR = 1.03, 95% CI = 1.0-1.1) was an independent risk factor for mortality. The receiver operating characteristic (ROC) curve showed that aPTT longer than 44.35 s was the cutoff value predicting mortality, with a sensitivity and specificity of 72% and 66.7%, respectively.MA-HLH had the lowest mortality rate, as most children died from the underlying malignant disease and not from HLH. Impaired liver and renal functions were related to mortality. Prolonged aPTT > 44.35 s is a strong predictive factor for mortality.
485	Severe cerebral involvement in adult-onset hemophagocytic lymphohistiocytosis.	32307298	The diagnosis of hemophagocytic lymphohistiocytosis (HLH) with cerebral involvement is challenging given the rarity of HLH and its resemblance to the much more common severe sepsis. Timely diagnosis and treatment may be lifesaving. We report two cases demonstrating different and rare forms of severe brain involvement in adult patients with HLH: acute necrotizing encephalopathy, and diffuse hemorrhagic disease due to disseminated intravascular coagulation. Severe HLH with brain involvement in adults is rare. HLH with cerebral involvement should be considered in patients presenting with severe systemic inflammatory response syndrome (SIRS) but negative cultures and unusual or unexpectedly severe clinical and/or radiologic signs of cerebral dysfunction. Similar brain injury may occur in patients with cytokine storm syndrome due to COVID-19. BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) presents with fevers, rash, organomegaly, cytopenia, and increased triglycerides and ferritin (Ramos-Casals et al., 2014) [1]. Neurologic abnormalities are reported in about one-third of patients (Cai et al., 2017), including a few cases of acute necrotizing encephalopathy (ANE) (Xiujuan et al., 2015). Coagulation abnormalities are frequent in HLH patients (Valade et al., 2015). OBJECTIVE: To raise awareness about the importance of early diagnosis and treatment of HLH with neurological involvement to prevent serious complications and demise.
486	HLH-30/TFEB Is a Master Regulator of Reproductive Quiescence.	32302543	All animals have evolved the ability to survive nutrient deprivation, and nutrient signaling pathways are conserved modulators of health and disease. In C. elegans, late-larval starvation provokes the adult reproductive diapause (ARD), a long-lived quiescent state that enables survival for months without food, yet underlying molecular mechanisms remain unknown. Here, we show that ARD is distinct from other forms of diapause, showing little requirement for canonical longevity pathways, autophagy, and fat metabolism. Instead it requires the HLH-30/TFEB transcription factor to promote the morphological and physiological remodeling involved in ARD entry, survival, and recovery, suggesting that HLH-30 is a master regulator of reproductive quiescence. HLH-30 transcriptome and genetic analyses reveal that Max-like HLH factors, AMP-kinase, mTOR, protein synthesis, and mitochondrial fusion are target processes that promote ARD longevity. ARD thus rewires metabolism to ensure long-term survival and may illuminate similar mechanisms acting in stem cell quiescence and long-term fasting.
487	Hemophagocytic Lymphohistiocytosis Occurring in Inflammatory Bowel Disease: Systematic Review.	32300936	Hemophagocytic lymphohistiocytosis (HLH) is a rare and aggressive syndrome of excessive cytokine requiring prompt recognition and aggressive therapy.We aimed to systematically characterize HLH in moderate-to-severe inflammatory bowel disease (IBD).We performed a systematic review of the literature (PubMED; EMBASE) and FDA Adverse Event Reporting System in accordance with the PRISMA statement. Use of biologics was used as a surrogate definition for disease severity (consistent with usual and contemporary clinical management), to enable identification of rare HLH cases with the highest fidelity.58 cases of HLH occurring in IBD patients are known (mean age: 26.0 years, 70% male, 83% with Crohn's disease, mean disease duration 7.0 years). 34.5% of patients were undergoing induction therapy at HLH diagnosis. All cases occurred on patients exposed to anti-TNF agents, but cases with anti-integrin or anti-IL-12/23 exposure were reported. 2/3 of cases did not report prior AZA/6MP exposure. Underlying opportunistic infection or lymphoma was found in > 80% of cases. Survival was 70% if promptly recognized and treated. Five patients restarted biologics after HLH resolved, and one patient developed recurrent HLH.HLH is rare among IBD patients exposed to biologic therapy. Most cases had an identifiable infection or malignancy at the time of diagnosis as well as history of immunomodulator use. Risk factors may include younger age, male gender, presence of Crohn's disease, and induction phase of treatment. Our study is not intended to assess risk of HLH with specific IBD therapies.
488	Hemophagocytic Lymphohistiocytosis in Adults: Associated Diagnoses and Outcomes, a Ten-Year Experience at a Single Institution.	32300462	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive systemic inflammation which causes tissue damage due to abnormal immune system activation and has a high fatality rate even with treatment. HLH continues to be a difficult diagnosis to make because of lack of awareness and its overlap with other illnesses. This disorder is well defined in pediatric patients under the age of 18, but there is also a paucity of data in the adult population. The goals of this study were to describe associated disorders, clinical course and outcomes, and to determine if additional clinical criteria can be used to help with the diagnosis of HLH in adult patients.Patients' electronic medical records from 2007 to 2017 (who were ≥ 18 years old at the time of the search) were screened by ICD 9 and ICD 10 codes which contain the diagnosis of HLH or hemophagocytic syndrome, infection-associated. We identified 41 adult cases of HLH that were treated at our medical center over the course of 10 years and assessed these patients using both the historical and newly proposed diagnostic criteria. We also identified underlying diagnoses related to the development of HLH and fatality rates.Median age at diagnosis was 55 years old (18 - 87 years old) and 22 were male. Twenty-two had an infection, 16 with malignancy, seven had an autoimmune disorder and one with Sweet syndrome. Fifteen patients were treated with steroids alone, 18 with steroids and chemotherapy, three with steroids and antiviral agents, and two with chemotherapy alone. Sixteen (39%) died and 25 (60%) survived discharge. Seven patients died after discharge. Median survival of all patients was 1,095 days.Our data show that HLH is primarily associated with infections and malignancies. Newly proposed diagnostic criteria are not as specific, but can be helpful in making the diagnosis of HLH. We also showed that the fatality rate at our institution was lower than currently published rates of adult HLH.
489	Macrophage Activation Syndrome Versus Hemophagocytic Lymphohistiocytosis: A Diagnostic Dilemma in a Patient With Still's Disease and Epstein-Barr Virus Viremia.	32300447	Macrophage activation syndrome (MAS) and hemophagocytic lymphohistiocytosis (HLH) are two overlapping, potentially fatal syndromes classified by disorganization and malfunction of the immune system that results in wide spread inflammation and end-organ damage. We present the case of a 22-year-old female with both underlying adult-onset still's disease and active Epstein-Barr virus (EVB) viremia who presented with criteria for MAS/HLH. She ultimately improved on an immunosuppressive regimen, and during follow-up was also found to be heterozygote carrier for a known genetic mutation that has been associated with "primary" HLH. This case thus highlights the clinical spectrum of HLH/MAS, the different treatment approaches, and the new investigations into the relationship between primary and secondary HLH.
490	Diagnosis and Management of Secondary HLH/MAS Following HSCT and CAR-T Cell Therapy in Adults; A Review of the Literature and a Survey of Practice Within EBMT Centres on Behalf of the Autoimmune Diseases Working Party (ADWP) and Transplant Complications Working Party (TCWP).	32296434	Introduction: Secondary haemophagocytic lymphohistiocytosis (sHLH) or Macrophage Activation Syndrome (MAS) is a life-threatening hyperinflammatory syndrome that can occur in patients with severe infections, malignancy or autoimmune diseases. It is also a rare complication of haematopoetic stem cell transplantation (HSCT), with a high mortality. It may be associated with graft vs. host disease in the allogeneic HSCT setting. It is also reported following CAR-T cell therapy, but differentiation from cytokine release syndrome (CRS) is challenging. Here, we summarise the literature and present results of a survey of current awareness and practice in EBMT-affiliated centres of sHLH/MAS following HSCT and CAR-T cell therapy. Methods: An online questionnaire was sent to the principal investigators of all EBMT member transplant centres treating adult patients (18 years and over) inviting them to provide information regarding: number of cases of sHLH/MAS seen in their centre over 3 years (2016-2018 inclusive); screening strategies and use of existing diagnostic/classification criteria and treatment protocols. Results: 114/472 centres from 24 different countries responded (24%). We report estimated rates of sHLH/MAS of 1.09% (95% CI = 0.89-1.30) following allogeneic HSCT, 0.15% (95% CI = 0.09-5.89) following autologous HSCT and 3.48% (95% CI = 0.95-6.01) following CAR-T cell therapy. A majority of centres (70%) did not use a standard screening protocol. Serum ferritin was the most commonly used screening marker at 78% of centres, followed by soluble IL-2 receptor (24%), triglycerides (15%), and fibrinogen (11%). There was significant variation in definition of "clinically significant" serum ferritin levels ranging from 500 to 10,000 μg/mL. The most commonly used criteria to support diagnosis were HLH-2004 (43%) and the H score (15%). Eighty percent of responders reported using no standard management protocol, but reported using combinations of corticosteroids, chemotherapeutic agents, cytokine blockade, and monoclonal antibodies. Conclusions: There is a remarkable lack of consistency between EBMT centres in the approach to screening, diagnosis and management. Further research in this field is needed to raise awareness of and inform harmonised, evidence-based approaches to the recognition and treatment of sHLH/MAS following HSCT/CAR-T cell therapy.
491	The genetics of macrophage activation syndrome.	32291394	Macrophage activation syndrome (MAS), or secondary hemophagocytic lymphohistiocytosis (HLH), is a cytokine storm syndrome associated with multi-organ system dysfunction and high mortality rates. Laboratory and clinical features resemble primary HLH, which arises in infancy (1 in 50,000 live births) from homozygous mutations in various genes critical to the perforin-mediated cytolytic pathway employed by NK cells and cytotoxic CD8 T lymphocytes. MAS/secondary HLH is about ten times more common and typically presents beyond infancy extending into adulthood. The genetics of MAS are far less defined than for familial HLH. However, the distinction between familial HLH and MAS/secondary HLH is blurred by the finding of heterozygous perforin-pathway mutations in MAS patients, which may function as hypomorphic or partial dominant-negative alleles and contribute to disease pathogenesis. In addition, mutations in a variety of other pathogenic pathways have been noted in patients with MAS/secondary HLH. Many of these genetically disrupted pathways result in a similar cytokine storm syndrome, and can be broadly categorized as impaired viral control (e.g., SH2P1A), dysregulated inflammasome activity (e.g., NLRC4), other immune defects (e.g., IKBKG), and dysregulated metabolism (e.g., LIPA). Collectively these genetic lesions likely combine with states of chronic inflammation, as seen in various rheumatic diseases (e.g., still disease), with or without identified infections, to result in MAS pathology as explained by the threshold model of disease. This emerging paradigm may ultimately support genetic risk stratification for high-risk chronic and even acute inflammatory disorders. Moving forward, continued whole-exome and -genome sequencing will likely identify novel MAS gene associations, as well as noncoding mutations altering levels of gene expression.
492	Hemophagocytic Lymphohistiocytosis and Graft Failure Following Unrelated Umbilical Cord Blood Transplantation in Children.	32287100	Hemophagocytic lymphohistiocytosis (HLH) following hematopoietic stem cell transplantation is closely correlated with graft failure and poor prognosis. Because of its rarity, the incidence, risk factors, and optimal treatment strategy are unclear. We analyzed data from cases of HLH following umbilical cord blood transplantation (UCBT) performed for pediatric patients at our center. Among 66 UCBT recipients, 5 developed HLH and imminent graft failure. The median time of diagnosis of HLH was 22 (range, 19 to 30) days after UCBT, and the cumulative incidence of HLH was 7.6% (95% confidence interval, 2.8-15.7) at day 60. In univariate analysis, the cumulative incidence of HLH was significantly higher in patients with infused CD34 cells <1.0×10/kg than in patients with higher CD34 cells. Patients with preengraftment infection showed a trend toward higher incidence of HLH compared with patients without any infection. All 5 patients with HLH received corticosteroids and low-dose etoposide (VP-16), with or without high-dose intravenous immunoglobulin. Following these treatments, successful engraftment was observed in 2 patients. Corticosteroids and low-dose VP-16 may be worthy of a trial before attempting salvage hematopoietic stem cell transplantation. Further analyses are required to identify risk factors and to develop methods for prophylaxis, diagnosis, and treatment of HLH.
493	Hematopoietic stem cell transplantation in children with Griscelli syndrome type 2: a single-center report on 35 patients.	32286505	In 2010, we reported the outcome of hematopoietic stem cell transplantation (HSCT) in 11 children with Griscelli syndrome type 2 (GS2). We report here the update on this cohort to include 35 patients. Twenty-seven (77%) patients received conditioning regimen including busulfan, cyclophosphamide with etoposide. Eight (23%) were given busulfan, fludarabine. Thiotepa was added to busulfan and fludarabine regimen in two patients; one received haploidentical marrow and one unrelated cord blood. Posttransplant clinical events included veno-occlusive disease (n = 7), acute (n = 8), or chronic (n = 1) graft-versus-host disease II-IV. With a mortality rate of 37.1% (n = 13) and a median follow-up of 87.7 months of the survivors, 5-year cumulative probability of overall survival (OS) for our cohort of patients was 62.7% (±8.2%). Cumulative probability of 5-year OS was significantly better in those who did not have hemophagocytic lymphohistiocytosis (HLH) prior to HSCT (100% vs. 53.3 ± 9.5%, P value: 0.042). Of the 16 patients with neurologic involvement before HSCT, 8 survived and 3 presented sequelae. OS at 5-year was 50 ± 12.5% and 73.3 ± 10.2% (P value: 0.320) in patients with and without CNS involvement, respectively. In conclusion, HSCT in patients with GS2 is potentially curative with long-term disease-free survival. Early HSCT before the development of the accelerated phase is associated with a better outcome.
494	TIM-3 deficiency presenting with two clonally unrelated episodes of mesenteric and subcutaneous panniculitis-like T-cell lymphoma and hemophagocytic lymphohistiocytosis.	32285995	This report offers novel clinical and diagnostic aspects of the association between germline mutations in HAVCR2 and subcutaneous panniculitis-like T-cell lymphoma (SPTCL). The patient presented with panniculitis-like T-cell lymphoma involving mesenteric fatty tissue associated with hemophagocytic lymphohistiocytosis (HLH). Five years later, he developed a clonally unrelated SPTCL and underwent hematopoietic stem cell transplantation. Retrospectively, he was found to carry germline mutations in HAVCR2 associated with reduced T-cell immunoglobulin mucin-3 (TIM-3) expression. We show that mesenteric fatty tissue localization of SPTCL can be the presenting manifestation of TIM-3 deficiency, that this condition predisposes to recurrent lymphoma, and that flow cytometry is a possible screening tool.
495	C9orf72/ALFA-1 controls TFEB/HLH-30-dependent metabolism through dynamic regulation of Rag GTPases.	32282804	Nutrient utilization and energy metabolism are critical for the maintenance of cellular homeostasis. A mutation in the C9orf72 gene has been linked to the most common forms of neurodegenerative diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we have identified an evolutionarily conserved function of C9orf72 in the regulation of the transcription factor EB (TFEB), a master regulator of autophagic and lysosomal genes that is negatively modulated by mTORC1. Loss of the C. elegans orthologue of C9orf72, ALFA-1, causes the nuclear translocation of HLH-30/TFEB, leading to activation of lipolysis and premature lethality during starvation-induced developmental arrest in C. elegans. A similar conserved pathway exists in human cells, in which C9orf72 regulates mTOR and TFEB signaling. C9orf72 interacts with and dynamically regulates the level of Rag GTPases, which are responsible for the recruitment of mTOR and TFEB on the lysosome upon amino acid signals. These results have revealed previously unknown functions of C9orf72 in nutrient sensing and metabolic pathways and suggest that dysregulation of C9orf72 functions could compromise cellular fitness under conditions of nutrient stress.
496	Rheumatologists' perspective on coronavirus disease 19 (COVID-19) and potential therapeutic targets.	3227736732388748	The ongoing pandemic coronavirus disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a matter of global concern. Environmental factors such as air pollution and smoking and comorbid conditions (hypertension, diabetes mellitus and underlying cardio-respiratory illness) likely increase the severity of COVID-19. Rheumatic manifestations such as arthralgias and arthritis may be prevalent in about a seventh of individuals. COVID-19 can result in acute interstitial pneumonia, myocarditis, leucopenia (with lymphopenia) and thrombocytopenia, also seen in rheumatic diseases like lupus and Sjogren's syndrome. Severe disease in a subset of patients may be driven by cytokine storm, possibly due to secondary hemophagocytic lymphohistiocytosis (HLH), akin to that in systemic onset juvenile idiopathic arthritis or adult-onset Still's disease. In the absence of high-quality evidence in this emerging disease, understanding of pathogenesis may help postulate potential therapies. Angiotensin converting enzyme 2 (ACE2) appears important for viral entry into pneumocytes; dysbalance in ACE2 as caused by ACE inhibitors or ibuprofen may predispose to severe disease. Preliminary evidence suggests potential benefit with chloroquine or hydroxychloroquine. Antiviral drugs like lopinavir/ritonavir, favipiravir and remdesivir are also being explored. Cytokine storm and secondary HLH might require heightened immunosuppressive regimens. Current international society recommendations suggest that patients with rheumatic diseases on immunosuppressive therapy should not stop glucocorticoids during COVID-19 infection, although minimum possible doses may be used. Disease-modifying drugs should be continued; cessation may be considered during infection episodes as per standard practices. Development of a vaccine may be the only effective long-term protection against this disease.Key Points• Patients with coronavirus disease 19 (COVID-19) may have features mimicking rheumatic diseases, such as arthralgias, acute interstitial pneumonia, myocarditis, leucopenia, lymphopenia, thrombocytopenia and cytokine storm with features akin to secondary hemophagocytic lymphohistiocytosis.• Although preliminary results may be encouraging, high-quality clinical trials are needed to better understand the role of drugs commonly used in rheumatology like hydroxychloroquine and tocilizumab in COVID-19.• Until further evidence emerges, it may be cautiously recommended to continue glucocorticoids and other disease-modifying antirheumatic drugs (DMARDs) in patients receiving these therapies, with discontinuation of DMARDs during infections as per standard practice.
497	Reactivity oscillation in the heavy-light-heavy Cl + CH4 reaction.	32277024	It has long been predicted that oscillatory behavior exists in reactivity as a function of collision energy for heavy-light-heavy (HLH) chemical reactions in which a light atom is transferred between two heavy atoms or groups of atoms, but direct observation of such a behavior in bimolecular reactions remains a challenge. Here we report a joint theoretical and crossed-molecular-beam study on the Cl + CH4 → HCl + CH3 reaction. A distinctive peak at a collision energy of 0.15 eV for the CH3(v = 0) product was experimentally detected in the backward scattering direction. Detailed quantum-dynamics calculations on a highly accurate potential energy surface revealed that this feature originates from the reactivity oscillation in this HLH polyatomic reaction. We anticipate that such reactivity oscillations exist in many HLH reactions involving polyatomic reagents.
498	ngn-1/neurogenin Activates Transcription of Multiple Terminal Selector Transcription Factors in the Caenorhabditis elegans Nervous System.	32273286	Proper nervous system development is required for an organism's survival and function. Defects in neurogenesis have been linked to neurodevelopmental disorders such as schizophrenia and autism. Understanding the gene regulatory networks that orchestrate neural development, specifically cascades of proneural transcription factors, can better elucidate which genes are most important during early neurogenesis. Neurogenins are a family of deeply conserved factors shown to be both necessary and sufficient for the development of neural subtypes. However, the immediate downstream targets of neurogenin are not well characterized. The objective of this study was to further elucidate the role of ngn-1/neurogenin in nervous system development and to identify its downstream transcriptional targets, using the nematode Caenorhabditis elegans as a model for this work. We found that ngn-1 is required for axon outgrowth, nerve ring architecture, and neuronal cell fate specification. We also showed that ngn-1 may have roles in neuroblast migration and epithelial integrity during embryonic development. Using RNA sequencing and comparative transcriptome analysis, we identified eight transcription factors (hlh-34/NPAS1, unc-42/PROP1, ceh-17/PHOX2A, lim-4/LHX6, fax-1/NR2E3, lin-11/LHX1, tlp-1/ZNF503, and nhr-23/RORB) whose transcription is activated, either directly or indirectly, by ngn-1 Our results show that ngn-1 has a role in transcribing known terminal regulators that establish and maintain cell fate of differentiated neural subtypes and confirms that ngn-1 functions as a proneural transcription factor in C. elegans neurogenesis.
499	Intravenous Anakinra for Macrophage Activation Syndrome May Hold Lessons for Treatment of Cytokine Storm in the Setting of Coronavirus Disease 2019.	32267072	Macrophage activation syndrome (MAS) and hemophagocytic lymphohistiocytosis (HLH) are increasingly recognized as being on a continuum of cytokine storm syndromes, with different initiating pathways culminating in cytotoxic dysfunction and uncontrolled activation and proliferation of T lymphocytes and macrophages. The activated immune cells produce large amounts of proinflammatory cytokines, including interleukin 1β (IL)-1β. Management depends on the recognized diagnosis. In the setting of a cytokine storm syndrome and infection, collaborative involvement of specialists, including infectious disease and rheumatology is ideal. Anakinra, a recombinant IL-1 receptor antagonist, has been used subcutaneously and intravenously in pediatric patients and is considered a first-line treatment for MAS and secondary HLH (sHLH) among many pediatric rheumatologists. Previous reports of anakinra used in adults for treatment of MAS or sHLH are limited to subcutaneous administration. In this issue, Moneagudo et al. present a series of adult patients with sHLH treated with intravenous anakinra, including patients in whom subcutaneous anakinra was insufficient. As the authors suggest, there is a potential therapeutic use for anakinra in sHLH or the cytokine storm syndrome triggered by COVID19. Trial design will be key, with the patient subpopulation, timing of intervention, and doses tested important.
500	Mitochondrial translation and dynamics synergistically extend lifespan in C. elegans through HLH-30.	3225919932434220	Mitochondrial form and function are closely interlinked in homeostasis and aging. Inhibiting mitochondrial translation is known to increase lifespan in C. elegans, and is accompanied by a fragmented mitochondrial network. However, whether this link between mitochondrial translation and morphology is causal in longevity remains uncharacterized. Here, we show in C. elegans that disrupting mitochondrial network homeostasis by blocking fission or fusion synergizes with reduced mitochondrial translation to prolong lifespan and stimulate stress response such as the mitochondrial unfolded protein response, UPRMT. Conversely, immobilizing the mitochondrial network through a simultaneous disruption of fission and fusion abrogates the lifespan increase induced by mitochondrial translation inhibition. Furthermore, we find that the synergistic effect of inhibiting both mitochondrial translation and dynamics on lifespan, despite stimulating UPRMT, does not require it. Instead, this lifespan-extending synergy is exclusively dependent on the lysosome biogenesis and autophagy transcription factor HLH-30/TFEB. Altogether, our study reveals the mechanistic crosstalk between mitochondrial translation, mitochondrial dynamics, and lysosomal signaling in regulating longevity.
501	The aim of the measurement of Epstein-Barr virus DNA in hydroa vacciniforme and hypersensitivity to mosquito bites.	32255500	Epstein-Barr virus (EBV) DNA load in the blood increases in posttransplant lymphoproliferative disorders and chronic active EBV infection. In this report, we analyzed the EBV DNA load in the peripheral blood mononuclear cells (PBMCs) and plasma of patients with hydroa vacciniforme (HV) and/or hypersensitivity to mosquito bites (HMB) to understand the clinical significance of EBV DNA load. All 30 patients showed high DNA loads in the PBMCs over the cut-off level. Of 16 plasma samples, extremely high in two samples obtained from patients with hemophagocytic lymphohistiocytosis (HLH). The amount of cell-free DNA in plasma was correlated to the serum levels of lactate dehydrogenase and inversely correlated to platelet counts. These results indicate that the EBV DNA load in PBMCs can provide one of the diagnostic indicators for HV and HMB and marked elevation of cell-free EBV DNA in plasma might be related to cytolysis such as that observed in HLH.
502	Lentiviral Gene Therapy for Familial Hemophagocytic Lymphohistiocytosis Type 3, Caused by UNC13D Genetic Defects.	32253931	Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a rare disease caused by mutations to the UNC13D gene and the subsequent absence or decreased activity of the Munc13-4 protein. Munc13-4 is essential for the exocytosis of perforin and granzyme containing granules from cytotoxic cells. Without it, these cells are able to recognize an immunological insult but are unable to execute their cytotoxic functions. The result is a hyperinflammatory state that, if left untreated, is fatal. At present, the only curative treatment is hematopoietic stem cell transplantation (HSCT), but eligibility and response to this treatment are largely dependent on the ability to control inflammation before HSCT. In this study, we describe an optimized lentiviral vector that can restore Munc13-4 expression and degranulation capacity in both transduced FHL3 patient T cells and transduced hematopoietic stem cells from the FHL3 (Jinx) disease model.
503	The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like Disease.	3225171732376396323764003237640332380318	Severe COVID-19 associated pneumonia patients may exhibit features of systemic hyper-inflammation designated under the umbrella term of macrophage activation syndrome (MAS) or cytokine storm, also known as secondary haemophagocytic lymphohistocytosis (sHLH). This is distinct from HLH associated with immunodeficiency states termed primary HLH -with radically different therapy strategies in both situations. COVID-19 infection with MAS typically occurs in subjects with adult respiratory distress syndrome (ARDS) and historically, non-survival in ARDS was linked to sustained IL-6 and IL-1 elevation. We provide a model for the classification of MAS to stratify the MAS-like presentation in COVID-19 pneumonia and explore the complexities of discerning ARDS from MAS. We discuss the potential impact of timing of anti-cytokine therapy on viral clearance and the impact of such therapy on intra-pulmonary macrophage activation and emergent pulmonary vascular disease.
504	Changes in Cytokine Profile during Initial Treatment of Pediatric Hemophagocytic Lymphohistiocytosis Associated with Epstein-Barr Virus.	32238729	Hemophagocytic lymphohistiocytosis (HLH) associated with Epstein-Barr virus (EBV) infection can be self-limiting, severe/aggressive, or fatal. We report a case of EBV-HLH with persistent fever, severe pancytopenia, hypertriglyceridemia, and hypofibrinogenemia in a 4-year-old boy. Levels of plasma cytokines and chemokines were measured with a Bio-Plex system at 1, 2, 3, 4, 5, and 8 days after hospital admission. Administration of steroid and high-dose intravenous immunoglobulin (1 g/kg) did not alleviate fever or reduce cytokine production; however, after administration of etoposide (an antineoplastic agent), fever decreased immediately, the patient's general condition improved, and levels of IL-6, IL-10, IL-8, MCP-1, IFN-γ, and TNF-α declined after etoposide administration. In particular, IFN-γ production sharply declined, from 1,104.1 pg/mL to 101.5 pg/mL, and IL-6 level decreased from 229.8 pg/mL to 11.0 pg/mL, on the day after initial etoposide administration. There was no later recurrence of symptoms during treatment with dexamethasone, etoposide, and cyclosporine A. This case suggests that early etoposide administration is critical for treatment success and indicates that etoposide promptly inhibits cytokine production.
505	[Good's syndrome developing hemophagocytic lymphohistiocytosis following thymectomy].	32224589	This report presents the case of a 68-year-old female patient previously diagnosed with thymoma by her local doctor. She was referred to our hospital for surgery, and the thymoma was removed and diagnosed as a World Health Organization (WHO) classification type AB thymoma. After surgery, she experienced general malaise, a loss of appetite, and weight loss, so she visited our hospital in May 2019. A blood test showed hypogammaglobulinemia and low B lymphocytes. A bone marrow examination revealed no morphological abnormalities. Flow cytometric analysis indicated a marked decrease in both the B cell-related surface markers CD19 and CD20 and the T cell-related surface marker CD4, and the CD4/CD8 ratio was also low. She was diagnosed with Good's syndrome, and immunoglobulin replacement therapy was administered. She subsequently developed hemophagocytic lymphohistiocytosis (HLH) due to infection and was treated according to the HLH2004 protocol, but she finally succumbed to multiple organ damage as a result of sepsis. Given that Good's syndrome is associated with both humoral and cellular immune dysfunctions, affected patients tend to develop severe infections and have a poor prognosis. In such cases, early detection, regular immunoglobulin replacement therapy, and infection prevention therapies are important.
506	Elevated serum myoglobin levels at hospital admission and the risk of early death among patients with hemophagocytic lymphohistiocytosis: evidence from 155 pediatric patients.	32221652	Patients with hemophagocytic lymphohistiocytosis (HLH) have high risk of early mortality. The purpose of this study was to test the hypothesis that the elevated level of serum myoglobin among patients with HLH is associated with disease severity and increased risk of mortality. We retrospectively investigated the serum myoglobin levels from 155 pediatric patients diagnosed with HLH in the Hunan Children's Hospital, China. The levels of myoglobin and creatine kinase at hospital admission among non-survivors and survivors were compared. The myoglobin level was dichotomized for the estimation of hazard ratio (HR) for mortality. Patients who died within 7 and 30 days of hospitalization had significantly higher myoglobin levels than did survivors (p < 0.05). The myoglobin level was negatively associated with the days of survival among non-survivors (Spearman correlation coefficient = - 0.29, p = 0.04). An elevated myoglobin level (> 90 ng/mL) was significantly associated with increased mortality (unadjusted HR = 2.66, 95%CI: 1.41, 5.00, p = 0.0024) and persisted after adjusting for age, Epstein-Barr virus infection, admission department, acute kidney injury, myocardial damage, and shock. In conclusion, an elevated serum myoglobin level was associated with increased risk of early death among pediatric patients with HLH, suggesting the potential of myoglobin to be used as a reference indicator for monitoring and managing of HLH.
507	Hemophagocytic Lymphohistiocytosis in Children: Clinical Profile and Outcome.	32218098	The objectives of this study were to describe the clinical and etiologic profile and outcomes of children with hemophagocytic lymphohistiocytosis (HLH) in a tertiary care hospital in South India.This is a combined 2-year prospective (2017 to 2018) and 5-year retrospective (2012 to 2016) descriptive study in which children from birth to 18 years who satisfied the HLH-2004 diagnostic criteria were included. Case details from patient records were analyzed.Fifty-three cases were enrolled of which 20 were prospective and 33 were retrospective. Fever, hepatomegaly, anemia, and hyperferritinemia were the common presentations. Infectious triggers were found in 33 (62%) cases. Five cases were secondary to rheumatic diseases, and 8 were primary HLH. Bacterial (14 cases) followed by viral infections (10 cases) were the leading triggers. Scrub typhus (6 cases) and dengue (4 cases) were the most common infectious agents. Major complications include febrile neutropenia (38%) and multiorgan dysfunction (26%). One child developed secondary malignancy. The most frequently used immunosuppressive drug for the treatment of HLH was steroid (70%), while 28% of cases recovered with only supportive therapy. The overall mortality was 41%.Infections were the most common triggers for HLH of which tropical infectious agents constituted the majority. Treatment with steroids alone or regimens without cytotoxic drugs may result in resolution of secondary HLH with mild to moderate disease activity. Without stem cell transplant, primary HLH has a high mortality rate.
508	Severe Recurrent Fever Episodes With Clinical Diagnosis of Hemophagocytic Lymphohistiocytosis, Incomplete Kawasaki Disease and Systemic-Onset Juvenile Idiopathic Arthritis: A Case Report and Literature Review.	32211357	The pathogeneses of recurrent fever are quite complicated when excluding repeated infections. Recurrent fever is a common symptom for autoinflammatory diseases, relapse of Systemic-onset juvenile idiopathic arthritis (SoJIA) and recurrent Kawasaki disease (KD). There are no specific diagnostic laboratory tests for the diseases. Some studies showed that KD was the precursor of hemophagocytic lymphohistiocytosis (HLH). Macrophage activation syndrome (MAS) is another form of HLH in SoJIA. Cytokine disturbances are considered to be involved in the pathogenesis of the diseases. We describe a Chinese female toddler that developed three separate fever episodes with eventual diagnose of SoJIA within about 10 months. The first episode was diagnosed as IKD, immunoglobulin nonresponsive KD, and HLH. The second and third episodes were diagnosed as IKD and SoJIA, respectively. The fever was hard to be relieved by antipyretics, and the peak axillary temperature was above 40°C. For every fever episode, infections were excluded. For the first episode, trends over time of hemoglobin, platelets, fibrinogen, and triglycerides indicated HLH, which was finally diagnosed and treated according to the HLH-2004 protocol. For the second episode 6 months later, after excluding an HLH relapse and infections, IKD was finally diagnosed. Oral aspirin was administered, and the HLH treatment was ceased. The third episode occurred 3 months later, and SoJIA was finally diagnosed. For each episode, except for relative tests, we only tested for cytokines interleukin-1β, interleukin-6, and interferon-γ, due to limited laboratory test availability. These cytokines were elevated during remission and rose much higher in the fever phases. The case showed the difficulty to differentiating the recurrent fever in clinical practice. Surveillance of routine laboratory parameters over time might reveal a trend that indicates possible disease, even when parameter values do not meet diagnostic criteria. Changes in cytokine profiles are promising markers for differentiating recurrent fever diseases in future. An unknown immunological defect for the case may contribute to the recurrent immunological insults, and we are following up the recurrence of fever episode.
509	Tuberculosis and Tumor Lysis Syndrome-Coincidence or Coexistent: A Case Report.	32205951	Extrapulmonary tuberculosis (TB) involving bone marrow can present with various manifestations, including pancytopenia, maturation arrest, hemophagocytic lymphohistiocytosis (HLH), or infiltration of the bone marrow by caseating or noncaseating granulomas causing reversible or irreversible fibrosis. Tumor lysis syndrome (TLS) is an oncologic emergency resulting from massive tumor cell lysis. Children with TB with bone marrow involvement may also present with laboratory features of TLS resulting from high catabolism and concomitant acute kidney injury (AKI), making the diagnosis difficult at times. We present a case of disseminated TB who presented to emergency with pancytopenia, AKI, and laboratory features of TLS.Swami V, Lalitha AV, TK Anjan kumar. Tuberculosis and Tumor Lysis Syndrome-Coincidence or Coexistent: A Case Report. Indian J Crit Care Med 2020;24(2):145-147.
510	Redox-mediated regulation of aging and healthspan by an evolutionarily conserved transcription factor HLH-2/Tcf3/E2A.	32203922	Physiological aging is a complex process, influenced by a plethora of genetic and environmental factors. While being far from fully understood, a number of common aging hallmarks have been elucidated in recent years. Among these, transcriptomic alterations are hypothesized to represent a crucial early manifestation of aging. Accordingly, several transcription factors (TFs) have previously been identified as important modulators of lifespan in evolutionarily distant model organisms. Based on a set of TFs conserved between nematodes, zebrafish, mice, and humans, we here perform a RNA interference (RNAi) screen in C. elegans to discover evolutionarily conserved TFs impacting aging. We identify a basic helix-loop-helix TF, named HLH-2 in nematodes (Tcf3/E2A in mammals), to exert a pronounced lifespan-extending effect in C. elegans upon impairment. We further show that its impairment impacts cellular energy metabolism, increases parameters of healthy aging, and extends nematodal lifespan in a ROS-dependent manner. We then identify arginine kinases, orthologues of mammalian creatine kinases, as a target of HLH-2 transcriptional regulation, serving to mediate the healthspan-promoting effects observed upon impairment of hlh-2 expression. Consistently, HLH-2 is shown to epistatically interact with core components of known lifespan-regulating pathways, i.e. AAK-2/AMPK and LET-363/mTOR, as well as the aging-related TFs SKN-1/Nrf2 and HSF-1. Lastly, single-nucelotide polymorphisms (SNPs) in Tcf3/E2A are associated with exceptional longevity in humans. Together, these findings demonstrate that HLH-2 regulates energy metabolism via arginine kinases and thereby affects the aging phenotype dependent on ROS-signaling and established canonical effectors.
511	Propionic acidemia: an extremely rare cause of hemophagocytic lymphohistiocytosis in an infant.	32199059	Hemophagocytic lymphohystiocytosis (HLH) may be primary (inherited/familial) or secondary to infections, malignancies, rheumatologic disorders, immune deficiency syndromes and metabolic diseases. Cases including lysinuric protein intolerance, multiple sulfatase deficiency, galactosemia, Gaucher disease, Pearson syndrome, and galactosialidosis have previously been reported. It is unclear how the metabolites trigger HLH in metabolic diseases. A 2-month-old infant with lethargy, pallor, poor feeding, hepatosplenomegaly, fever and pancytopenia, was diagnosed with HLH and the HLH-2004 treatment protocol was initiated. Analysis for primary HLH gene mutations and metabolic screening tests were performed together; primary HLH gene mutations were negative, but hyperammonemia and elevated methyl citrate were detected. Propionic acidemia was diagnosed with tandem mass spectrometry in neonatal dried blood spot. We report this case of HLH secondary to propionic acidemia. Both metabolic disorder screening tests and gene mutation analysis may be performed simultaneously especially for early diagnosis in infants presenting with HLH.La linfohistiocitosis hemofagocítica (LHH) puede ser primaria (hereditaria) o secundaria a infecciones, tumores malignos, trastornos reumatológicos, síndromes de inmunodeficiencia y metabolopatías. Se informaron casos de intolerancia a la proteína lisinúrica, deficiencia de múltiples sulfatasas, galactosemia, enfermedad de Gaucher, síndrome de Pearson y galactosialidosis. No se sabe cómo se desencadena la LHH en las metabolopatías. Se diagnosticó LHH en un lactante de 2 meses con letargo, palidez, alimentación deficiente, hepatoesplenomegalia, fiebre y pancitopenia, y se instauró el protocolo HLH-2004. Se realizaron, en conjunto, análisis para detectar mutaciones genéticas y pruebas metabólicas; los resultados fueron negativos para las mutaciones genéticas de LHH primaria, pero se detectaron hiperamoniemia y concentración elevada de metilcitrato. Se diagnosticó acidemia propiónica. Aquí informamos sobre un caso de LHH secundaria a acidemia propiónica. Es posible la realización simultánea de pruebas de detección de trastornos metabólicos y de mutaciones genéticas para el diagnóstico temprano en los lactantes con LHH.
512	Associations between PRF1 Ala91Val polymorphism and risk of hemophagocytic lymphohistiocytosis: a meta-analysis based on 1366 subjects.	32198610	Perforin (PRF1) gene mutation can cause the onset of hemophagocytic lymphohistiocytosis (HLH). It has reported that PRF1 Ala91Val polymorphism was related with HLH risk. In the meta-analysis, we aim to evaluate the association between PRF1 Ala91Val polymorphism and HLH risk.We accomplished a meta-analysis of six published case-control studies including 391 patients with HLH and 975 controls. We evaluated the quality of each study through Newcastle-Ottawa Scale (NOS). Data analysis was performed with Stata software.In general, all studies were of high quality (NOS score higher than 7). There were statistically significant between the PRF1 Ala91Val polymorphism and HLH risk though the pooled analysis [for Ala/Val vs. Ala/Ala: pooled odds ratio (OR) = 3.22, 95% confidence interval (CI) 1.08-9.56, P = 0.035, random model; for Ala/Val + Val/Val vs. Ala/Ala: pooled OR = 2.96, 95% CI 1.14-7.69, P = 0.025, random model]. Furthermore, sensitivity analysis also revealed a relationship between PRF1 Ala91Val polymorphism and HLH risk (for Ala/Val vs. Ala/Ala: pooled OR = 5.236, 95% CI 2.72-10.08, P < 0.000, I2 = 12.1%, Pheterogeneity = 0.332; for Ala/Val + Val/Val vs. Ala/Ala, pooled OR = 4.856, 95% CI 2.66-8.85, P < 0.000, I2 = 5.9%, Pheterogeneity = 0.373). Funnel plot and Egger's test did not indicate obvious published bias (P = 0.841 for Ala/Val vs. Ala/Ala; P = 0.284 for Ala/Val + Val/Val vs. Ala/Ala).This meta-analysis indicated that PRF1 Ala91Val polymorphism affects the factor for developing HLH and future studies of PRF1 Ala91Val on the onset of HLH will be guaranteed.
513	Hemophagocytic syndrome with severe sepsis caused by Capnocytophaga canimorsus.	32197716	Capnocytophaga canimorsus, commonly transmitted by dog bites, can cause severe sepsis, and the mortality rate is very high. We experienced a case of hemophagocytic lymphohistiocytosis (HLH) complicated by severe sepsis caused by C. canimorsus. A 58-year-old man with no remarkable medical history was admitted to another hospital with fever and mild consciousness disorder developed 3 days after being bitten by his dog. The next day, the patient developed disseminated intravascular coagulation and shock and was transferred to our emergency medical center. Blood tests showed hyperferritinemia and cytopenia, and bone marrow aspiration was performed. As a result, we diagnosed severe sepsis and HLH. Once antibiotic and steroid therapy was started, the patient's infection and cytopenia improved. Unfortunately, the patient's fingers and toes required amputation, but his life was saved, and he was discharged from hospital. Because HLH may be hidden in such cases, it may be necessary to measure serum ferritin and perform bone marrow aspiration if hyperferritinemia is suspected.
514	Adult Hemophagocytic Lymphohistiocytosis in Sub-Saharan Area: A Retrospective Study of 26 cases.	32195069	Introduction and background Hemophagocytic lymphohistiocytosis (HLH) is a condition caused by inappropriate stimulation of macrophage cells with hemophagocytosis. This paper aims to describe its diagnostic specifics and etiology and seeks to identify the factors that affect its prognosis in the black African adult population. Methods  A retrospective multicentre study was carried out in three medical units in Senegal: Department of Internal Medicine at Pikine Teaching Hospital, and Department of Internal Medicine and Department of Nephrology at Aristide Le Dantec Teaching Hospital; the study covered the period from January 1, 2012 to March 30, 2015. This study included patients aged 18 years and older with a Hemophagocytosis Score (HScore) of ≥202 (with probabilities of acquired HLH of >90%). The data was obtained through medical records. Results In total, 26 patient files were included. The average age of the patients was 41 years, with a male-to-female ratio of 2.25:1. Fever and cytopenia were frequent. Other clinical signs included peripheral lymphadenopathy (69.2%), hepatomegaly (53.8%), splenomegaly (34.6%), neurological disorders (34.5%), and respiratory disorders (15.3%). Thrombocytosis was noted in three cases. Renal involvement was present in eight patients, with one case of collapsing glomerulopathy. The bone marrow aspirate revealed myelodysplasia in 12 patients. The dominant etiologies of HLH were hematological malignancies and infections. The mortality rate of HLH was 73%. Male gender and non-etiological targeted therapy were significantly associated with mortality. However, the age of <40 years in patients and current systemic disease in some cases were correlated with survival. The use of etoposide had no significant impact on the prognosis of our patients. Conclusion A high rate of male predominance, important central nervous system disorders, myelodysplasia, and paradoxical thrombocytosis were found to be the distinct features of adult HLH in our study population.
515	Molecular characterization and functional analysis of IKKα in orange-spotted grouper (Epinephelus coioides).	32194248	Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as EcIKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKβ-NEMO-binding domain, was identified in EcIKKα. Phylogenetic analysis suggested that EcIKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with EcIKKα plasmid. Moreover, overexpression of EcIKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that EcIKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens.
516	COVID-19: consider cytokine storm syndromes and immunosuppression.	32192578322202783223897632378064324235853263183632668194	NaN
517	Unusual accumulation of a wide array of antimicrobial resistance mechanisms in a patient with cytomegalovirus-associated hemophagocytic lymphohistiocytosis: a case report.	32192451	Infections with multidrug-resistant organisms (MDRO) pose a serious threat to patients with dysregulated immunity such as in hemophagocytic lymphohistiocytosis (HLH), but such infections have rarely been comprehensively characterized. Here, we present a fatal case of HLH secondary to cytomegalovirus (CMV) infection complicated by both anti-viral drug resistance and sepsis from multiple MDROs including pandrug-resistant superbug bacteria.A previously healthy, six-year-old boy presented with a 45-day history of fever prior to a diagnosis of hemophagocytic lymphohistiocytosis and hemorrhagic colitis, both associated with CMV. On hospital admission, the patient was found to be colonized with multiple, multidrug-resistant (MDR) bacteria including vancomycin-resistant enterococci (VRE) and carbapenamase-producing organisms (CPO). He eventually developed respiratory, urine and bloodstream infections with highly drug-resistant, including pandrug-resistant bacteria, which could not be controlled by antibiotic treatment. Antiviral therapy also failed to contain his CMV infection and the patient succumbed to overwhelming bacterial and viral infection. Whole genome sequencing (WGS) of the MDR bacteria and metagenomic analysis of his blood sample revealed an unusual accumulation of a wide range of antimicrobial resistance mechanisms in a single patient, including antiviral resistance to ganciclovir, and resistance mechanisms to all currently available antibiotics.The case highlights both the risk of acquiring MDR superbugs and the severity of these infections in HLH patients.
518	Hemophagocytic lymphohistiocytosis with advanced malignant melanoma accompanied by ipilimumab and nivolumab: A case report and literature review.	32191382	Combination therapy with nivolumab + ipilimumab was recently approved for treating unresectable cases of malignant melanoma. In spite of the high response rate, it is associated with a high incidence of serious adverse events, including immune-related hemophagocytic syndrome/hemophagocytic lymphohistiocytosis (irHPS/HLH), a difficult to diagnose rare disease. This is the first report of this disease in an Asian malignant melanoma patient treated with nivolumab + ipilimumab. A 69-year-old Japanese woman with unresectable malignant melanoma was treated with nivolumab + ipilimumab. Following the combined therapy, her fever and symptoms of malaise occurred, and she visited to our hospital's emergency department. Blood tests revealed significant liver dysfunction, anemia, and thrombocytopenia. We suspected irHPS/HLH, based on tests revealing decreased fibrinogen and significantly increased ferritin. Bone marrow biopsy revealed numerous macrophages and high hemophagocytosis levels. After 50 mg prednisolone (1 mg/kg per day) was administered, fever and cytopenia markedly improved. irHPS/HLH has a high rate of coagulation abnormalities accompanied by hypertriglyceridemia and hypofibrinogenemia, which are unlikely to occur in adult HPS/HLHs. Because irHPS/HLH responds better to steroids than other secondary HPS/HLHs, we expect a complete cure with steroids. Quick diagnosis and appropriate treatment based on clinical symptoms and laboratory tests are needed in suspected cases.
519	Ldb1 is required for Lmo2 oncogene-induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia.	32181817	Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.
520	Coagulation Disorders in Hemophagocytic Lymphohistiocytosis/Macrophage Activation Syndrome.	32172822	Hemophagocytic lymphohistiocytosis (HLH) is a rare and severe condition that can lead patients to the intensive care unit. HLH diagnosis may be challenging, as it relies on sets of aspecific criteria. Several organ dysfunctions have been described during HLH, including hemostasis impairment found in more than half of the patients. The most frequently reported anomaly is a decrease in the fibrinogen level, which has been associated with higher mortality rates. Coagulation impairment study in patients with HLH represents an interesting field of research, as little is known about the mechanism leading to hypofibrinogenemia.
521	Q fever as a rare cause of hemophagocytic lymphohistiocytosis: Case report.	32171685	Hemophagocytic Lymphohistiocytosis (HLH) is a reactive disorder of the mononuclear phagocytic system characterized by increased histiocytic proliferation, activation and hemaphagocytosis. The underlying etiology may be genetic (primary) or acquired (secondary). Secondary causes include drugs, autoimmune diseases, malignancies and infections of which EBV is the most common. A 28-year old male patient who was a shepherd with no known concomitant comorbid disease was admitted to the Emergency Department with the complaints of abdominal pain, fever, severe fatigue. Physical examination revealed high fever, hepatosplenomegaly and laboratory examination revealed pancytopenia, hyperferritinemia and hypertriglyceridemia. Hemophagocytes were observed in the bone marrow biopsy and the patient was diagnosed as HLH. The patient was treated with cyclosporine A, dexamethasone, intravenous immunoglobulin (IvIg) and etoposide according to the HLH 2004 protocol. Coxiella burnetii was detected in the serological evaluation of the etiology and doxycycline was added to the current treatment. Fever was controlled in the second week of the treatment and the patient was discharged after complete recovery of the cytopenia in the fourth week. In the outpatient setting, treatment was completed in 8 weeks and follow-up of the patient is still ongoing without medication. To the best of our knowledge, this is the first case from Turkey of HLH secondary to Q-fever which was treated and managed successfully. Since the mortality of HLH is quite high, the etiology should be determined as soon as possible to be able to provide appropriate treatment.
522	Development and validation of the prognostic value of ferritin in adult patients with Hemophagocytic Lymphohistiocytosis.	32164748	Hemophagocytic Lymphohistiocytosis (HLH) is a rare clinical syndrome with high mortality rate. The diagnosis of HLH draws on a constellation of clinical and laboratory abnormalities including extremely high serum ferritin levels. However, no biomarker has been firmly established as a clinically useful prognostic tool in HLH patients. We aimed to perform a retrospective analysis of two independent cohorts to explore the prognostic value of discharge serum ferritin for newly diagnosed adult HLH patients who recently started treatment. The prognostic value of serum ferritin levels at discharge (will be called as post-treatment ferritin level) was initially evaluated in a "test cohort" of 161 previously untreated consecutive adult HLH patients. It was then validated in a second cohort of 68 consecutive previously untreated patients (validation cohort).Multivariate analysis revealed that significantly high post-treatment serum ferritin levels (>1050 μg/L) were associated with a higher risk of death and poor overall survival in the test cohort (hazard ratio (HR): 3.176, 95% confidence interval (CI) 1.468-6.869, P = 0.003), and the validation cohort (HR: 13.412, 95%CI 1.716-104.816, P = 0.013). At 6-month follow-up period in the test cohort, patients with a > 81% decrease in the serum ferritin level had a significantly higher probability of survival when compared with the patients with ≥14% increase in the serum ferritin level (94% vs. 31%, P < 0.001). Similar findings were observed on the analysis of the decrease in the serum ferritin level in the validation cohort.These results suggest that the serum ferritin level can be used as an independent prognostic marker in the adult HLH patients.
523	Checkmate for EBV-HLH.	3216355931914172	NaN
524	Clinical outcomes of adults with hemophagocytic lymphohistiocytosis treated with the HLH-04 protocol: a retrospective analysis.	32157935	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of pathologic immune activation in children that is increasingly being recognized in adults. Efficacy data for the HLH-04 protocol in adults is lacking. This study retrospectively analyzed 31 adult patients, median age 46 years, who received HLH-04 from 1/1/2004 to 5/1/2018. HLH etiology included malignancy (n = 9), autoimmune (n = 8), infection (n = 8), and idiopathic (n = 6). Eighteen patients were evaluable for response at week 4 with 7 having no response, 11 reaching partial response, and 0 reaching complete response (CR). Six patients eventually achieved CR at a median 195 days. The 1-year overall survival (OS) was 35% and median OS was 3.2 months. Univariate analysis showed shorter survival for hemoglobin <9 g/dL (HR 4.29, p = 0.003), platelets <100 × 109/L (HR 4.06, p = 0.027), ANC <1 × 109/L (HR 5.24, p = 0.001), and total bilirubin >1.2 mg/dL (HR 3.30, p = 0.022). Outcomes of adults treated with HLH-04 remain dismal and newer treatment modalities are needed.
525	A neonatal case of coxsackievirus B3 vertical infection with symptoms of hemophagocytic lymphohistiocytosis.	32154109	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of excessive immune activation that most commonly affects infants. We report the case of a term female neonate with HLH associated with coxsackievirus B3 Infection. Her mother was hospitalized due to high fever 4 days before the delivery. The patient was delivered by vaginal delivery after the induction of labor. She was admitted to the neonatal care unit due to continuous high fever and poor sucking on her 4th day of life. She developed apnea on her 5th day of life. Laboratory findings on the patient's 7th day of life indicated severe thrombocytopenia, liver dysfunction, coagulation abnormality and hyperferritinemia. Coxsackievirus B3 was isolated from all cultured specimens by the PCR method. She received intravenous transfusion of platelets and immunoglobulin. Her platelet count gradually increased to the normal range by her 14th day of life and she was discharged without any sequelae on her 25th day of life. To the best of our knowledge, this is the first case report of neonatal HLH associated with a vertical transmission of coxsackievirus B3. Coxsackievirus is an important virus that can cause HLH in neonates. An early diagnosis and timely treatment are crucial.
526	Hemophagocytic Lymphohistiocytosis Related to Tuberculosis Disease.	32148351	Hemophagocytic lymphohistiocytosis (HLH) is a rare, albeit potentially fatal, condition in which fever, hepatosplenomegaly, and cytopenia predominate the clinical picture. Although it may be primary, it may also develop secondary to various etiologies. Herein, we aimed to report a patient who was diagnosed with pulmonary tuberculosis, developed fever and cytopenia during follow-up, and received immunomodulatory therapy together with antituberculosis therapy for the diagnosis of HLH. Sequencing of PRF1 showed heterozygous mutation. Although primary HLH has been detected in infants and children, genetic mutation of genes should be considered a differential diagnosis of HLH even in the adolescent.Erdoğan S, Çakır D, Bozkurt T, Karakayalı B, Kalın S, Koç B, et al. Hemophagocytic Lymphohistiocytosis Related to Tuberculosis Disease. Indian J Crit Care Med 2020;24(1):63-65.
527	[Analysis of five cases of hepatitis associated aplastic anemia presenting with hemophagocytic lymphohistiocytosis at onset].	32135593	Objective: To discuss the clinical characteristics and management approaches to hepatitis associated aplastic anemia (HAAA) presenting as hemophagocytic lymphohistiocytosis (HLH) at onset. Methods: The clinical data and laboratory results of hospitalized 5 HAAA patients presenting as HLH at onset in Beijing Children's Hospital from January 2017 to May 2019 were analyzed retrospectively. Results: Among 5 cases, there were 4 males and 1 female. The age of onset was 6.0 (2.7-12.7) years. All patients presented with high fever, hepatomegaly, hepatic dysfunction (aspartate aminotransferase 1 716 (1 409-2 570) U/L, alanine aminotransferase 1 699 (937-2 540) U/L) at onset. After admission, the laboratory results showed pancytopenia (white blood cell 1.2 (0.6-6.7) ×10(9)/L, haemoglobin 94 (65-111) g/L, blood platelet 29 (10-41) ×10(9)/L), decreased fibrinogen (1.3 (1.1-2.5) g/L), significantly elevated triglyceride (4.0 (2.8-5.1) mmol/L), ferritin (1 766 (399-5 253) μg/L) and soluble CD25 (27 457 (9 625-44 000) ng/L). Hemophagocytosis was found in the bone marrow smears of all 5 patients. The diagnosis of acute hepatitis and HLH was confirmed. During the treatment of HLH, the blood cells remain below normal level and the further biopsy of bone marrow (iliac bone) indicated low myeloproliferation. After exclusion of congenital bone marrow failure syndromes and other pancytopenic diseases, HAAA was confirmed. After the diagnosis of HAAA, 1 patient received antithymocyte globulin (ATG) and cyclosporin treatment in our hospital, 1 patient received allogeneic stem cell transplantation (HSCT) in other hospital, 2 patients received ATG in other hospitals. Only 1 patient died of severe infection. Conclusions: HAAA can present as HLH at onset. It is mainly manifested by high fever, acute severe hepatitis, pancytopenia, elevated ferritin and hemophagocytosis in the bone marrow. The diagnosis of HAAA should be considered whenever cytopenia could not completely corrected while apparent improvement of HLH and hepatitis related complications were improved after immunosuppressive therapy. ATG or HSCT treatment should be performed as soon as the diagnosis of severe or transfusion dependent aplastic anemia is confirmed.目的： 探讨以噬血细胞综合征（HLH）为首发表现的肝炎相关性再生障碍性贫血（HAAA）的临床特点及诊治要点。 方法： 回顾性分析2017年1月至2019年5月于首都医科大学附属北京儿童医院住院治疗的5例以HLH为首发表现的HAAA患儿的临床病例资料。 结果： 5例患儿中男4例、女1例，发病年龄6.0（2.7~12.7）岁。所有患儿在发病初期均表现为不明原因的高热、肝肿大、入院前血生化提示肝功能损伤[天冬氨酸转氨酶1 716（1 409~2 570）U/L、丙氨酸转氨酶1 699（937~2 540）U/L]，入院后查血细胞减少[白细胞计数1.2（0.6~6.7）×10(9)/L、血红蛋白94（65~111）g/L、血小板计数29（10~41）×10(9)/L]、纤维蛋白原降低[1.3（1.1~2.5）g/L]，甘油三酯[4.0（2.8~5.1） mmol/L]、铁蛋白[1 766（399~5 253） μg/L]、可溶性CD25[27 457 (9 625~44 000 ng/L）]明显升高,患儿骨髓涂片中都可见吞噬现象，故急性肝炎、HLH诊断明确。在HLH治疗过程中，患儿血细胞持续减低不缓解，完善骨髓（髂骨）活检提示骨髓增生低下，在除外先天骨髓衰竭性疾病及其他全血细胞减少性疾病后，HAAA诊断成立。HAAA确诊后，1例患儿在北京儿童医院行抗人胸腺免疫球蛋白（ATG）和环孢素治疗，1例外院行造血干细胞移植，2例明确诊断后外院行ATG治疗，1例患儿死亡。 结论： HAAA可以HLH为首发表现，临床表现以高热、肝功能损伤、血细胞减少、铁蛋白升高及骨髓吞噬现象为主。若经免疫抑制治疗，HLH以及肝炎相关指标均好转而血常规持续不恢复应考虑HAAA可能。当HAAA符合重型再生障碍性贫血诊断标准或依赖输注时，应尽快行ATG或者造血干细胞移植治疗。.
528	How can an internal medicine specialist save a patient with hemophagocytic lymphohistiocytosis (HLH)?	32134401	Hemophagocytic lymphohistiocytosis (HLH; also, hemophagocytic syndrome) occurs when an inflammatory reaction cannot stop on its own but continues to self‑ accelerate with positive feedback loops. If not interrupted, this pathomechanism leads to death. HLH in adults is usually diagnosed based on the HLH‑2004 criteria, but its confirmation should not stop the diagnostic process. Finding the triggering factor (especially malignancy) is of utmost importance. Treatment strongly depends on the established trigger and it is often based on the etoposide HLH‑94 protocol (adjusted for adults). Diagnostic workup should not unnecessarily delay the treatment since patients in severe or quickly deteriorating clinical condition require its fast initiation. Considering the progressive nature of HLH, time is extremely important. Prompt diagnosis and treatment, frequently made by an internal medicine specialist, is life‑saving. The aim of this review is to raise HLH awareness among internal medicine specialists and to provide advice on HLH management tailored for this group of physicians. Suggested approach is based on the latest recommendations by the Histiocyte Society and include novel insights from the authors' experience.
529	Adult-onset Primary Hemophagocytic Lymphohistiocytosis: Reporting a Rare Case with Review of Literature.	32133251	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon, aggressive hematological syndrome. It is caused by an increased and unchecked proliferation of T lymphocytes and histiocytes. These cells secrete a large number of inflammatory cytokines and infiltrate various tissues causing multi-organ system failure. HLH may be primary or associated with different types of infections, autoimmune disorders, or malignancies. Primary or familial HLH is fatal and is frequently considered a disorder of infants and young children. Only a few cases of primary HLH have been reported in adults. We present a case of a 37-year-old man who presented with fever, pancytopenia, and hepatosplenomegaly. Lymph node biopsy showed collections of histiocytes with lymphoplasmacytic cells. After excluding all the secondary causes a final diagnosis of primary HLH was made. The patient was started on HLH-2004 protocol (etoposide, cyclosporin A, dexamethasone) along with empiric antituberculous drugs as necrotic granulomas were also noted in the biopsy. HLH has a very poor prognosis and familiarity with clinical symptoms, and diagnostic criteria can aid in timely diagnosis.
530	Hemophagocytic lymphohistiocytosis resulting from a cytokine storm triggered by septicemia in a child with chronic granuloma disease: a case report and literature review.	32126983	Hemophagocytic lymphohistiocytosis (HLH) is a rare potentially fatal illness characterized by impaired natural killer and cytotoxic T cell function. Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by a defect in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients display an increased susceptibility to infection with bacteria and fungi. Repeated infections lead to an increased risk for developing HLH. The case of CGD with repeated Salmonella septicemia complicated with HLH is very rare, and the CGD mutation identified has not been reported.A 3-year-old boy was admitted to our hospital for fever, hepatosplenomegaly and pancytopenia. According to the clinical manifestations and laboratory results, hemophagocytic lymphohistiocytosis (HLH) was diagnosed. Blood and bone marrow culture confirmed septicemia due to Salmonella Typhimurium. On the basis of antiinfection treatment, methylprednisolone was used to control HLH. After treatment, the clinical symptoms and laboratory results improved. Gene analysis showed a novel hemizygous CYBB gene mutation: c.302A > G (p.H101P). Combined with a past history of recurrent infection, the child was diagnosed with HLH secondary to CGD triggered by septicemia.In case of a known (or highly suspected) CGD with a documented infection, clinical or biological features of HLH should encourage the physician to make possible to confirm or not the HLH. Therefore, to initiate the adequate treatment in association with anti-infective therapy.
531	Pediatric hemophagocytic lymphohistiocytosis.	32107531	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome describing patients with severe systemic hyperinflammation. Characteristic features include unremitting fever, cytopenias, hepatosplenomegaly, and elevation of typical HLH biomarkers. Patients can develop hepatitis, coagulopathy, liver failure, central nervous system involvement, multiorgan failure, and other manifestations. The syndrome has a high mortality rate. More and more, it is recognized that while HLH can be appropriately used as a broad summary diagnosis, many pediatric patients actually suffer from an expanding spectrum of genetic diseases that can be complicated by the syndrome of HLH. Classic genetic diseases in which HLH is a typical and common manifestation include pathogenic changes in familial HLH genes (PRF1, UNC13D, STXBP2, and STX11), several granule/pigment abnormality genes (RAB27A, LYST, and AP3B1), X-linked lymphoproliferative disease genes (SH2D1A and XIAP), and others such as NLRC4, CDC42, and the Epstein-Barr virus susceptibility diseases. There are many other genetic diseases in which HLH is an infrequent complication of the disorder as opposed to a prominent manifestation of the disease caused directly by the genetic defect, including other primary immune deficiencies and inborn errors of metabolism. HLH can also occur in patients with underlying rheumatologic or autoinflammatory disorders and is usually designated macrophage activation syndrome in those settings. Additionally, HLH can develop in patients during infections or malignancies without a known (or as-yet-identified) genetic predisposition. This article will attempt to summarize current concepts in the pediatric HLH field as well as offer a practical diagnostic and treatment overview.
532	A Case of Hemophagocytic Lymphohistiocytosis following Refractory Kawasaki Disease.	32097970	Hemophagocytic lymphohistiocytosis (HLH) is a systemic inflammatory disorder characterized by uncontrolled histiocytic proliferation, hemophagocytosis, macrophage activation, and up-regulation of inflammatory cytokines (Grom AA., Current opinion in rheumatology 2003; 15: 587-590). HLH is usually divided into two types: primary (familial) HLH and secondary (reactive) HLH. Primary HLH is associated with primary immune deficiencies in which specific gene mutations play an important role, such as perforin defects.
533	Hemophagocytic lymphohistiocytosis in adults: collaborative analysis of 137 cases of a nationwide German registry.	32076823	Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome emerging from a deregulated immune response due to various triggers. In adults, systematic data are sparse, which is why recommendations on diagnosis and management have been adopted from pediatric guidelines. A nationwide clinical registry with associated consulting service as collaborative initiative of HLH-specialized pediatricians and hematologists was initiated to better characterize HLH in adults.Patients with proven or suspected HLH were registered by 44 institutions. Both HLH-2004 diagnostic criteria and the HScore (www.saintantoine.aphp.fr/score/) were used to confirm HLH diagnosis. Data referring to underlying disease, treatment, outcome, clinical presentation and laboratory findings were recorded.The study included 137 patients and provides the first systematic data on adult HLH in Germany. Median age was 50 years with a wide range (17-87 years), 87 patients (63.5%) were male. Most common triggering diseases were infections in 61 patients (44.5%) and malignancies in 48 patients (35%). Virtually all patients had elevated ferritin concentrations, and 74% had peak concentrations greater than 10,000 µg/l. At time of analysis, 67 of 131 patients (51%) had died. Patients with malignancy-associated HLH had the shortest median survival (160 days), however no statistically significant difference between subgroups was observed (p = 0.077). Platelets under 20*109/l and low albumin concentrations (< 20 g/l) were associated with poor overall and 30-day survival.Close multidisciplinary case consultation and cooperation is mandatory when treating adult HLH patients. Early contact with reference centers is recommended, especially in relapsing or refractory disease.
534	FHLdb: A Comprehensive Database on the Molecular Basis of Familial Hemophagocytic Lymphohistiocytosis.	32076423	Background: Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders. The lack of comprehensive disease-specific mutation databases may hinder or delay classification of the genetic variants found in samples from these patients. This is especially true for familial hemophagocytic lymphohistiocytosis (FHL), a life-threatening PID classically considered an autosomal recessive condition, but with increasingly demonstrated genetic heterogeneity. Objective: The aim of this study was to build an open-access repository to collect detailed information on the known genetic variants reported in FHL. Methods: We manually reviewed more than 120 articles to identify all reported variants related to FHL. We retrieved relevant information about the allelic status, the number of patients with the same variant, and whether functional assays were done. We stored all the data retrieved in a PostgreSQL database and then built a website on top of it, using the Django framework. Results: The database designed (FHLdb) (https://www.biotoclin.org/FHLdb) contains comprehensive information on reported variants in the 4 genes related to FHL (PRF1, UNC13D, STXBP2, STX11). It comprises 240 missense, 69 frameshift, 51 nonsense, 51 splicing, 10 in-frame indel, 7 deep intronic, and 5 large rearrangement variants together with their allelic status, carrier(s) information, and functional evidence. All genetic variants have been classified as pathogenic, likely pathogenic, uncertain significance, likely benign or benign, according to the American College of Medical Genetics guidelines. Additionally, it integrates information from other relevant databases: clinical evidence from ClinVar and UniProt, population allele frequency from ExAC and gnomAD, and pathogenicity predictions from well-recognized tools (e.g., PolyPhen-2, SIFT). Finally, a diagram depicts the location of the variant relative to the gene exon and protein domain structures. Conclusion: FHLdb includes a broad range of data on the reported genetic variants in familial HLH genes. It is a free-access and easy-to-use resource that will facilitate the interpretation of molecular results of FHL patients, and it illustrates the potential value of disease-specific databases for other PIDs.
535	Pulmonary Lymphomatoid Granulomatosis With Hemophagocytic Lymphohistiocytosis as the Initial Manifestation.	32064234	Lymphomatoid granulomatosis (LYG) is an extremely rare angio-centric and angio-destructive B-cell lymphoproliferative disease. Driven by Epstein-Barr virus (EBV), LYG predominantly involves the bilateral lungs. Commonly presenting as multiple nodules in the lung, pulmonary LYG can masquerade as various infectious diseases, vasculitis, lung cancer, or other metastatic neoplasm. It is difficult to be diagnosed and is always neglected by clinicians. No standardized therapeutic regimens for LYG has been established yet now. Hemophagocytic lymphohistiocytosis (HLH), a life-threatening condition caused by abnormal activation of macrophages and T-cells, is characterized by fever, hepatosplenomegaly, pancytopenia, hypercytokinemia, and the presence of hemophagocytosis within the bone marrow, liver, spleen, or other lymphatic tissue. We herein report a 55-year-old man with recurrent fever, severe jaundice, and multiple high-density opacities and nodules in both lungs, who was finally diagnosed with pulmonary LYG (Grade 3) manifested with secondary HLH. Administration of HLH-1994 protocol led to the rapid control of the symptoms caused by HLH. Rituximab-based combination therapy was useful yet LYG (Grade 3) progressed rapidly. This case demonstrates that tissue biopsy is essential for early pathological diagnosis and effective treatment of LYG.
536	Molecular and functional characterization of two DELLA protein-coding genes in litchi.	32061763	DELLA proteins are members of the plant-specific GRAS family, acting as negative regulators of plant growth. In this study, we identified two DELLA protein-coding genes in litchi, denoted as LcGAI and LcRGL1. Motif analysis showed that LcGAI and LcRGL1 proteins both contain a conserved DELLA and TVHYNP motif at the N-terminus as well as LHR1, VHIID, LHR2, PFYRE, and SAW motifs at the C terminus. The fused proteins of LcGAI-GFP and LcRGL1-GFP were both localized in the nucleus. Overexpression of LcGAI and LcRGL1 in Arabidopsis substantially inhibits leaf growth. Expression analysis showed that HLH factors, PRE1 and PRE5, were restrained, whereas gibberellin (GA) receptors GID1a and LcGID1b were enhanced in LcGAI and LcRGL1 overexpression lines. Results of the yeast two-hybrid assay showed that LcGAI and LcRGL1 interact with LcGID1b/LcGID1c in a GA dose-dependent manner, whereas LcGAI and LcRGL1 had a greater binding capacity to LcGID1b than LcGID1c. These observations suggested that LcGAI and LcRGL1 proteins are nuclear growth repressors.
537	Hemophagocytic Lymphohistiocytosis in Children.	3205619432451828	To study the profile of children with Hemophagocytic Lymphohistiocytosis (HLH) in a tertiary care hospital for children.A retrospective analysis of case records of 52 children diagnossed with HLH was performed.Of the 52 children 13% (n = 7) had Familial HLH and 87% (n = 45) had secondary HLH (sHLH). Common manifestations were fever (100%), organomegaly (87%), respiratory distress (54%), neurological symptoms (31%) and skin rashes (26.2%). Anemia and thrombocytopenia were present in 51% and 73% respectively. Hyperferritinemia was present in 96% and hypofibrinogenemia in 42% and high lactate dehydrogenase (LDH) in 91%. Bone marrow examination showed hemophagocytosis in 80%. Most common etiology among infections was viral infections (67%), of which Dengue was the most common (52%). Among children with sHLH 51% received supportive care only. Thirty-seven percent (n = 17) received intravenous (IV) immunoglobulin and steroids. Of these 77% (n = 35) recovered completely. Children with familial HLH were initiated on HLH 2004 protocol but all of them expired due to disease progression.Identifying HLH early and managing it, poses a significant challenge. Prompt recognition and initiation of immunosuppressive therapy is extremely important for the better outcome; hence high clinical suspicion and structured work up including immunological, and genetic studies is required. It may be difficult to differentiate primary and secondary HLH in many instances unless genetic analysis is done. Identification of familial HLH is necessary for early referral to Hematopoietic Stem Cell Transplantation (HSCT). Hence screening for primary HLH needs to be considered in all children with HLH.
538	Hemophagocytic Lymphohistiocytosis in a Patient With Advanced HIV and Cytomegalovirus Infection.	32054345	Hemophagocytic lymphohistiocytosis (HLH) is a rare, aggressive, and, if not treated, fatal disorder that is characterized by excessive immune system activation. This disorder can be precipitated by different triggers including malignancies, infections, and autoimmune disorders. Diagnosis is made by fulfilling criteria that was last updated in 2004, and treatment frequently includes management of the underlying trigger but can also include chemotherapy. In this article, we report a case of HLH in a 27-year-old male, who had been diagnosed with advanced untreated HIV, who presented to the hospital with fever and generalized fatigue with no obvious etiology. Infectious workup revealed cytomegalovirus viremia, and the patient met HLH criteria with impressive hyperferritinemia of 15 432 ng/mL. The patient was started on treatment for cytomegalovirus infection that led to resolution of HLH. Our report highlights the importance of early detection of HLH in special populations, and that treating the presumptive trigger can lead to resolution of HLH.
539	Hemophagocytic Lymphohistiocytosis Induced Cytokine Storm Secondary to Human Immunodeficiency Virus Associated Miliary Tuberculosis.	32051801	Hemophagocytic lymphohistiocytosis (HLH) is a rarely diagnosed fatal inflammatory disease associated with an overactive immune system. It occurs in a host of conditions, with human immunodeficiency virus (HIV) being a rare but serious cause, usually occurring in patients with acquired immunodeficiency syndrome (AIDS). The diagnosis of HLH can be very difficult, as it presents with vague signs and symptoms, which can be present in multiple diseases. This case highlights the diagnostic dilemma faced when treating this potentially fatal condition. Usually, treating the underlying trigger for HLH is sufficient to counteract the overwhelming inflammatory response; however, this can prove to be difficult, as demonstrated in our patient. We present a case of miliary tuberculosis in the setting of HIV/AIDS, complicated by HLH in a young male patient. Whether it was due to delays in treatment or the rapidly fulminant nature of the disease, our patient had a poor clinical outcome. Although rare, tuberculosis-associated HLH must be considered as a cause of secondary HLH in all patients, especially those who are immunosuppressed.
540	A Brief Report and Mini-Review of Visceral Leishmaniasis-associated Hemophagocytic Lymphohistiocytosis in Children.	32049769	We present the case of a 7-year-old boy who fulfilled the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH). Prompt visualization of his bone marrow confirmed the diagnosis of visceral leishmaniasis (VL). He responded well to treatment with liposomal amphotericin-B. The patient had a false-negative enzyme-linked immunosorbent assay for Leishmania infantum and a false-positive immunoglobulin M test for Epstein Barr virus (EBV). Because age at presentation is similar in children with VL and familial HLH for whom EBV is the usual trigger, ruling out VL is extremely important because nonspecific serologic tests for EBV can lead to the inappropriate diagnosis of EBV-driven primary HLH and to the administration of unnecessary immunochemotherapy.
541	Status Epilepticus as a Consequence of Hemophagocytic Lymphohistiocytosis in a Previously Healthy Infant.	32042537	Hemophagocytic lymphohistiocytosis (HLH) is a rare and fatal entity with an incidence rate of 1.2 cases per million people per year. HLH is explained as a highly destructive inflammatory consequence of rampant hypercytokinemia due to excessive lymphocyte-mediated activation of macrophages and histiocytes. Primary HLH is a product of genetic dysfunction and could be familial (five subtypes), syndromic immunodeficiency, or as a consequence of mutations predisposing a person to Epstein Barr Virus (EBV) infection. With secondary HLH, there is an identifiable cause provoking the inflammatory reaction, whether it is an infection, an autoimmune disease, or malignancy (particularly hematological). As a result of widespread cytokine deposition, systemic manifestations are seen with a variety of manifestations that can vary between cases. This is a case of a patient who initially presented to the emergency department with fever, altered mentation, and gastroenteritis. Initial investigations showed non-anion gap metabolic acidosis, high white cell count, and deteriorating renal function. Further laboratory tests, bone marrow biopsies, and neurological imaging were conducted throughout the course of admission as the patient further deteriorated systemically. However, it's important to note the abundant neurological manifestations with a worsening level of consciousness and seizures, some of which were categorized as status epilepticus.
542	EBV-positive T/NK-associated lymphoproliferative disorders of childhood: A complete autopsy report.	32031127	Epstein-Barr Virus (EBV)-associated systemic T-cell lymphoproliferative disorder of childhood is a rare but severe manifestation of chronic EBV infection. Despite several case reports characterizing this rare hematological neoplasm, the literature describes extensive heterogeneity in the presentation of this disease.Here we present a complete autopsy of a 16-year-old girl who ultimately succumbed to EBV-associated systemic T-cell lymphoproliferative disorder of childhood. Her clinical presentation demonstrated a non-specific pharyngitis with positive mono spot test, evolving into fulminant multi-organ failure, disseminated intravascular coagulopathy, sepsis, and ultimately death.Post-mortem findings included extensive hemorrhage, and infiltration of the liver, spleen, lymph nodes and bone marrow with neoplastic T-cells. There was extensive hemophagocytic lymphohistiocytosis (HLH) within these organs, suggesting overlap between the EBV-associated systemic T-cell lymphoproliferative disorder of childhood and EBV-associated HLH. We hope these findings provide a more comprehensive overview of several possible manifestations of EBV-associated systemic T-cell lymphoproliferative disorder of childhood.
543	A case of subcutaneous emphysema/mediastinal emphysema during the use of humidified high-flow nasal cannula.	32026981	Heated, humidified, high-flow nasal cannula (HHFNC) oxygen therapy allows optimal humidification of inspired gas at high flows and creates a distending pressure similar to nasal continuous positive airway pressure [1]. It has been safely used in adults with moderate hypoxemia with few complications [2, 3]. Hereby, we report serious complications occurred during HHFNC oxygen therapy.A 53-year-old female with hemophagocytic lymphohistiocytosis (HLH) was admitted to the intensive care unit because of respiratory failure. After weaning from mechanical ventilation which lasted for 2 weeks, HHFNC therapy at 40 L/min with an FiO2 of 0.5 was started for hypoxemia. Four days later, dyspnea and hypoxemia occurred and chest X-ray and CT scan revealed localized pneumothorax, subcutaneous emphysema, and massive pneumomediastinum. After cessation of HHFNC, respiratory condition improved.Subcutaneous emphysema, pneumothorax, and pneumomediastinum should be notified as a serious complication during HHFNC therapy.
544	Pembrolizumab-induced Hemophagocytic Lymphohistiocytosis: an immunotherapeutic challenge.	32025343	As the number of indicated malignancies for which immune checkpoint inhibitor therapy such as pembrolizumab grows the descriptions of associated immune-related adverse events (irAEs) increases as well. On rare occasions immunotherapy can lead to development of Hemophagocytic Lymphohistiocytosis (HLH) which is a potentially lethal inflammatory disorder characterized by histiocyte activation and cytokine storm. At this time no cases of HLH developing in head and neck squamous cell carcinoma (HNSCC) patients receiving pembrolizumab have been reported.Here we describe the first documented case of pembrolizumab-induced HLH in a 61 year-old male with metastatic HNSCC after having received multiple prior cycles of pembrolizumab without event. Following cycle 14 the patient developed fever associated with new pancytopenia and transaminitis prompting hospital admission. Infectious workup was negative, his metastatic lesions were found to be stable, and there was no evidence of new malignancy. Further workup demonstrated hyperferritinemia and bone marrow biopsy demonstrated hemophagocytosis concerning for pembrolizumab-induced HLH. Etoposide and dexamethasone therapy was initiated leading to clinical improvement and safe discharge.Immunotherapy is a groundbreaking therapeutic intervention for patients with malignancy, however by nature of their mechanism carry a risk of inflammatory side effects. In rare circumstances these inflammatory reactions include potentially deadly syndromes such as HLH. As immunotherapeutics such as pembrolizumab become more widely utilized increased awareness of complications such as HLH is clinically relevant.
545	Macrophage activation syndrome in pediatrics.	32017214	Macrophage activation syndrome (MAS) is a serious, potentially life-threatening, hyperinflammatory condition, which belongs to the spectrum of hemophagocytic lymphohistiocytosis (HLH) and can complicate several immunologic and rheumatic disorders. MAS is characterized by a dysfunctional immune response that is similar to that seen in other forms of HLH. Because MAS may pursue a rapidly fatal course, prompt recognition of its clinical and laboratory features and immediate therapeutic intervention are fundamental. Recently, a set of classification criteria for MAS complicating sJIA has been developed through a multinational collaborative effort. High-dose parenteral corticosteroids remain the mainstay of treatment of MAS.
546	Adult macrophage activation syndrome-haemophagocytic lymphohistiocytosis: 'of plasma exchange and immunosuppressive escalation strategies' - a single centre reflection.	32013725	In the context of systemic autoimmunity, that is systemic lupus erythematosus (SLE) or adult-onset Still's disease (AOSD), secondary haemophagocytic lymphohistiocytosis (HLH; also referred to as macrophage activation syndrome (MAS) or more recently MAS-HLH) is a rare and potentially life-threatening complication. Pathophysiological hallmarks are aberrant macrophage and T cell hyperactivation and a systemic cytokine flare, which generate a sepsis-like, tissue-damaging, cytopenic phenotype. Unfortunately, for adult MAS-HLH we lack standardized treatment protocols that go beyond high-dose corticosteroids. Consequently, outcome data are scarce on steroid refractory cases. Aside from protocols based on treatment with calcineurin inhibitors, etoposide, cyclophosphamide and anti-IL-1, favourable outcomes have been reported with the use of intravenous immunoglobulin (IvIG) and plasma exchange (PE).Here we report a retrospective series of steroid refractory MAS-HLH, the associated therapeutic regimes and outcomes.In this single-centre experience, 6/8 steroid refractory patients survived (median follow-up: 54.4 (interquartile range: 23.3-113.3) weeks). All were initially treated with PE, which induced partial response in 5/8 patients. Yet, all patients required escalation of immunosuppressive therapies. One case of MAS-HLH in new-onset AOSD had to be escalated to etoposide, whereas most SLE-associated MAS-HLH patients responded well to cyclophosphamide. Relapses occurred in 2/8 cases.Together, early use of PE is at most a supportive measure, not a promising monotherapy of adult MAS-HLH. In refractory cases, conventional cytoreductive therapies (i.e. cyclophosphamide and etoposide) constitute potent and reliable rescue approaches, whereas IvIG, anti-thymoglobulin, and biologic agents appear to be less effective.
547	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in a small child: A case report.	32011461	Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially lethal disorder, characterized by a dysregulation of the immune response, leading to a severe inflammatory syndrome. Epstein-Barr virus (EBV)-associated HLH is a form of secondary HLH, a fulminant presentation of an otherwise benign viral infection.We report the case of a 3-year-old girl who presented with fever, signs of accute upper respiratory tract infection and spontaneous, disseminated ecchymoses. Initial laboratory tests revealed pancytopenia. A bone marrow aspirate was performed, which revealed megaloblasts and numerous macrophages, with abundant foamy cytoplasm. Megaloblastic anemia was excluded, as the levels of vitamin B12 and folic acid were both within normal ranges.Hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, and splenomegaly were relevant criteria for the diagnosis of HLH, in accordance with the bone marrow specimen. Positive immunoglobulin M antibodies for EBV were supportive of an acute EBV infection, which was the most probable trigger of HLH. The patient's evolution was complicated by a massive epistaxis, in the context of thrombocytopenia which required plasma, thrombocyte, and erythrocyte substitutes.The patient was started on a treatment regimen of 8 weeks with etoposide and dexamethasone.Her evolution was favorable, the treatment being successful in remission induction.Our case emphasizes the diagnostic challenges of HLH, in a patient with EBV infection whose evolution was hindered by a severe epistaxis, with potentially fatal outcome.
548	The role of allogeneic hematopoietic stem cell transplantation and Epstein-Barr virus infection on the treatment for child primary hemophagocytic lymphohistiocytosis patients with X-linked lymphoproliferative disease: A rare case report and family survey study.	32011062	XLP-2 is known as a rare primary immunodeficiency disease, which is characterized by the susceptibility to EBV infection and potential development into the pHLH. The existing studies believe that the dysfunction of XIAP represents one of the most significant pathogenic mechanisms of XLP-2, and allo-HSCT is regarded as a crucial treatment for the long-term survival in XLP-2 patients. In our present study, a 2-year-old male patient was diagnosed with XLP-2. After receiving chemotherapy by using HLH-2004 without allo-HSCT, he reached a complete remission, and his EBV load was brought under control. Our family survey revealed a novel frameshift mutation in the XIAP gene in this patient, as well as in his cousin and grandfather. Until now, the patient has been followed up for 22 months with no recurrence reported yet. Based on these findings, it is believed that for child pHLH patients with XLP-2, the treatment by controlling symptoms alone without allo-HSCT and with regular monitoring of EBV load could be conducive to a long-term survival.
549	Natural Killer Cells in Systemic Autoinflammatory Diseases: A Focus on Systemic Juvenile Idiopathic Arthritis and Macrophage Activation Syndrome.	32010140	Natural killer (NK) cells are innate immune lymphocytes with potent cytolytic and immune-regulatory activities. NK cells are well-known for their ability to kill infected and malignant cells in a fast and non-specific way without prior sensitization. For this purpose, NK cells are equipped with a set of cytotoxic molecules such as perforin and apoptosis-inducing proteins. NK cells also have the capacity to produce large amounts of cytokines and chemokines that synergize with their cytotoxic function and that ensure interaction with other immune cells. A less known feature of NK cells is their capacity to kill non-infected autologous cells, such as immature dendritic cells and activated T cells and monocytes. Via the release of large amounts of TNF-α and IFN-γ, NK cells may contribute to disease pathology. Conversely they may exert a regulatory role through secretion of immuno-regulatory cytokines such as GM-CSF, IL-13, and IL-10. Thus, NK cells may be important target and effector cells in the pathogenesis of autoinflammatory diseases, in particular in those disorders associated with a cytokine storm or in conditions where immune cells are highly activated. Key examples of such diseases are systemic juvenile idiopathic arthritis (sJIA) and its well-associated complication, macrophage activation syndrome (MAS). sJIA is a chronic childhood immune disorder of unknown etiology, characterized by arthritis and systemic inflammation, including a daily spiking fever and evanescent rash. MAS is a potentially fatal complication of autoimmune and autoinflammatory diseases, and most prevalently associated with sJIA. MAS is considered as a subtype of hemophagocytic lymphohistiocytosis (HLH), a systemic hyperinflammatory disorder characterized by defective cytotoxic pathways of cytotoxic T and NK cells. In this review, we describe the established features of NK cells and provide the results of a literature survey on the reported NK cell abnormalities in monogenic and multifactorial autoinflammatory disorders. Finally, we discuss the role of NK cells in the pathogenesis of sJIA and MAS.
550	Hemophagocytic Lymphohistiocytosis in a Patient with Rheumatoid Arthritis on Pembrolizumab for Lung Adenocarcinoma.	32009093	Immune checkpoint inhibitors have changed the landscape of classic cancer treatment. However, their use is associated with the emergence of new adverse events. An elderly man with rheumatoid arthritis was started on pembrolizumab for newly diagnosed advanced lung cancer. He subsequently developed hemophagocytic lymphohistiocytosis (HLH), which is potentially fatal but has not been properly established as an immune checkpoint inhibition-induced event. We herein report the case of a patient with pembrolizumab-induced HLH.
551	Comprehensive analysis of inhibitor of differentiation/DNA-binding gene family in lung cancer using bioinformatics methods.	32003423	The inhibitor of differentiation/DNA-binding (ID) is a member of the helix-loop-helix (HLH) transcription factor family, and plays a role in tumorigenesis, invasiveness and angiogenesis. The aims were to investigate the expression patterns and prognostic values of individual ID family members in lung cancer, and the potential functional roles. The expression levels of ID family were assessed using the Oncomine online database and GEPIA database. Furthermore, the prognostic value of ID family members was evaluated using the Kaplan-Meier plotter database. The genetic mutations of ID family members were investigated using the cBioPortal database. Moreover, enrichment analysis was performed using STRING database and Funrich software. It was found that all the ID family members were significantly down-regulated in lung cancer. Prognostic results indicated that low mRNA expression levels of ID1 or increased mRNA expression levels of ID2/3/4 were associated with improved overall survival, first progression and post progression survival. Additionally, genetic mutations of ID family members were identified in lung cancer, and it was suggested that amplification and deep deletion were the main mutation types. Furthermore, functional enrichment analysis results suggested that ID1/2/4 were significantly enriched in 'regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism' for biological process, 'transcription factor activity' for molecular function and 'HLH domain' for protein domain. However, it was found that ID3 was not enriched in the above functions. The aberrant expression of ID family members may affect the occurrence and prognosis of lung cancer, and may be related to cell metabolism and transcriptional regulation.
552	Extreme hyperferritinemia: etiological spectrum and impact on prognosis.	31995958	Hyperferritinemia can be the result of inflammation, infection, iron overload, or other uncommon pathologies including hemophagocytic lymphohistiocytosis (HLH). The significance of its elevation and its association with poor prognosis and critical clinical situations is unclear. To study the spectrum of diagnosis associated with elevated serum ferritin, we made a retrospective review of patients admitted to our center from 2015 and 2017 with serum ferritin levels above 2000 μg L-1. The H score was retrospectively assessed in all cases to evaluate the possible presence of HLH. The degree of ferritinemia found was compared with the evaluation of the undelying diagnosis and the results of laboratory examinations. A total of 77 patients were identified with a serum ferritin level >2000 μg L-1. Hematological malignancy was the most prevalent diagnosis with n=20; severe infection was next with n=14. Eleven patients were diagnosed with HLH. The hemophagocytosis pictures on bone marrow smear and mortality rate were significantly correlated with ferritin level above 6000 μg L-1 (p<0.01). The comparison of the HLH subgroup with the rest of the cohort showed that fever, cytopenia (anemia, leucopenia, neutropenia and thrombocytopenia), hemophagocytosis pictures, and major liver disturbances were significantly increased in the HLH subgroup. The H score was significantly elevated in the subgroup of patients with ferritin above 6000 μg L-1, with a significatively higher probability of HLH (p<0.01). The mortality rate at 3 months was significantly increased in the HLH subgroup. Extreme hyperferritin cannot be considered as a specific marker for the diagnosis. The cut off of 6000 μg L-1 is significantly associated with HLH diagnosis. The H score is an interesting screening tool that physicians should use to rule out the probability of HLH when facing critical clinical situations.
553	Post-Transfusion Hemophagocytosis Without Hemophagocytic Lymphohistiocytosis.	31993572	Hemophagocytosis refers to ingestion of hematopoietic elements or mature blood cells by another cell, typically by cells conventionally associated with phagocytic capacity. Although the finding of hemophagocytosis as a prominent feature in a patient's bone marrow might prompt consideration of a hemophagocytic syndrome (HPS) such as hemophagocytic lymphohistiocytosis (HLH) in a clinician's or pathologist's differential diagnosis, this morphologic feature can be nonspecific in the absence of other clinical and laboratory features of pathologic immune activation, which is the sine qua non of HPS/HLH. We describe three patients whose clinical presentations included transfusion-dependent anemia and whose bone marrow aspirates showed unexpectedly brisk hemophagocytosis of mature red blood cells. Despite striking morphologic hemophagocytosis, no patient met criteria for diagnosis of an HPS. Transfusion-associated hemophagocytosis and hyperferritinemia must be carefully distinguished from HLH through clinical and laboratory assessment. Biomarkers of pathologic immune activation are important diagnostic aids.
554	Reevaluating the role of ferritin in the diagnosis of adult secondary hemophagocytic lymphohistiocytosis.	31991015	The standard diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) include hyperferritinemia to >500 ng/mL. This ferritin threshold is based on pediatric data, and evidence for its application among adults with secondary HLH is lacking.We conducted a retrospective study assessing the relationship between extreme hyperferritinemia and adult secondary HLH at our institution. All adult inpatients seen over a 10-year period, with serum ferritin >5000 ng/mL, were included.Among 1055 patients with serum ferritin >5000 ng/mL, there were 69 cases of HLH (HLH prevalence of 6.5%). Mean ferritin among HLH patients was 70 398 ng/mL (SD 122 908), median 40 019 ng/mL (IQR 16 051-68 326). The prevalence of HLH only reached 50% as serum ferritin approached 90 000 ng/mL. A variety of conditions were contributory to hyperferritinemia, most commonly bacterial sepsis (33%), hematologic malignancy (29%), renal failure (24%), and liver injury (18%). The optimal cutoff ferritin for diagnosis of HLH was 16 000 ng/mL (sensitivity 79.4%, specificity 79.2%, PPV 20.9%, and NPV 98.2%).The threshold ferritin levels used in diagnostic criteria for adult secondary HLH are too low to be clinically relevant, and efforts should be undertaken to revise them upward. Similar reappraisals should be taken of the other criteria used to diagnose adult HLH.
555	Incorporation of thiotepa in a reduced intensity conditioning regimen may improve engraftment after transplant for HLH.	31989587	NaN
556	Liver Failure Among Young Saudi Infants: Etiology, Clinical Presentation, and Outcome.	31978013	The published data on early infantile liver failure (EILF) are scarce and limited to Caucasians. We conducted this study to describe the etiology and outcome of EILF among Arabs and identify prognostic factors.We retrospectively reviewed our database of 524 infants presenting with liver impairment from 2008 to 2018, and identified cases of EILF defined as presence of biochemical pattern of liver disease and INR ≥2 (unresponsive to vitamin K) with onset before 3 months of life. Primary outcomes included death or liver transplantation (LT) (poor outcome group) and survival with native liver (good outcome group).Forty-two cases of EILF (22 girls) were identified (8%). The etiology was indeterminate in 14 (33.3%) and established in 27 (64.3%): galactosemia (7 cases, 16.6%), tyrosinemia (5, 12%), neonatal hemochromatosis (NH), and hemophagocytic lymphohistiocytosis (HLH) (4 each, 9.5%]) mitochondrial hepatopathy (3, 7%), and miscellaneous (5, 12%). LF resolved in 15 cases (35.7%), either spontaneously or in response to specific therapy, 23 (54.7%) died, and 4 underwent LT (9.5%). ROC analysis for the best cut-off value of serum total bilirubin for prediction of study outcomes was 120 μmol/L (sensitivity 81.5%, specificity 80%). Among the diagnostic groups, galactosemia and tyrosinemia predicted good outcome, whereas the idiopathic diagnosis predicted poor outcome (OR = 13).Similar to Western countries, galactosemia, tyrosinemia, NH, HLH, and mitochondrial hepatopathy are the main players in EILF in Saudi Arabia. Galactosemia and tyrosinemia predict good prognosis and idiopathic diagnosis predicts poor prognosis.
557	Serum soluble VSIG4 as a surrogate marker for the diagnosis of lymphoma-associated hemophagocytic lymphohistiocytosis.	31960419	Lymphoma-associated haemophagocytic lymphohistiocytosis (L-HLH) is characterized by excessively activated macrophages and cytotoxic T lymphocytes, but few reliable markers for activated macrophages are available clinically. This study, designed to discover novel biomarkers for the diagnosis of lymphoma patients with L-HLH, was initiated between 2016 and 2018. Fifty-seven adult lymphoma patients were enrolled - 39 without HLH and 18 with HLH. The differential serum protein expression profile was first screened between lymphoma patients with and without L-HLH by a quantitative mass spectrometric approach. Soluble V-set and immunoglobulin domain-containing 4 (sVSIG4), specifically expressed by macrophages, was significantly upregulated in the L-HLH group. Subsequently, sVSIG4 concentration was confirmed by enzyme-linked immunosorbent assay to be significantly increased in lymphoma patients with L-HLH. When it was exploited for the diagnosis of lymphoma patients with L-HLH, the area under a receiver operating characteristic curve was 0·98 with an optimal cut-off point of 2195 pg/ml and the corresponding sensitivity and specificity were 94·44% and 94·87% respectively. In addition, the one-year overall survival was significantly worse in patients with a sVSIG4 concentration above 2195 pg/ml compared with those below 2195 pg/ml (5·3% vs. 72·2%, P < 0·0001). sVSIG4 may be a surrogate marker of activated macrophages for the diagnosis of lymphoma patients with L-HLH.
558	Hemophagocytic lymphohistiocytosis associated with oxcarbazepine.	31951347	Kırık S, Güneş H, Yurttutan S, Sarışık N, Acıpayam C, Kırık Y. Hemophagocytic lymphohistiocytosis associated with oxcarbazepine. Turk J Pediatr 2019; 61: 297-300. Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening multisystem disorder. Reports of the disorder as a side effect of drugs are extremely rare. We report the case of a 3-year-old boy with a history of epileptic seizures in which oxcarbazepine was added to treatment for the last 35 days and dose had been increased. For 10 days he had a fever, hepatosplenomegaly, rash, edema and other systemic symptoms. He was diagnosed with HLH after bone marrow examination. Oxcarbazepine treatment was terminated after the intravenous immunoglobulin treatment. The next day, clinical and laboratory results had improved. This is the first HLH report of an association with oxcarbazepine. Bone marrow aspiration may be indicated to confirm the diagnosis when facing a patient with systemic symptoms after newly added antiepileptic drug treatment.
559	Adult haemophagocytic lymphohistiocytosis: a Review.	31943120	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder characterized by hyperimmune response. The mortality is high despite progress being made in the diagnosis and treatment of the disease.This review aimed to update knowledge on adult HLH pathophysiology, identifiy the numerous causes, and help clinicians make early diagnosis and initiate treatment.Using Embase, we searched relevant articles published from January 1, 2010 to October 31, 2019, with the MESH term « hemophagocytic lymphohistiocytosis; macrophagic activation syndrome, adult ».The mean age at presentation is about 50 years, with a male predominance. The most frequent disease associations are haematological diseases, viral or bacterial infections, and autoimmune diseases. The pathophysiologic mechanism is probably the combination of inherited genetic mutations and extrinsic triggers. The mortality rate is 26.5% to 74.8%. H-score is more efficient than HLH-2004 criteria to identify HLH, with diagnostic sensitivity and specificity 90% and 79%, respectively.18F-FDG PET/CT is potentially useful for detecting underlying disease and the extent of secondary HLH. Disease-specific treatment should be given as soon as possible. Treatment with corticosteroids combined or not with etoposide is the mainstay of treatment. Monoclonal antibodies and JAK pathway inhibitors show promise of being effective.In adult HLH, infectious diseases, autoimmune disease and malignancy should be suspected so that disease-specific treatment can be given promptly. Treatment with corticosteroids combined or not with etoposide is the mainstay of treatment, but new therapies show promise of being effective.
560	Hemophagocytic lymphohistiocytosis complicating invasive pneumococcal disease: a pediatric case report.	31931763	Hemophagocytic lymphohistiocytosis (HLH) is an infrequent but life-threatening disease due to excessive immune activation. Secondary HLH can be triggered by infections, autoimmune diseases, and malignant diseases. Streptococcus pneumoniae is a pathogenic bacterium responsible for invasive pneumococcal disease (IPD) such as meningitis and bacteremia. Although the pneumococcal conjugate vaccine (PCV) has led to reductions in IPD incidence, cases of IPD caused by serotypes not included in PCV are increasing. There are few reports of secondary HLH caused by IPD in previously healthy children. We herein report a rare case of a previously healthy boy with secondary HLH complicating IPD of serotype 23A, which is not included in the pneumococcal 13-valent conjugate vaccine (PCV-13).An 11-month-old boy who had received three doses of PCV-13 was hospitalized with prolonged fever, bilateral otitis media, neutropenia and elevated C-reactive protein (CRP) levels. Blood culture on admission revealed S. pneumoniae, leading to a diagnosis of IPD. HLH was diagnosed based on a prolonged fever, neutropenia, anemia, hepatosplenomegaly, hemophagocytosis in the bone marrow, and elevated serum levels of triglycerides, ferritin, and soluble interleukin-2 receptor. He received broad-spectrum antibiotics and intravenous immunoglobulins for IPD and high-dose steroid pulse therapy and cyclosporine A for HLH; thereafter, his fever resolved, and laboratory findings improved. The serotype of the isolated S. pneumoniae was 23A, which is not included in PCV-13.It is important to consider secondary HLH as a complication of IPD cases with febrile cytopenia or hepatosplenomegaly, and appropriate treatment for HLH should be started without delay.
561	Folate-conjugated Helix lucorum hemocyanin - preparation, stability, and cytotoxicity.	31926108	This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance-Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 μg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 μg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.
562	Nivolumab treatment of relapsed/refractory Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in adults.	3191417232163559	Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening hyperinflammatory syndrome triggered by EBV infection. It often becomes relapsed or refractory (r/r), given that etoposide-based regimens cannot effectively clear the virus. r/r EBV-HLH is invariably lethal in adults without allogeneic hematopoietic stem cell transplantation. Here, we performed a retrospective analysis of 7 r/r EBV-HLH patients who were treated with nivolumab on a compassionate-use basis at West China Hospital. All 7 patients tolerated the treatment and 6 responded to it. Five of them achieved and remained in clinical complete remission with a median follow-up of 16 months (range, 11.4-18.9 months). Importantly, both plasma and cellular EBV-DNAs were completely eradicated in 4 patients. Single-cell RNA-sequencing analysis showed that HLH syndrome was associated with hyperactive monocytes/macrophages and ineffective CD8 T cells with a defective activation program. Nivolumab treatment expanded programmed death protein-1-positive T cells and restored the expression of HLH-associated degranulation and costimulatory genes in CD8 T cells. Our data suggest that nivolumab, as a monotherapy, provides a potential cure for r/r EBV-HLH, most likely by restoring a defective anti-EBV response.
563	Effectiveness of therapeutic plasma exchange in a critically ill child with secondary hemophagocytic lymphohistiocytosis.	31896925	Currently, the ASFA has not included TPE in the management of HLH but many cases reports have reported successful role of TPE in HLH. Here we are presenting a case in which HLH was managed successfully with TPE. Diagnosis of HLH is based on the HLH 2004 diagnostic criteria proposed by HLH society. TPE was done using COM. TEC (Fresenius Kabi, Germany). Patient required three sessions of TPE. After three sessions of TPE patient's clinical condition improved remarkably and he was switched to IV Dexamethasone as maintenance treatment. One standard TPE procedure was 1.5 plasma volume exchanges. In view of deranged coagulation profile fresh frozen plasma was used as a replacement fluid. During follow up after one month of discharge, patient was absolutely normal. In developing countries like India, where infections are still a prime concern to the physicians, making an accurate diagnosis of HLH is a great concern. High suspicion, timely diagnosis and early start of TPE can be life saving in such patients.
564	Clinical features and outcomes of patients with hemophagocytic lymphohistiocytosis at onset of systemic autoinflammatory disorder and compare with Epstein-Barr virus (EBV)-related hemophagocytic lymphohistiocytosis.	31895784	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease. In clinical practice, we have observed that some HLH patients who have features of systemic autoinflammatory diseases (SAIDs) exhibit unique clinical manifestations and outcomes different from other HLH patients.We analyzed data from 25 HLH patients who were considered to have SAIDs; data were collected from patients of our center between January 1, 2015 and September 1, 2018.The median age of the patients was 1.75 years. In the early phase, all patients had a fever and 92% of patients had a rash; 96% of patients had high white blood cell count (WBC), C-reaction protein, and erythrocyte sedimentation rate. With progression, the above laboratory results decreased gradually. During the HLH period, we compared SAIDs-related HLH and Epstein-Barr virus (EBV)-related HLH and found that rash was more common (92%, P < .001) and splenomegaly was less common (64%, P = .023) in SAIDs-related HLH. Further, WBC, ferritin, and Interleukin-6 levels in SAIDs-related HLH patients were higher than those in EBV-related HLH patients. In contrast, hemoglobin, triglyceride, sCD25, Interleukin-10, and interferon-γ levels in SAIDs-related HLH patients were lower compared with those in EBV-related HLH patients. SAIDs-related HLH patients received a modified HLH-2004 protocol at our center. Most patients had a good prognosis.We provide a summary of the unique clinical and laboratory features, treatment protocols, and outcomes of SAIDs patients with HLH at onset. The findings indicate that these patients had a better response to corticosteroids and cyclosporin compared with EBV-related HLH patients.
565	Toxoplasma-induced hemophagocytic lymphohistiocytosis after haploidentical allogeneic stem cell transplantation.	31895492	Hemophagocytic lymphohistiocytosis (HLH) is a disorder of immune regulation, manifested by fever, pancytopenia, hyperferritiniemia, hypertriglyceridemia, and extensive hemophagocytosis involving the bone marrow and spleen. HLH can occur in adults with an underlying hematopoietic malignancy, or with systemic infections. HLH following hematopoietic stem cell transplantation (HSCT) is unusual, and the diagnosis may be challenging particularly because the diagnostic criteria in the HLH-2004 guidelines overlap with common post-transplant complications such as engraftment syndrome, graft-vs-host disease, and infections. HLH is commonly triggered by viral, bacterial and, less commonly, parasitic infections. Following HSCT, patients with latent Toxoplasma infection may develop systemic disease secondary to reactivation, and rarely this may lead to a HLH physiology, with a very high mortality rate. Herein we describe the successful management of disseminated toxoplasmosis associated with life-threatening HLH using tocilizumab and antimicrobial therapy.
566	Hepatic sinusoidal hemophagocytosis with and without hemophagocytic lymphohistiocytosis.	31887162	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life threatening hyperinflammatory syndrome. Sinusoidal hemophagocytosis is occasionally observed on liver biopsy in patients who do not have clinical suspicion of HLH. We aimed at comparing the clinical characteristics and outcomes of patients with signs of hemophagocytosis on liver biopsy meeting and not meeting the HLH diagnostic criteria.We reviewed the clinical, laboratory features and outcomes of all adult patients consecutively admitted in our center between 08/2011 and 08/2017 presenting with liver histology showing sinusoidal hemophagocytosis and of critically ill patients presenting with severe liver disease in whom hemophagocytosis was histologically confirmed. The characteristics of patients fulfilling and not fulfilling the diagnostic criteria of HLH were compared.We identified 12 cases (58% male, median age 61, 75% with a chronic underlying disease) with liver histology showing sinusoidal hemophagocytosis. All had at least some of the clinical features typically associated with HLH. Six were critical ill patients. In 4 cases with insufficient laboratory and clinical criteria, liver biopsy allowed to confirm the HLH diagnosis. Six patients died, of which four met the diagnostic criteria for HLH. Two patients with chronic liver disease died despite not fulfilling the diagnostic criteria of HLH.Hemophagocytosis on liver biopsy may contribute to confirming a diagnosis of HLH in suspected cases with indeterminate clinical and laboratory findings. Sinusoidal hemophagocytosis in patients with cirrhosis was associated with bad outcome.
567	Midkine-a Is Required for Cell Cycle Progression of Müller Glia during Neuronal Regeneration in the Vertebrate Retina.	31882403	In the retina of zebrafish, Müller glia have the ability to reprogram into stem cells capable of regenerating all classes of retinal neurons and restoring visual function. Understanding the cellular and molecular mechanisms controlling the stem cell properties of Müller glia in zebrafish may provide cues to unlock the regenerative potential in the mammalian nervous system. Midkine is a cytokine/growth factor with multiple roles in neural development, tissue repair, and disease. In midkine-a loss-of-function mutants of both sexes, Müller glia initiate the appropriate reprogramming response to photoreceptor death by increasing expression of stem cell-associated genes, and entering the G1 phase of the cell cycle. However, transition from G1 to S phase is blocked in the absence of Midkine-a, resulting in significantly reduced proliferation and selective failure to regenerate cone photoreceptors. Failing to progress through the cell cycle, Müller glia undergo reactive gliosis, a pathological hallmark in the injured CNS of mammals. Finally, we determined that the Midkine-a receptor, anaplastic lymphoma kinase, is upstream of the HLH regulatory protein, Id2a, and of the retinoblastoma gene, p130, which regulates progression through the cell cycle. These results demonstrate that Midkine-a functions as a core component of the mechanisms that regulate proliferation of stem cells in the injured CNS.SIGNIFICANCE STATEMENT The death of retinal neurons and photoreceptors is a leading cause of vision loss. Regenerating retinal neurons is a therapeutic goal. Zebrafish can regenerate retinal neurons from intrinsic stem cells, Müller glia, and are a powerful model to understand how stem cells might be used therapeutically. Midkine-a, an injury-induced growth factor/cytokine that is expressed by Müller glia following neuronal death, is required for Müller glia to progress through the cell cycle. The absence of Midkine-a suspends proliferation and neuronal regeneration. With cell cycle progression stalled, Müller glia undergo reactive gliosis, a pathological hallmark of the mammalian retina. This work provides a unique insight into mechanisms that control the cell cycle during neuronal regeneration.
568	An atypical HLH transcriptional regulator plays a novel and important role in strawberry ripened receptacle.	31881835	In soft fruits, the differential expression of many genes during development and ripening is responsible for changing their organoleptic properties. In strawberry fruit, although some genes involved in the metabolic regulation of the ripening process have been functionally characterized, some of the most studied genes correspond to transcription factors. High throughput transcriptomics analyses performed in strawberry red receptacle (Fragaria x ananassa) allowed us to identify a ripening-related gene that codes an atypical HLH (FaPRE1) with high sequence homology with the PACLOBUTRAZOL RESISTANCE (PRE) genes. PRE genes are atypical bHLH proteins characterized by the lack of a DNA-binding domain and whose function has been linked to the regulation of cell elongation processes.FaPRE1 sequence analysis indicates that this gene belongs to the subfamily of atypical bHLHs that also includes ILI-1 from rice, SlPRE2 from tomato and AtPRE1 from Arabidopsis, which are involved in transcriptional regulatory processes as repressors, through the blockage by heterodimerization of bHLH transcription factors. FaPRE1 presented a transcriptional model characteristic of a ripening-related gene with receptacle-specific expression, being repressed by auxins and activated by abscisic acid (ABA). However, its expression was not affected by gibberellic acid (GA3). On the other hand, the transitory silencing of FaPRE1 transcription by agroinfiltration in receptacle produced the down-regulation of a group of genes related to the ripening process while inducing the transcription of genes involved in receptacle growth and development.In summary, this work presents for the first time experimental data that support an important novel function for the atypical HLH FaPRE1 during the strawberry fruit ripening. We hypothesize that FaPRE1 modulates antagonistically the transcription of genes related to both receptacle growth and ripening. Thus, FaPRE1 would repress the expression of receptacle growth promoting genes in the ripened receptacle, while it would activate the expression of those genes related to the receptacle ripening process.
569	Ruxolitinib treatment for SR-aGVHD in patients with EBV-HLH undergoing allo-HSCT.	31879790	Ruxolitinib is a promising option for treating steroid-refractory acute graft-versus-host disease (SR-aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we describe ruxolitinib treatment for SR-aGVHD in HSCT patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) to evaluate its effectiveness. We evaluated the outcomes of 12 patients who received ruxolitinib for SR-aGVHD between January 2017 and March 2019. Of the 12 patients who received ruxolitinib, 7 patients achieved a complete response (CR), 3 had a partial response (PR), and 2 experienced treatment failure (TF). OS and CR rates were 83.3% and 58.3%, respectively. Moreover, CR was achieved by the six patients who had aGVHD with skin involvement. The mean time of steroid application in the patients who received ruxolitinib was 28.1 days. Median survival after HSCT was 64.6 weeks. The adverse effects of ruxolitinib included grades 3 to 4 neutropenia (n = 7) and grades 3 to 4 thrombocytopenia (n = 6). Cytomegalovirus reactivation was observed in three patients. A high rate of CR and short steroid application time of ruxolitinib as a salvage treatment were observed in HSCT patients with EBV-HLH. Consequently, from this study, it was determined that ruxolitinib is an optimal choice to treat SR-aGVHD in patients with EBV-HLH.
570	Neuroimaging features of CNS histiocytosis syndromes.	31874337	Histiocytosis syndromes (HS) are group of heterogeneous disorders characterized by abnormal accumulation and infiltration of histiocytes, cells derived from hematopoietic cells of monocyte/macrophage lineage. Overall these disorders are rare. When they do occur they involve many organ systems including the central nervous system (CNS). While imaging findings can provide important clues, diagnosis of this disorder is challenging and definitive diagnosis often necessitates pathologic examination. In this review, we describe imaging features of HS involving the CNS, with the aim to increase our understanding of these disorders. The entities discussed in this review will include: Langerhans cell histiocytosis (LCH), Rosai-Dorfman Disease (RDD), Erdheim Chester Disease (ECD), hemophagocytic lymphohistiocytosis (HLH), and crystal-storing histiocytosis (CSH).
571	Molecular analysis of the novel L243R mutation in STXBP2 reveals impairment of degranulation activity.	31865540	The presence of mutations in PRF1, UNC13D, STX11 and STXBP2 genes in homozygosis or compound heterozygosis results in immune deregulation. Most such cases lead to clinical manifestations of haemophagocytic lymphohistiocytosis (HLH). In the present study, we analyzed degranulation and cytotoxicity in a pediatric patient with a late presentation of HLH associated with Epstein-Barr virus infection. Remarkably, the results of the degranulation assay showed reduction of CD107a median fluorescence intensity (MFI) and absent cytotoxicity. Genetic analysis identified compound heterozygous mutations in STXBP2 gene: a previously reported splicing defect in exon 15 (c.1247-1G>C, p.V417LfsX126) and a novel missense mutation in exon 9 (c.728T>G, p.L243R). Transfection experiments of STXBP2-L243R or STXBP2-WT constructs showed an undetectable protein expression of the STXBP2-L243R mutation. The residue L243 is highly preserved evolutionarily; moreover, computational analysis of its structure revealed its participation in the rich network of interactions that stabilizes domains 2 and 3 of the protein. Altogether, we demonstrated by molecular and in silico analysis that the new L243R mutation in STXBP2 plays a pathogenic role that, together with the p.Val417Leufsc mutation, shows the synergistic negative effect of these two mutations on STXBP2 function, leading to a decrease of degranulatory activity in vivo.
572	Intravascular Large B-cell Lymphoma Associated with Systemic and Central Nervous System Hemophagocytic Lymphohistiocytosis: A Case Report.	31852057	We present a case of an Asian variant of intravascular large B-cell lymphoma associated with systemic and central nervous system hemophagocytic lymphohistiocytosis (HLH) with multiple neurologic manifestations.A 68-year-old, previously healthy, Korean woman presented with a 4-week history of fever and weight loss. She had pancytopenia with neutropenia, a ferritin level of 5030 μg/L, and elevated liver enzyme levels. Bone marrow, liver biopsy specimens, and cerebrospinal fluid demonstrated hemophagocytosis. The patient was treated with the HLH-2004 protocol, but her disease relapsed 3 months later. A repeated liver biopsy specimen confirmed the initially missed diagnosis of intravascular large B-cell lymphoma, a known driver of HLH in patients of Asian origin. She was then treated with lymphoma-directed therapy and had a prompt resolution of fever and neurologic symptoms as well as normalization of her blood cell counts and ferritin level.This case serves as a reminder that patients with an Asian variant of intravascular large B-cell lymphoma frequently present with HLH and neurologic manifestations, including seizures, strokes, and cognitive deficits. A high index of suspicion is needed for prompt diagnosis and initiation of lymphoma-directed therapy.
573	The interferon-gamma pathway is selectively up-regulated in the liver of patients with secondary hemophagocytic lymphohistiocytosis.	31846457	Aim of this study was to investigate the activation of the IFNγ pathway in the affected liver and in the blood of patients with secondary hemophagocytic lymphohistiocytosis (sHLH). To this purpose, the mRNA expression levels of IFNG and IFNγ-inducible genes as well as Tyrosine (701)-phosphorylated signal transducer and activator of transcription 1 (STAT1) protein levels were evaluated in the liver and in peripheral blood mononuclear cells (PBMCs) of three patients with sHLH with predominant liver involvement. The mRNA expression levels of IFNG and IFNγ-inducible genes were markedly higher in patient livers compared to control livers and to one disease control liver. Conversely, slight differences in the expression levels of Type I IFN-inducible genes and other classical inflammatory cytokine genes were found. Further supporting the activation of the IFNγ pathway, higher protein levels of phosphorylated and total STAT1 were detected in patient livers compared to control livers. When the expression of the same genes analysed in liver tissues was evaluated in PBMCs collected from 2 out of 3 patients before the liver biopsy, we found that mRNA levels of IFNγ-inducible genes were markedly increased. Accordingly, high circulating levels of IFNγ-inducible CXCL9 were observed in patients. Altogether, these data demonstrate the selective and marked up-regulation of the IFNγ pathway in the liver tissue and blood of patients with active sHLH. Finally, we show that measurement of circulating CXCL9 levels and evaluation of IFNγ-inducible gene expression levels in PBMCs may represent a new valid tool to better identify patients with suspected HLH with predominant liver involvement.
574	An Immune-Stimulatory Helix-Loop-Helix Peptide: Selective Inhibition of CTLA-4-B7 Interaction.	31841301	Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix-loop-helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4-B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a KD of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4-B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4-B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.
575	Talaromycosis-Associated Secondary Hemophagocytic Lymphohistiocytosis in Nine Human Immunodeficiency Virus-Negative Patients: A Multicenter Retrospective Study.	31824178	Talaromyces marneffei (T.M) is an intracellular opportunistic fungus that causes invasive mycosis in patients with or without human immunodeficiency virus (HIV) infection. Hemophagocytic lymphohistiocytosis (HLH) caused by T.M infection is extremely rare. Here, we analyzed the clinical features, immune mechanisms, treatment, and prognosis related to this comorbidity.This retrospective study was conducted between August 2012 and February 2019 at multiple research centers. Patients who presented with culture and/or histopathological proof of talaromycosis-associated HLH were included.HIV-negative patients (n = 126) were enrolled. Of nine patients with T.M infection combined with secondary HLH, six were preschool children (five boys and one girl), and three were adults (two men and one woman). Seven of these nine had underlying diseases or recurrent infections. The most common symptoms were fever, anemia, hypoproteinemia, cough, weight loss, oral thrush, lymphadenopathy, hepatomegaly, splenomegaly, digestive symptoms, joint pain, and dyspnea. All patients showed reduced hemoglobin concentrations and platelet numbers. Liver dysfunction, hyperferritinemia, elevated lactate dehydrogenase, and low natural killer cell numbers were observed. Eight of nine patients received antifungal therapy, one patient did not receive therapy, and two of nine patients received anti-HLH therapy. Four died during treatment.T.M fungemia associated with HLH was related to high mortality. Once diagnosed, timely and effective antifungal treatments and supportive care are essential.
576	A rare complication of systemic lupus erythematosus in a 9-year-old girl: Questions.	31823041	Serious renal involvement in systemic diseases is common and generally constitutes a pivotal prognostic factor, making those pathology frequently seen in nephrology departments. Authors describe the case of a nine-year-old girl with lupus nephritis. After admission the patient's state deteriorated over a period of a few days, with an unremitting high-grade fever, significant weakness and drowsiness, generalized erythema, and decrease of the kidney function to eGFR nadir of 56 ml/min/1,73m 2. Treatment with pulsed methylprednisolone was started. After the first pulse the general state of the patient improved slightly, although laboratory tests showed an alarming evolution, with the exacerbation of anemia, leukopenia, neutropenia, increase of serum CRP concentration, extremely high D-dimer concentration and increase in activity of lactate dehydrogenase. The concentration of ferritin rose reaching the level of 540 μg/l, triglicerydes level was also high. Intravenous cyclophosphamide pulse therapy was added to the ongoing steroid treatment, and resulted in a radical patient improvement. Authors underline that it seems important to be aware of rare, non-renal, but potentially devastating complications of systemic diseases, like in this clinical case: the secondary hemophagocytic lymphohistiocytosis (HLH). When HLH complicates a rheumatic disease, it is also referred to as macrophage activation syndrome (MAS). Unfortunately treatment of MAS is still based on reports provided by individual centres and gathered own experiences so drawing up unambiguous diagnostic criteria will be valuable in future. The treatment should be individually tailored, and more specific evidence-based recommendations are needed.
577	Novel AP3B1 compound heterozygous mutations in a Japanese patient with Hermansky-Pudlak syndrome type 2.	31820501	Hermansky-Pudlak syndrome type 2 (HPS2) is an extremely rare autosomal recessive inherited disease characterized by partial oculocutaneous albinism (OCA), bleeding diathesis due to a storage pool deficiency and immunodeficiency. The disorder is caused by disruption of the adapter protein 3 complex, which is involved in impaired intracellular vesicle transport. Here, we report the first case of a 1-year-old girl with HPS2 in Asia. She had no specific symptoms other than OCA and neutropenia. We analyzed her platelet function using transmission electron microscopy and a platelet aggregation test, cytotoxic degranulation assay of her natural killer (NK) cells and bleeding time, the results of which led to the diagnosis of HPS2. Although her NK-cell cytotoxic degranulation was impaired, she had not developed signs of hemophagocytic lymphohistiocytosis (HLH) or fibrosing lung disease. Molecular genetic analyses showed novel heterozygous mutations (c.188T>A [p.M63K] and c.2546>A [p.L849X]) in AP3B1. When examining patients with OCA, blood tests should be performed to confirm neutrophil count, bleeding time and platelet agglutination. When HPS2 is suspected, detailed immunological tests should be considered, and attention should be paid to HLH and pulmonary lesions immediately and over the long term.
578	Hodgkin's lymphoma with HLH and complete remission with brentuximab-based therapy.	31818889	A 63-year-old man presented to the hospital with generalised weakness, fatigue and a 22 kg weight loss 4 months after being diagnosed with sarcoidosis on a mediastinal lymph node biopsy, with minimal improvement in symptoms on prednisone and methotrexate therapy. On arrival, he was found to have a haemoglobin of 57 g/L and platelet count of 82×109/L. Further work-up revealed six of eight diagnostic criteria for haemophagocytic lymphohistiocytosis (HLH): fever >38.9°C, splenomegaly, cytopaenia, hypertriglyceridaemia, haemophagocytosis and elevated ferritin >31 000 ng/mL. He was also found to have Epstein-Barr viraemia with greater than 17 000 copies. Bone marrow biopsy showed the presence of haemophagocytic histiocytes and evidence of classic Hodgkin's lymphoma. He was started on HLH-94 protocol. Later treatment was switched to lymphoma-directed therapy and he finished six cycles of A+AVD (brentuximab vedotin, doxorubicin, vinblastine and dacarbazine) with end-of-treatment positron emission tomography/CT and bone marrow negative for lymphoma.
579	Clinical features of infantile subcutaneous panniculitis-like T-cell lymphoma.	31808238	NaN
580	Type 5 Familial Hemophagocytic Lymphohistiocytosis in a Seven-year-old Girl Post Second Bone Marrow Transplantation with Failure to Thrive: STXBP2 Novel Mutation.	31807395	Familial hemophagocytic lymphohistiocytosis (HLH) is a fatal autosomal recessive disorder resulting in an exaggerated and ineffective immune response. Genetic defects in familial HLH can lead to the impaired function of the secretory lysosome-dependent exocytosis pathway. We report an STXBP2 homozygous missense mutation c.1139A>G, p.(Gln380Arg) consistent with a genetic diagnosis of familial hemophagocytic lymphohistiocytosis type 5 associated with chronic diarrhea in a seven-year-old girl. She was diagnosed with HLH and achieved remission by the HLH-2004 protocol and allogeneic matched bone marrow transplantation (BMT) from her sibling. However, six years later, she had a relapse of HLH, which required a second BMT. Ever since then, she continued to have persistent chronic watery diarrhea and failure to thrive. Patients with familial HLH type 5 due to STXBP2 gene mutation can manifest as either with or without chronic diarrhea. This unusual relationship directs toward a specific gene mutation of STXBP2 as the cause of chronic diarrhea in familial HLH. The prevalence of familial HLH in Saudi Arabia is underestimated. Due to the high rate of consanguinity and the local customs of marrying within the same community, clinicians should consider familial HLH as a cause of persistent, unexplained, chronic diarrhea among the pediatric age group.
581	A developmental gene regulatory network for C. elegans anchor cell invasion.	31806663	Cellular invasion is a key part of development, immunity and disease. Using an in vivo model of Caenorhabditis elegans anchor cell invasion, we characterize the gene regulatory network that promotes cell invasion. The anchor cell is initially specified in a stochastic cell fate decision mediated by Notch signaling. Previous research has identified four conserved transcription factors, fos-1 (Fos), egl-43 (EVI1/MEL), hlh-2 (E/Daughterless) and nhr-67 (NR2E1/TLX), that mediate anchor cell specification and/or invasive behavior. Connections between these transcription factors and the underlying cell biology that they regulate are poorly understood. Here, using genome editing and RNA interference, we examine transcription factor interactions before and after anchor cell specification. Initially, these transcription factors function independently of one another to regulate LIN-12 (Notch) activity. Following anchor cell specification, egl-43, hlh-2 and nhr-67 function largely parallel to fos-1 in a type I coherent feed-forward loop with positive feedback to promote invasion. Together, these results demonstrate that the same transcription factors can function in cell fate specification and differentiated cell behavior, and that a gene regulatory network can be rapidly assembled to reinforce a post-mitotic, pro-invasive state.
582	Pediatric macrophage activation syndrome, recognizing the tip of the Iceberg.	31804174	Macrophage activation syndrome (MAS) is the name given to secondary hemophagocytic lymphohistiocytosis (sHLH) associated with rheumatic diseases. Previously, MAS has been best studied in children with systemic juvenile idiopathic arthritis (sJIA), who are at high risk of developing MAS. MAS/sHLH is a cytokine storm that results in multi-organ system failure and is frequently fatal. Early diagnosis and treatment is critical for improving survival. Various diagnostic tools have been developed for identifying MAS in the setting of sJIA, as well as for all forms of MAS/sHLH. These are largely based on clinical (e.g., fever) and laboratory features (e.g., cytopenias). None are perfectly sensitive and specific, however, increasing awareness of this condition is also paramount in making the diagnosis. Rare familial forms of HLH can also be diagnosed based on homozygous mutation in genes largely involved in perforin-mediated cytolytic function of lymphocytes (natural killer cells and CD8 T cells). Intriguingly, heterozygous defects in these same genes are frequently identified in patients with sHLH and MAS. Decreased cytolytic function results in prolonged interaction of the lytic lymphocytes and their target antigen presenting cells, thus resulting in the pro-inflammatory cytokine storm believed responsible for the multi-organ failure. Novel cytokine-targeted therapies are currently being explored for a less toxic yet effective alternative to chemotherapeutic approaches to treating children with sHLH/MAS. As increased recognition and diagnosis of MAS is on the rise, an earlier and cytokine-targeted approach to therapy will likely save many lives of children with this disorder.
583	Parvovirus B19-induced hemophagocytic lymphohistiocytosis: Case report and review of the literature.	31788255	HLH is a catastrophic and likely underdiagnosed pathology with multiple triggers including infection. PVB19 can cause persistent marrow infection leading to HLH despite negative acute serologic markers making timely diagnosis difficult. Increased awareness of PVB19-HLH is warranted given its potentially lethal nature and the careful interpretation required with serologic markers.
584	Comparison between clinical features and prognosis of malignancy- and non-malignancy-associated pediatric hemophagocytic lymphohistiocytosis.	31783813	The differences between the clinical characteristics and survival time in malignancy- and non-malignancy-associated secondary hemophagocytic lymphohistiocytosis (HLH) are unclear. Here, we describe the clinical characteristics, prognostic factors, and survival outcomes of malignancy-associated HLH compared to that of non-malignancy-associated HLH.We retrospectively analyzed 91 pediatric patients with HLH (age < 14 years) at the Affiliated Hospital of Qingdao University Pediatric Department between January 2005 and October 2016. The patients were divided into the malignancy-associated group (n = 22) and non-malignancy-associated group (n = 69, also considered the control group). The clinical features were compared using the Mann-Whitney U and χ2 tests. The overall survival time was compared using log rank and Mann-Whitney U tests.Hemoglobin (HGB; p = 0.004), alanine aminotransferase (ALT; p = 0.002), and aspartate aminotransferase (AST; p = 0.001) levels in the malignancy-associated group differed from that in the non-malignancy-associated group. The mean survival times were 26.9 ± 3.82 months (malignancy-associated HLH) and 35.03 ± 2.19 months (non-malignancy-associated HLH). The overall survival time between the two groups was not statistically significantly different (p = 0.055). Univariate analysis showed that disseminated intravascular coagulation (DIC) score > 5 (p = 0.001), albumin < 25 g/L (p = 0.000), HGB < 60 g/L (p = 0.001), and platelet count (PLT) < 30 × 109/L (p = 0.042) correlated with prognosis. Multivariate Cox analysis showed that albumin < 25 g/L (p = 0.017), HGB < 60 g/L (p = 0.027), and bone marrow hemophagocytosis (p = 0.034) correlated with worse prognosis.Patients with non-malignancy-associated HLH do not have better survival, although their prognosis is relatively better in clinical practice. A higher DIC score at diagnosis and lower albumin, HGB, and PLT levels are negative prognostic factors in malignancy-associated HLH.
585	Hemophagocytic lymphohistiocytosis in a patient with glioblastoma: a case report.	31777271	Adult onset hemophagocytic lymphohistiocytosis (HLH) is a rare condition, usually secondary to either a precipitating infective or hematologic malignancy. We present a case of Epstein-Barr virus associated HLH in a 55-year-old female receiving treatment for a glioblastoma (GBM). It is possible that HLH is under recognized, as patients with GBM often have features of a nonspecific systemic inflammatory response syndrome, multiorgan failure and cognitive decline. A high index of suspicion and increased awareness can help improve timeliness of diagnosis. Therapeutically, Epstein-Barr virus associated HLH in patients with solid organ malignancy poses significant challenges. An individualized, multidisciplinary approach is essential when managing adult-onset HLH and providers will need to be mindful of the high mortality rate despite treatment.
586	Familial hemophagocytic lymphohistiocytosis in a girl with a novel homozygous mutation of STX11: A case report.	31770233	Familial hemophagocytic lymphohistiocytosis (FHL) is a rare fatal autosomal recessively inherited disease and can be divided into five types. The mortality of untreated patients is up to 95% and it can be healed only after immunochemotherapy for disease control and hematopoietic stem cell transplantation. Clinical data of a girl with late-onset and recurrent hemophagocytic lymphohistiocytosis (HLH) was retrospectively analyzed to determine the etiology and potential pathogenic gene.The proband was a female child patient from a consanguineous marriage family who was 11 years old, and clinically manifested delayed (onset at the age of 4 years and 6 months) and recrudescent HLH. Both of her elder brothers died at the ages of 4 and 5 years, respectively. The patient had a degranulation function defect of CD107a in natural killer (NK) cells, and the degranulation function of cytotoxic T lymphocytes (CTL) obviously declined (ΔMFI: 1.4%, normal ≧2.8%); the degranulation function of NK cells and CTL of her father was also obviously reduced. To identify possible underlying genetic causes, gene mutation analysis was undertaken. A novel homozygous nonsense mutation in STX11 (c.49C>T, p.Q17X) was documented, arising from both her parents.According to the clinical manifestations and detection results of STX11, the diagnosis of FHL-type 4 was confirmed and her parents were heterozygotic carriers.Good responses to HLH-2004 chemotherapy had been achieved for each onset, and the maximum remission duration (without taking any drug) was 23 months. The patient has been alive for 82 months since the onset, and has stopped taking dexamethasone and etoposide, but is still on oral cyclosporine to maintain the treatment. She has performed HLA matching and now is actively looking for a donor to prepare hematopoietic stem cell transplantation.Relevant gene detections should be implemented at the earliest for young patients from consanguineous marriages and with a family history of HLH so as to provide a basis for etiological diagnosis and radical treatment by hematopoietic stem cell transplantation and provide accurate genetic counseling for family members.
587	Haemophagocytic lymphohisticytosis-an underrecognized hyperinflammatory syndrome.	31769857	Haemophagocytic lymphohisticytosis (HLH) is a syndrome of uncontrolled, severe systemic inflammation (hyperinflammation) arising either from a genetic immune system defect [primary (pHLH)] or triggered as a complication of malignancy, infection, or rheumatologic disease [secondary (sHLH)]. Patients with HLH often have non-specific symptoms and become progressively and critically unwell, with fever, cytopenia and multi-organ failure. Untreated, HLH is almost universally fatal, but even when treated, mortality is high, particularly when HLH complicates malignancy. HLH is managed with immunosuppression, and this can seem difficult to justify in such unwell patients. This review aims to examine the diagnostic and treatment challenges posed by sHLH and to improve recognition among rheumatologists who, being expert in the management of multisystem diseases and in the use of immunosuppression, are ideally placed to deliver care and build an evidence base for better disease characterization and treatment.
588	Epstein-Barr virus-associated T- and NK-cell lymphoproliferative diseases: an update and diagnostic approach.	31767131	Epstein-Barr virus (EBV)-positive T-cell and natural killer (NK)-cell lymphoproliferative diseases (EBV-TNKLPD) are a group of uncommon disorders characterised by EBV infection of T- and NK-cells. As a group, EBV-TNKLPD are more commonly encountered in Asians and Native Americans from Central and South America compared to Western populations. They encompass a spectrum of entities that range from non-neoplastic lesions such as EBV-associated haemophagocytic lymphohistiocytosis (EBV-HLH) to more chronic conditions with variable outcomes such as chronic active EBV infections (CAEBV) of T- and NK-cell type (cutaneous and systemic forms) and malignant diseases such as systemic EBV-positive T-cell lymphoma of childhood, aggressive NK-cell leukaemia, extranodal NK/T-cell lymphoma, nasal-type, and primary EBV-positive nodal T/NK-cell lymphoma. Due to their rarity, broad clinicopathological spectrum and significant morphological and immunophenotypic overlap, the diagnosis and precise classification of EBV-TNKLPD often pose a challenge to clinicians and pathologists. Correct classification of this group of rare diseases relies heavily on the age of onset, disease presentation, duration of symptoms and cell of origin (T- vs NK-cell lineage). In this review, we provide an update on the clinicopathological and molecular features of the various EBV-TNKLPD entities occurring in non-immunocompromised patients and present a practical algorithmic approach for the general pathologist who is confronted with these disorders in routine clinical practice.
589	[A case of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in an elderly woman].	31761859	An 84-year-old woman who had diabetes mellitus and atrial fibrillation presented to our hospital with appetite loss and difficulty in standing up. Her cervical, axillary, and mediastinal lymph nodes were swollen. At first, malignant lymphoma was suspected, but positron emission tomography-computed tomography and cervical lymph node biopsy did not support this diagnosis. She was admitted to our hospital 41 days after the first consultation because of appetite loss and fever. We suspected a disorder related to Epstein-Barr virus infection because Epstein-Barr virus-encoded small RNAs were found in the lymph node. She was diagnosed with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) based on the diagnostic criteria of the Japanese Society for Pediatric Infectious Diseases. Her symptoms, including appetite loss and fever, were alleviated after the administration of prednisolone (20 mg per day). She was discharged 90 days after admission. At two weeks after discharge, she was readmitted for respiratory failure, and died five days after readmission.To the best of our knowledge there are no previous reports of EBV-HLH in patients over 80 years of age, and the optimal treatment strategy is unknown. This case suggested that prednisolone may alleviate the symptoms of EBV-HLH. When a patient presents with swollen lymph nodes, clinicians should keep the possibility of EBV-HLH in mind.
590	X-linked lymphoproliferative syndrome in mainland China: review of clinical, genetic, and immunological characteristic.	31754776	X-linked lymphoproliferative syndrome (XLP) is a rare primary immunodeficiency disease that can be divided into two types: SAP deficiency (XLP1) and XIAP deficiency (XLP2), caused by mutations in the SH2D1A and XIAP genes, respectively. Few cases of XLP (particularly XIAP deficiency) have been reported in mainland China; hence, little is known about the characteristics of Chinese patients with XLP. We identified 13 and 7 patients with SAP and XIAP deficiency, respectively, in our center. Of our 20 patients, 19/20 (95%) presented with disease symptoms at a very early age: six in infancy and 13 in childhood. One XIAP- and three SAP-deficient patients died, while 3/7(42.9%) and 4/13(30.8%), respectively, developed hemophagocytic lymphohistiocytosis (HLH). Epstein-Barr virus (EBV) infection was significantly more common in SAP-deficient 10/13 (76.9%) than XIAP-deficient 2/7 (28.6%) patients, as was hypogammaglobulinemia (10/13 (76.9%) vs. 1/7 (14.3%)). None of the seven XIAP-deficient patients had colitis or lymphoma. Nine SAP-deficient patients and five XIAP-deficient patients showed markedly deficient SAP and XIAP expression, respectively, in lymphocytes. Significantly reduced levels of switched memory B cells were observed in six SAP-deficient patients with persistent hypogammaglobulinemia. One of 13 (7.7%) SAP-deficient patients and 1 of 7 (12.3%) XIAP-deficient patients have received HSCT treatment and are now alive and well; the other alive patients were waiting for HSCT. We also summarized clinical, genetic, and immunological characteristics of all 55 patients (including our 20 patients) reported in the literature in mainland China today.Conclusion: The overall characteristics of SAP deficiency in mainland China were consistent with those in previous reports, whereas manifestations of XIAP deficiency varied significantly. None of inflammatory bowel disease (IBD) has been reported among XIAP-deficient patients in our center; however, whether Chinese XIAP-deficient patients will develop colitis in the future warrants further investigation. HSCT is the only curative therapy for XLP and this therapy should be urgently considered.What is Known:• SAP and XIAP deficiencies share common clinical feature, HLH, whereas they also have their own specific manifestations.• IBD affects 25-30% of XIAP-deficient patients, which has been reported in other countries especially in European country and Japan.What is New:• This is the largest patient cohort study of XLP in China.• We firstly summarized the clinical features and outcomes of Chinese XIAP-deficient patients and found only 1 in 22 patients developed IBD and diet background may contribute to it; Asian SAP-deficient patients carrying SH2D1A R55X mutation were more prone to HLH.
591	Epidemiologic features of children with Epstein-Barr virus associated diseases in Hangzhou, China.	31750545	Epstein-Barr virus (EBV) infection is the causative agent of multiple diseases. EBV DNA in blood is a useful diagnostic marker of primary EBV infection and reactivation. This study aimed to provide epidemiological information on children with EBV-associated diseases identified by positive EBV DNA in Hangzhou, a city in East China. All children admitted to the Children's Hospital of Zhejiang University School of Medicine from 2010 to 2015 with suspected EBV-related diseases and serum EBV DNA tested by quantitative real-time polymerase chain reaction were included. Of the 10 470 children, 1205 were determined to have positive EBV DNA, and the positive rate was 11.5%. 15.8% (973 of 6162) of the illnesses of patients aged 1 to 7 years were caused by EBV as compared to that of 6.6% (179 of 2708) of children older than 7 years (P < .01) and 3.3% (53 of 1600) of of that of infants <1 year of age (P < .01). Among positive EBV DNA patients, 80.7% of EBV infections occurred in children at the age stage of 1 to 7 years. IM was the most common EBV-related disease, accounting for 75.7% of 581 hospitalized patients. Children aged 1 to 3 years were the age group most commonly hospitalized with EBV-IM (32.7% of the cohort) and EBV-hemophagocytic lymphohistiocytosis (HLH) (52.6%), while EBV-lymphoma was more common in children over 9-year old (58.3% of the cohort). The serum EBV-DNA load was much higher in patients with EBV-HLH than in patients with IM (P < .05). This is a large sample study, which revealed the epidemiological characteristics of children with EBV-associated diseases, including age, monthly and disease distribution.
592	Platelet-derived alpha-granules are associated with inflammation in patients with NK/T-cell lymphoma-associated hemophagocytic syndrome.	31739215	Due to the variable overlap of multiple symptoms, accurate early diagnosis of NK/T-cell lymphoma-associated hemophagocytic syndrome (NK/T-LAHS) is difficult, making the prognosis extremely poor. Hemophagocytic syndrome (HPS) is now diagnosed primarily based on the hemophagocytic lymphohistiocytosis (HLH)-2004 diagnostic criteria, and platelet count is one of the baseline evaluations. However, in our study, the data showed that decreased platelets were not only a clinical feature of HPS but also the key cells that regulate inflammation by releasing α-granules containing upregulated platelet factor 4 (PF4) and downregulated platelet-derived growth factors (PDGFs). Furthermore, we found that angiopoietin-4 (ANG-4), which has significant differential expression, has been less reported, that may affect hematopoiesis and proinflammatory responses and can be used as diagnostic biomarkers together with PF4 and PDGFs.
593	Hemophagocytic Lymphohistiocytosis in Langerhans Cell Histiocytosis: A Case Report and Review of the Literature.	31725543	A toddler undergoing treatment for refractory Langerhans cell histiocytosis (LCH) developed concurrent hemophagocytic lymphohistiocytosis (HLH). These are thought to be distinct histiocytic disorders, with different pathophysiologies, diagnostic criteria, and treatments. HLH in a patient with LCH is thought to be quite rare. In this report, we review the presentation of our patient, as well as review the existing literature of other pediatric patients who have been diagnosed with both LCH and HLH.
594	Successful Treatment of Veno-occlusive Disease, Transplantation-Associated Thrombotic Microangiopathy, and Acute Graft-vs-Host Disease in a Patient with Relapsed Epstein-Barr Hemophagocytic Lymphohistiocytosis After Haploidentical Hematopoietic Stem Cell Transplantation: A Case Report.	31711585	Allogenic hematopoietic stem cell transplantation may be the best currently available method to treat relapsed hemophagocytic lymphohistiocytosis (HLH) related to Epstein-Barr virus. The high rate of transplantation-related complications was initially the main obstacle preventing the wider adoption of this protocol; however, the previously more common complications, such as infection and graft failure, have fallen to very low levels with the development of new drugs and methods. Some other complications, such as veno-occlusive disease and transplantation associated thrombotic microangiopathy, are rare after allogenic hematopoietic stem cell transplantation, but the morbidity and mortality associated with them are very high.A patient with relapsed HLH related to Epstein-Barr virus showed the sequential severe complications of veno-occlusive disease, transplantation-associated thrombotic microangiopathy, and acute graft-vs-host disease after haploidentical transplantation. This patient was successfully treated by stopping administration of calcineurin inhibitors and instead treating with defibrotide, rituximab, CD25 monoclonal antibody, atorvastatin calcium tablets, methylprednisolone, budesonide, continuous plasma exchange, and bedside ultrafiltration. At the last follow-up, the patient had been living disease free for 2 years without any other complications.Epstein-Barr virus related-HLH patients have severe clinical features and currently poor prognosis. Allogenic hematopoietic stem cell transplantation may be the best way to treat this disease; however, the management of related complications is vital in the improvement of long-term survival.
595	A research on the effects of successful aging on the acceptance and use of technology of the elderly.	31710261	The aim of the study is to analyze the effects of successful aging on technology acceptance and use behaviors, via developing a model. This study was conducted with 687 participants in five Retirement Rest Homes in Turkey. A model was developed to meet the study's research objectives using the scales of successful aging and the second unified theory of acceptance and use of technology. A path analysis was performed with Partial Least Squares (PLS) structural equation model. Struggle Against Difficulties (SAD) significantly predicted performance expectancy, effort expectancy, social influence, facilitating conditions, hedonic motivation, price value, habit, and behavior intention, even though SAD did not predict USE. HLH significantly predicted performance expectancy, social influence and facilitating conditions; HLH did not predict effort expectancy, hedonic motivation, price 15 value, habit, behavior intention, and USE. It was revealed that successful aging affected the technology acceptance model in general except the use of technology. It is recommended for future studies that successful aging should be evaluated with varied model studies.
596	Haematopoietic Stem Cell Transplantation for Primary Haemophagocytic Lymphohistiocytosis.	31709205	Haematopoietic stem cell transplantation currently remains the only curative treatment of primary forms of haemophagocytic lymphohistiocytosis (HLH). Rapid diagnosis, efficient primary treatment of hyperinflammation, and conditioning regimens tailored to this demanding condition have substantially improved prognosis in the past 40 years. However, refractory hyperinflammation, central nervous system (CNS) involvement, unavailability of matched donors, susceptibility to conditioning-related toxicities, and a high frequency of mixed chimaerism remain a challenge in a substantial proportion of patients. Gene therapeutic approaches for several genetic defects of primary HLH are being developed at pre-clinical and translational levels.
597	Identification, characterization, and expression analysis of a serotonin receptor involved in the reproductive process of the Pacific abalone, Haliotis discus hannai.	31696430	Serotonin receptor (5-HT) is a biogenic amine acting as a neurotransmitter and neuromodulator that mediates various aspects of reproduction and gametogenesis. The full-length nucleotide sequence of Haliotis discus hannai encodes a protein of 417 amino acids with a predicted molecular mass of 46.54 kDa and isoelectric point of 8.94. The structural profile of 5-HTHdh displayed key features of G protein-coupled receptors, including seven hydrophobic transmembrane domains, putative N-linked glycosylation sites, and several phosphorylation consensus motifs. It shares the highest homology of its amino acid sequence with the 5-HT receptor from Haliotis asinina, and to lesser extent of human 5-HT receptor. The cloned sequence possesses two cysteine residues (Cys-115 and Cys-193), which are likely to form a disulfide bond. Phylogenetic comparison with other known 5-HT receptor genes revealed that the 5-HTHdh is most closely related to the 5-HTHa receptor. The three-dimensional structure of the 5-HTHdh showed multiple alpha helices which is separated by a helix-loop-helix (HLH) structure. Quantitative PCR demonstrated that the receptor mRNA was predominantly expressed in the pleuropedal ganglion. Significant differences in the transcriptional activity of the 5-HTHdh gene were observed in the ovary at the ripening stage. An exclusive expression was detected in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000 °C). In situ hybridization showed that the 5-HTHdh expressing neurosecretory cells were distributed in the cortex of the pleuropedal ganglion. Our results suggest that 5-HTHdh synthesized in the neural ganglia may be involved in oocyte maturation and spawning of H. discus hannai.
598	Serum high mobility group box protein 1 (HMGB1) levels reflect clinical features of childhood hemophagocytic lymphohistiocytosis.	31695540	Hemophagocytic lymphohistiocytosis (HLH) is a potentially lethal hyperinflammatory disorder. For further understanding of the pathogenesis of HLH, we examined serum levels of high mobility group box protein 1 (HMGB1) in children with HLH.Serum HMGB1 levels were measured in 28 patients with HLH and 6 normal controls using a quantitative enzyme-linked immunosorbent assay. The patients were 21 boys and 7 girls, aged from 10 days to 21 years, with a median age of 8.5 years. The underlying conditions of HLH were infection-associated HLH in 18 patients, malignancy-associated HLH in 7 patients, and genetic HLH in 3 patients. The relations between serum HMGB1 levels and clinical symptoms and laboratory parameters were analyzed.Serum HMGB1 levels were significantly higher in patients with HLH than in normal controls (median, 6.5 ng/mL, interquartile range, 4.25-13.1). The serial serum HMGB1 levels in one patient fell to reflect the disease activity. Serum HMGB1 levels were significantly higher in patients with disseminated intravascular coagulation (DIC) than in patients without DIC (p<0.001) and were also significantly higher in patients with central nervous system (CNS) complications than in patients without CNS complications (p<0.01). Serum HMGB1 levels were positively correlated with aspartate aminotransferase (rs =0.48, p<0.01, Spearman's rank correlation coefficient) and negatively correlated with fibrinogen (rs = -0.475, p=0.011) and hemoglobin (rs = -0.465, p=0.013).Serum HMGB1 levels reflect clinical features of childhood HLH. HMGB1 is a potential mediator involved in the pathogenesis and determining the clinical findings of HLH.
599	A Pediatric Case Report of Epstein-Barr Virus-associated Hemophagocytic Lymphohistiocytosis With Pericardial Effusion and Multiple Coronary Artery Aneurysms.	31688637	Pediatric coronary artery aneurysms (CAAs) are mainly detected in Kawasaki disease and in chronic active Epstein-Barr virus (EBV) infection sometimes, and cardiac complications are rare in viral-associated hemophagocytic lymphohistiocytosis (HLH) patients. Here, we report a pediatric case of EBV-associated HLH with pericardial effusion and multiple CAAs, whereas the patient did not fulfill the diagnostic criteria of Kawasaki disease or chronic active EBV. The case indicates that CAAs may occur in EBV-HLH. Specifically, in a patient with a long-term fever and a high EBV DNA copy number, the detection of cardiac complications may help signal the possible occurrence of HLH, and CAAs may affect the prognosis for high risk of cardiac events.
600	Epstein-Barr Virus Primary Infection Complicated by Hemophagocytic Lymphohistiocytosis and Plasmablastic Lymphoma in a HIV-Negative Patient.	31687232	EBV (Epstein-Barr virus) viremia causes immune dysregulation through various mechanisms, and we are understanding more that mutations in B, T, and NK (natural killer) cell signaling pathways allow EBV complications such as HLH (hemophagocytic lymphohistiocytosis) and lymphomas to arise. Here, we report a 20-year-old previously healthy, HIV- (human immunodeficiency virus-) negative male who presented with fevers, sore throat, and lymphadenopathy (LAD). He was found to have EBV viremia, pancytopenia, and elevated LFTs (liver function tests) suspicious for HLH. Bone marrow biopsy and elevated IL-2 (interleukin) receptor confirmed this diagnosis. Additionally, gastric biopsy confirmed diagnosis of plasmablastic lymphoma (PBL), a rare, aggressive HIV- and EBV-associated lymphoma. Both bone marrow and gastric biopsy showed evidence of EBV. Patients with EBV complications should have a rigorous workup to characterize the full extent of immune dysregulation including genetic testing at a high-volume center.
601	Hemophagocytic Lymphohistiocytosis in an AIDS Patient with Kaposi Sarcoma: A Treatment Dilemma.	31687230	Hemophagocytic lymphohistiocytosis (HLH) is a result of an abnormal activation of immune cells (T lymphocytes, natural killer cells, and macrophages) resulting in cytokine overproduction and immune destruction of cells, eventually resulting in multiorgan failure. Genetic causes are responsible for primary hemophagocytosis, but malignancies, infections, and autoimmunity underlie most of the secondary cases. We present an unusual case of a patient with AIDS and disseminated Kaposi sarcoma who was commenced on highly active antiretroviral therapy (HAART) but developed HLH secondary to immune reconstitution inflammatory syndrome (IRIS). We report this case to highlight the difficulty in managing this patient given the complex interplay of immunosuppression due to AIDS, immune reconstitution following initiation of HAART, and immune overdrive manifesting as HLH.
602	Heightened Immune Response to Presumed Loxosceles reclusa Envenomation.	31685322	Loxoscelism is a systemic inflammatory reaction in response to a brown recluse spider bite (BRSB). In this case we describe a patient with a heightened inflammatory response to a presumed BRSB, with Coomb's positive hemolysis, cytoplasmic antineutrophil cytoplasmic antibody (cANCA) vasculitis, and features of hemophagocytic lymphohistiocytosis (HLH). A 24-y-old female presented with sudden pain and swelling to her lower back, nausea, fever, and tachycardia after a presumed BRSB. Hemolysis began on hospital day 3 (15.9 g·dL-1) with a nadir on hospital day 5 (6.3 g·dL-1). She had an lactate dehydrogenase of 1415 U·L-1, ferritin of 15534 ng·mL-1, persistent fever, and signs of bone marrow suppression despite hemolysis, with thrombocytopenia (100,000 μL-1) and an inadequate reticulocyte response (1.7%) suggestive of HLH. The patient's blood was Coomb's and cANCA/antiproteinase 3 positive. She had signs of toxin-induced vasculitis, with respiratory failure requiring bilevel positive airway pressure, radiographs with bilateral pulmonary infiltrates, and a desquamating rash. She received 6 U of packed red blood cells, furosemide for pleural and pericardial effusions, antibiotics, and symptomatic treatment during the acute phase of her illness. Hemolysis improved without glucocorticoids by hospital day 6. The patient demonstrated a dysregulated immunologic and complement-mediated response to the presumed BRSB. The triad of Coomb's positive hemolysis, cANCA vasculitis, and HLH-like reaction associated with a presumed BRSB is described for the first time in the literature and brings up questions for future research regarding the role of immune modulators and complement inhibitors in the treatment of severe loxoscelism as well as the genetic factors that predispose certain individuals to such reactions.
603	Natural Killer Cell Function Tests by Flowcytometry-Based Cytotoxicity and IFN-γ Production for the Diagnosis of Adult Hemophagocytic Lymphohistiocytosis.	31671661	Although natural killer (NK) cell function is a hallmark of hemophagocytic lymphohistiocytosis (HLH), there is no standard method or data on its diagnostic value in adults. Thus, we performed a single-center retrospective study of 119 adult patients with suspected HLH. NK cell function was determined using both flowcytometry-based NK-cytotoxicity test (NK-cytotoxicity) and NK cell activity test for interferon-gamma (NKA-IFNγ). NK cell phenotype and serum cytokine levels were also tested. Fifty (42.0%) HLH patients showed significantly reduced NK cell function compared to 69 non-HLH patients by both NK-cytotoxicity and NKA-IFNγ (p < 0.001 and p = 0.020, respectively). Agreement between NK-cytotoxicity and NKA-IFNγ was 88.0% in HLH patients and 58.0% in non-HLH patients. NK-cytotoxicity and NKA-IFNγ assays predicted HLH with sensitivities of 96.0% and 92.0%, respectively. The combination of NKA-IFNγ and ferritin (>10,000 µg/L) was helpful for ruling out HLH, with a specificity of 94.2%. Decreased NK-cytotoxicity was associated with increased soluble IL-2 receptor levels and decreased CD56dim NK cells. Decreased NKA-IFNγ was associated with decreased serum cytokine levels. We suggest that both NK-cytotoxicity and NKA-IFNγ could be used for diagnosis of HLH. Further studies are needed to validate the diagnostic and prognostic value of NK cell function tests.
604	Characterization of Retinal Lesions Secondary to Hemophagocytic Lymphohistiocytosis With OCTA.	31671199	Hemophagocytic lymphohistiocytosis (HLH) is a high-mortality primary immunologic or genetic disorder that rarely presents with ocular symptoms. This is a case report of a 30-year-old Asian female with quiescent HLH in whom retinal lesions were identified during the third trimester of pregnancy. Multimodal imaging, including the first use of optical coherence tomography angiography (OCTA) in HLH, was used to characterize these lesions. OCTA was useful for distinguishing chronic ocular HLH from other uveitic syndromes affecting the retina. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:653-655.].
605	Discovery of BAY-298 and BAY-899: Tetrahydro-1,6-naphthyridine-Based, Potent, and Selective Antagonists of the Luteinizing Hormone Receptor Which Reduce Sex Hormone Levels in Vivo.	31670515	The human luteinizing hormone receptor (hLH-R) is a member of the glycoprotein hormone family of G-protein-coupled receptors (GPCRs), activated by luteinizing hormone (hLH) and essentially involved in the regulation of sex hormone production. Thus, hLH-R represents a valid target for the treatment of sex hormone-dependent cancers and diseases (polycystic ovary syndrome, uterine fibroids, endometriosis) as well as contraception. Screening of the Bayer compound library led to the discovery of tetrahydrothienopyridine derivatives as novel, small-molecule (SMOL) hLH-R inhibitors and to the development of BAY-298, the first nanomolar hLH-R antagonist reducing sex hormone levels in vivo. Further optimization of physicochemical, pharmacokinetic, and safety parameters led to the identification of BAY-899 with an improved in vitro profile and proven efficacy in vivo. BAY-298 and BAY-899 serve as valuable tool compounds to study hLH-R signaling in vitro and to interfere with the production of sex hormones in vivo.
606	A Case of Human Immunodeficiency Virus -triggered Hemophagocytic Lymphohistocytosis Presenting with Severe Bleeding Tendency.	31668025	Human immunodeficiency virus (HIV) is one of the less common triggers of secondary hemophagocytic lymphohistiocytosis (HLH) in which coagulation disorder is a frequent manifestation. Here, we present a case of HIV-triggered secondary HLH presenting with severe bleeding tendency and fever. Despite high-dose dexamethasone infusion (10 mg/body surface area/day), progressive disseminated intravascular coagulation and thrombocytopenia resulted in massive hemathochezia: the bleeding episode ceased after endoscopic hemoclipping. After then, he took a highly-active antiretroviral therapy (HAART). Eventually, body temperature and overall laboratory findings normalized in response to HAART. Clinicians should not overlook HIV infection as a possible trigger of secondary HLH. In such cases, HAART is the core treatment.
607	Diagnostic biomarkers for adult haemophagocytic lymphohistiocytosis in critically ill patients (HEMICU): a prospective observational study protocol.	31666276	Haemophagocytic lymphohistiocytosis (HLH) in adults is characterised by toxic immune activation and a sepsis-like syndrome, leading to high numbers of undiagnosed cases and mortality rates of up to 68%. Early diagnosis and specific immune suppressive treatment are mandatory to avoid fatal outcome, but the diagnostic criteria (HLH-2004) are adopted from paediatric HLH and have not been validated in adults. Experimental studies suggest biomarkers to sufficiently diagnose HLH. However, biomarkers for the diagnosis of adult HLH have not yet been investigated.The HEMICU (Diagnostic biomarkers for adult haemophagocytic lymphohistiocytosis in critically ill patients) study aims to estimate the incidence rate of adult HLH among suspected adult patients in intensive care units (ICUs). Screening for HLH will be performed in 16 ICUs of Charité - Universitätsmedizin Berlin. The inclusion criteria are bicytopaenia, hyperferritinaemia (≥500 µg/L), fever or when HLH is suspected by the clinician. Over a period of 2 years, we expect inclusion of about 100 patients with suspected HLH. HLH will be diagnosed if at least five of the HLH-2004 criteria are fulfilled, together with an expert review; all other included patients will serve as controls. Second, a panel of potential biomarker candidates will be explored. DNA, plasma and serum will be stored in a biobank. The primary endpoint of the study is the incidence rate of adult HLH among suspected adult patients during ICU stay. Out of a variety of measured biomarkers, this study furthermore aims to find highly potential biomarkers for the diagnosis of adult HLH in ICU. The results of this study will contribute to improved recognition and patient outcome of adult HLH in clinical routine.The institutional ethics committee approved this study on 1 August 2018 (Ethics Committee of Charité - Universitätsmedizin Berlin, EA4/006/18). The results of the study will be disseminated in an international peer-reviewed journal and presented at international conferences.NCT03510650.
608	Histoplasmosis-Associated Hemophagocytic Lymphohistiocytosis: A Review of the Literature.	31662810	Histoplasmosis is an endemic fungal disease with diverse clinical presentations. Histoplasmosis-associated hemophagocytic lymphohistiocytosis (HLH) is a rare disorder with limited data regarding treatment and outcome. We described the clinical features, treatment, and outcomes of five patients in our institution with histoplasmosis-associated HLH. This review also summarizes the current literature about presentation, treatment, and outcome of this infection-related HLH entity.We searched the electronic medical records for patients with histoplasmosis-associated HLH at our institution from 1/1/2006 to 9/30/2017. Diagnosis of HLH was confirmed by chart review using the HLH-04 criteria. We also searched the current literature for case reports and case series.Five cases of histoplasmosis-associated HLH were included from our institution. All five patients were diagnosed after 2010. The literature review yielded 60 additional cases of histoplasmosis-associated HLH. The most common underlying condition was HIV in 61% of cases. The majority of histoplasmosis patients (81%) were treated with amphotericin B formulations. Documented specific treatments for HLH were as follows: nine patients received steroids only, six patients received intravenous immunoglobulin (IVIG) only, three patients received dexamethasone and etoposide, two patients received etoposide, dexamethasone, and cyclosporine, two patients received steroids and IVIG, and one patient received Anakinra and IVIG. The inpatient case fatality rate was 31% with most of the deaths occurring within two weeks of hospital admission.Histoplasmosis-associated HLH among adults is an uncommon but serious complication with high associated mortality. Early antifungal therapy with a lipid formulation amphotericin B is critical. The initiation of immunosuppressive therapy with regimens like HLH-04 in this disease entity should be individualized.
609	The Significance of the Intrinsically Disordered Regions for the Functions of the bHLH Transcription Factors.	31653121	The bHLH proteins are a family of eukaryotic transcription factors regulating expression of a wide range of genes involved in cell differentiation and development. They contain the Helix-Loop-Helix (HLH) domain, preceded by a stretch of basic residues, which are responsible for dimerization and binding to E-box sequences. In addition to the well-preserved DNA-binding bHLH domain, these proteins may contain various additional domains determining the specificity of performed transcriptional regulation. According to this, the family has been divided into distinct classes. Our aim was to emphasize the significance of existing disordered regions within the bHLH transcription factors for their functionality. Flexible, intrinsically disordered regions containing various motives and specific sequences allow for multiple interactions with transcription co-regulators. Also, based on in silico analysis and previous studies, we hypothesize that the bHLH proteins have a general ability to undergo spontaneous phase separation, forming or participating into liquid condensates which constitute functional centers involved in transcription regulation. We shortly introduce recent findings on the crucial role of the thermodynamically liquid-liquid driven phase separation in transcription regulation by disordered regions of regulatory proteins. We believe that further experimental studies should be performed in this field for better understanding of the mechanism of gene expression regulation (among others regarding oncogenes) by important and linked to many diseases the bHLH transcription factors.
610	Familial hemophagocytic lymphohistiocytosis type 5 in a Chinese Tibetan patient caused by a novel compound heterozygous mutation in STXBP2.	31651895	Familial hemophagocytic lymphohistiocytosis (FHL) is a fatal autosomal recessive immunodeficiency disease whose rapid and accurate diagnosis is paramount for appropriate treatment. Mutations in STXBP2 gene have been associated with FHL type 5 (FHL-5). Here, we report the first Tibetan Chinese patient diagnosed with FHL-5 caused by a novel compound heterozygous mutation in STXBP2.A 9-year-old girl who presented with recurrent fever, splenomegaly, pancytopenia, hypofibrinogenemia, and conspicuous bone marrow hemophagocytosis was diagnosed with haemophagocytic lymphohistiocytosis (HLH).FHL mutation analysis of the patient and her parents revealed that she presented compound heterozygosity for STXBP2: a novel missense mutation c.663G > C (p.Glu221Asp) and the known pathogenic splice-site mutation c.1247-1G > C (p.Val417LeufsX126). Bioinformatics analyses predicted that the new mutation was pathogenic and the FHL-5 diagnosis was confirmed.Upon diagnosis, HLH-2004-directed chemotherapy was instituted, but there was a relapse. Allogeneic hematopoietic stem cell transplantation (HSCT) was performed.After transplantation, the patient presented implantation dysfunction, chronic graft-versus-host disease, and 5 episodes of pancreatitis. A follow-up after 5 years revealed that the patient had died of pancreatitis.This finding expands the spectrum of FHL-5-related mutations in Chinese patients and indicates a clear genotype-phenotype correlation of FHL-5 in China.
611	Proinflammatory Cytokine Secretion in a Patient With Recurrent Neuroblastoma Related to the Onset of Malignancy-associated Hemophagocytic Lymphohistiocytosis.	31651727	The onset of malignancy-associated hemophagocytic lymphohistiocytosis (M-HLH) may be associated with the secretion of proinflammatory cytokines from malignant cells. We here report a patient with recurrent neuroblastoma who developed hemophagocytic lymphohistiocytosis after surgery for brain metastases and relapsed after chemotherapy. The neuroblastoma cells in the recurrent tumor were positive for tumor necrosis factor-α and interleukin-6, whereas the primary site was negative. The secretion of proinflammatory cytokines from neuroblastoma cells may have been related to the pathogenesis of M-HLH in our patients. We should pay attention to possible development of M-HLH when treating advanced neuroblastoma.
612	Different Clinical Presentation of 3 Children With Familial Hemophagocytic Lymphohistiocytosis With 2 Novel Mutations.	31651726	Although familial hemophagocytic lymphohistiocytosis (FHL) generally manifest with a combination of unremitting fever, hepatosplenomegaly, and pancytopenia; unusual presentations should also be taken into account. Herein, we present 3 FHL cases with 2 novel mutations with different initial presentations. The first patient bearing a homozygous truncation mutation in UNC13D (c.2650C>T.p.Gln884Ter) presented with central nervous system involvement and skin rash. The patient responded to the HLH-2004 protocol, and allogenic hematopoietic stem cell transplantation was performed from her healthy sister. The second and third patients with homozygous splice site mutation (c.430-1G>A) in STXBP2 were siblings who presented at birth with fevers, elevated aspartate aminotransferase, alanine aminotransferase, and hyperferritinemia but did not fulfill FHL criteria. The last 2 infants died despite intervention. Hematologists should be vigilant about the different presentation of FHL in children.
613	Stevens-Johnson syndrome complicated by fatal hemophagocytic lymphohistiocytosis.	31649973	NaN
614	KLF15-activating Twist2 ameliorated hepatic steatosis by inhibiting inflammation and improving mitochondrial dysfunction via NF-κB-FGF21 or SREBP1c-FGF21 pathway.	31648561	Twist-related protein 2 (TWIST2) is identified as a basic helix-loop-helix (b-HLH) transcription repressor by dimerizing with other b-HLH proteins. The significance of TWIST2 has been emphasized in various tumors; however, few studies report its functions in metabolism and metabolic diseases. Here we aimed to explore the novel role and regulation mechanism of TWIST2 in hepatic steatosis. Our results showed that Twist2 knockdown caused mice obesity, insulin resistance, and hepatic steatosis, which were accompanied with inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction. In vitro, TWIST2 overexpression ameliorated hepatocellular steatosis, inhibited inflammation, and improved mitochondrial content and function with a fibroblast growth factor 21 (FGF21)-dependent pattern. NF-κB negatively regulated FGF21 transcription by directly binding to FGF21 promoter DNA, which was eliminated by TWIST2 overexpression by inhibiting NF-κB expression and translocation to nucleus. TWIST2 overexpression decreased intracellular reactive oxygen species level, increased mitochondrial DNA and biogenesis, and enhanced ATP production and antioxidation ability. Additionally, TWIST2 expression was repressed by insulin-targeting sterol regulatory element-binding protein 1c (SREBP1c) and forkhead box protein O1 and was enhanced by dexamethasone targeting Krüppel-like factor 15, which directly interacted with Twist2 promoter DNA. Together, our studies identify an important role and regulation mechanism of TWIST2 in maintaining hepatic homeostasis by ameliorating steatosis, inflammation, and oxidative stress via the NF-κB-FGF21 or SREBP1c-FGF21 pathway, which may provide a new therapeutic scheme for nonalcoholic fatty liver disease.-Zhou, L., Li, Q., Chen, A., Liu, N., Chen, N., Chen, X., Zhu, L., Xia, B., Gong, Y., Chen, X. KLF15-activating Twist2 ameliorated hepatic steatosis by inhibiting inflammation and improving mitochondrial dysfunction via NF-κB-FGF21 or SREBP1c-FGF21 pathway.
615	Hemophagocytic lymphohistiocytosis: epidemiological, clinical and biological profile	31648438	Hemophagocytic lymphohistiocytosis (HLH) is a clinical, biological, and pathological entity that is rare but has certain morbidity that may be life-threatening. This work aims to establish a focus on the hemophagocytic lymphohistiocytosis and analyze different aspects of diagnosis while emphasizing the biological data.We report the results of a retrospective study conducted in the hematology department of Avicenna Hospital in Marrakesh. Thirty-one patients with hemophagocytic lymphohistiocytosis were enrolled.The clinical presentation was dominated by fever and deterioration of the general state for almost all our patients. Splenomegaly was objectified in 90% of the patients. Hepatomegaly, lymphadenopathy, and hemorrhagic manifestations were observed in almost 50% of the patients. Biological assessments revealed bi- or pancytopenia in 96% of the patients, and coagulation disorders in 51% of the patients. On the other hand, hyperferritinemia was found in 84% of the patients, and hepatic cytolysis and hypertriglyceridemia in half of the patients. Hemophagocytosis was observed in all bone marrow samples taken from our patients. Concerning the evolution of patients, in 38.5% of the patients, the evolution was favorable with regression of clinical and biological signs. Twenty six percent of the patients had died, mainly from multiple organ failure and disseminated intravascular coagulation.HLH is a diverse condition with many causes and is likely to be under-recognized, which contributes to its high morbidity and mortality. Clinicians need to be able to recognize the signs and symptoms commonly seen in HLH and actively pursue this diagnosis in the cases of undiagnosed febrile illness with multiorgan dysfunction. Early recognition is crucial for any reasonable attempt at curative therapy to be made.
616	Japanese Spotted Fever with Hemophagocytic Lymphohistiocytosis.	31645532	Japanese spotted fever (JSF) is an uncommon but potentially fatal infection transmitted by tick bites. We herein report a fulminant case of JSF infection that occurred in an immunocompetent adult that was complicated by disseminated intravascular coagulation and hemophagocytic lymphohistiocytosis (HLH). We discuss the difficulty in making the diagnosis and identifying the complication of HLH in our patient. HLH is a rare complication of rickettsiosis, and this is the first reported case in English of JSF complicated by HLH in an immunocompetent adult. Secondary HLH caused by rickettsiosis requires a different treatment from primary HLH. Rickettsiosis must therefore be considered in patients with HLH.
617	Successful treatment of diffuse large B-cell lymphoma with secondary hemophagocytic lymphohistiocytosis by R-CHOP-E regimen: a case report.	31642356	Hemophagocytic lymphohistiocytosis (HLH) is a rare fatal clinical syndrome characterized by a hyperinflammatory condition caused by aberrantly activated macrophages and cytotoxic T cells, resulting in a cytokine storm and organ impairment. Lymphoma, especially B-cell lymphoma in Japan, is a common trigger of secondary HLH. In China, however, most cases of HLH secondary to lymphoma occur in patients with T-cell/natural killer-cell lymphoma or Hodgkin`s lymphoma; HLH is relatively uncommon in patients with B-cell non-Hodgkin's lymphoma. We herein describe a man with diffuse large B-cell lymphoma (DLBCL) and secondary HLH who was successfully treated by R-CHOP-E chemotherapy. All symptoms resolved and laboratory indications of HLH normalized, and complete remission of the lymphoma was achieved. This rare case highlights not only the possibility of HLH secondary to DLBCL but also the importance of early initiation of R-CHOP-E chemotherapy.
618	A human immune dysregulation syndrome characterized by severe hyperinflammation with a homozygous nonsense Roquin-1 mutation.	3163626731745085	Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation.
619	Hemophagocytic Lymphohistiocytosis Associated With Parvovirus B19 in a Patient With Acquired Immunodeficiency Syndrome.	31635495	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening condition characterized by widespread inflammation due to massive immune activation and cytokine release. It is of 2 types, primary or familial and secondary or acquired. Diagnosis is made by fulfilling 5 of 8 criteria as determined by the Histiocyte Society. Treatment includes etoposide, dexamethasone, with or without intrathecal methotrexate in the presence of neurologic involvement as well as treating the underlying cause in secondary HLH. We present a case of a 23-year-old female with congenital human immunodeficiency virus (HIV) infection who presents with nonspecific signs and symptoms of cough, fever, leukopenia, and anemia, and a high-serum parvovirus B19 DNA, later diagnosed with HLH and treated with etoposide and dexamethasone. She made clinical improvements and was successfully discharged to home after 26 days of admission.
620	Serial analysis of cytokine and chemokine profiles and viral load in severe fever with thrombocytopenia syndrome: Case report and review of literature.	31626125	Severe fever with thrombocytopenia syndrome (SFTS) is a recently recognized fatal infectious disease caused by the SFTS virus, and severe cases are complicated by the presence of hemophagocytic lymphohistiocytosis (HLH) associated with a cytokine storm. Herein, we report on serial changes of serum cytokine levels and viral load in a severe case of SFTS.A 63-year-old Japanese woman presented with high-grade fever, abdominal pain, diarrhea, impaired consciousness, leukocytopenia, and thrombocytopenia.SFTS was diagnosed based on a positive serum test for SFTS virus RNA and electroencephalogram (EEG) findings of encephalopathy.The patient was treated with supportive therapy, including steroid pulse therapy (intravenous methylprednisolone 1 g/d for 3 days) for HLH and intravenous recombinant thrombomodulin 19200 U/d for 7 days for disseminated intravascular coagulation.Treatment for 7 days improved both symptoms and abnormal EEG findings, and SFTS virus RNA disappeared from the serum at day 10 from the onset of symptoms. The serum cytokines and chemokines analysis during the clinical course revealed 2 distinct phases: the acute phase and the recovery phase. The cytokines and chemokines elevated in the acute phase included interleukin (IL)-6, IL-10, interferon (IFN)-α2, IFN-γ, tumor necrosis factor-α, interferon-γ-induced protein-10, and fractalkine, while the IL-1β, IL-12p40, IL-17, and vascular endothelial growth factor levels were higher in the recovery phase.These observations suggest that the cytokines and chemokines elevated in the acute phase may reflect the disease severity resulted in a cytokine storm, while those in the recovery phase may be attributed to T-cell activation and differentiation.
621	Hemophagocytic Lymphohistiocytosis in Critically Ill Patients.	31626037	Hemophagocytic lymphohistiocytosis (HLH), an uncontrolled overactivation of the immune system, is well characterized in pediatric patients, yet, much less is known about this life-threatening condition in adult patients. As HLH is often complicated by organ failure, patients will require admission to the intensive care unit for organ support therapy. However, recognition of HLH patients in the intensive care unit (ICU) is challenged by the clinical overlap with sepsis. Here, we analyze HLH patients to better understand its clinical presentation, diagnosis, and treatment.For the purpose of this retrospective observational study, we searched for suspected and diagnosed adult HLH of all patients admitted to at least one adult surgical, anesthesiological or medical ICU between January 2006 and August 2018 at the university hospital Charité - Universitätsmedizin Berlin. All cases were reviewed by two HLH experts, who confirmed or declined the diagnosis.Of 6,340 ICU patients with ferritin measurement, 40 suffered from HLH (0.63%). Of these, in-hospital mortality was 60.0% over all cases, which was highest in malignancy-associated HLH (71.4%). Infections were identified as most common triggers (42.5%). A variety of 19 different treatment strategies were applied. Non-survivors showed higher ferritin at diagnosis compared with survivors (P = 0.021), which was also seen in multivariable analyses. A minimum ferritin of 4083 μg/L after diagnosis was most predictive for 30-day mortality (AUC 0.888, 95% CI 0.771-1.000; sensitivity 93.8%, specificity 78.9%).Mortality in adult HLH patients in the ICU is high, particularly in malignancy-associated HLH. Infections are the most frequent HLH triggers in critically ill patients. At present, there is no standardized treatment for HLH in adult patients available. Assessment of ferritin is valuable for diagnosis, prognosis, and treatment monitoring.The study was registered with www.ClinicalTrials.gov (NCT02854943) on August 1, 2016.
622	Hemophagocytic lymphohistiocytosis complicated by polyserositis: A case report.	31624756	Hemophagocytic lymphohistiocytosis (HLH) is a rare group of disorders of immune dysregulation characterized by clinical symptoms of severe inflammation. There are basically two types of clinical scenarios: Familial HLH and sporadic HLH. It is thought that the syndrome is implicated in the development of infections, malignancies, and autoimmune diseases. HLH, whether primary or secondary, is characterized by activated macrophages in hematopoietic organs, hepatosplenomegaly, cytopenia, and fever; however, HLH complicated with polyserositis (PS) has never been reported.We present a case of fever in a 46-year-old previously healthy Chinese woman complicated by pericardial, pleural, and abdomen effusions. She had no contact with sick individuals, recent travel, illicit drug use, or new sexual contacts. She did not consume alcohol or tobacco and lacked a family history of other diseases. Antibiotics were prescribed for suspected infection, and acute liver injury subsequently occurred. Contrast-enhanced computed tomography showed mild pericardial effusion, pleural effusion, hepatosplenomegaly, and a large amount of ascites. A full blood count revealed leukopenia and thrombocytopenia. Increased ferritin and triglyceride levels were observed. The test for Epstein-Barr (EB) virus DNA was positive. This suggests that EB virus replication and EB virus infection existed. Additional studies showed hemophagocytosis in bone marrow biopsy specimens. The patient's condition progressed rapidly. After providing symptomatic support treatment, eliminating immune stimuli, and administering comprehensive cyclosporine and dexamethasone treatment, the patient's condition continued to progress, and the patient's family members decided to stop treatment; the patient subsequently died.This case shows the significance of considering HLH as part of the evaluation of unexplained fever and PS of unknown origin.
623	Hemophagocytic Lymphohistiocytosis: Management and Special Consideration in Human Immunodeficiency Virus Positive Patients with Immune Reconstitution Syndrome.	31620325	The human body is capable of reacting to multiple aggressors by developing an inflammatory response with the secretion of inflammatory cytokines. The worrisome clinical manifestations occur when this inflammatory response is disproportionate. Hemophagocytic lymphohistiocytosis (HLH) is a rare and severe condition characterized by an overwhelming inflammatory response that may result in end-organ damage and might be fatal. Correspondingly, immune reconstitution inflammatory syndrome (IRIS) is another well-known disorder, seen commonly in human immunodeficiency virus (HIV)-infected patients after the commencement of highly active antiretroviral therapy (HAART). Both entities share a similar clinical presentation and a dismal prognosis. Due to widespread clinical manifestations and laboratory abnormalities, diagnosis is often missed at the time of presentation. There is little consensus on the treatment of secondary HLH, which is usually handled on a case-by-case basis. Rapid curbing of the widespread inflammatory response is the main goal of treatment. To the best of our knowledge, there is scarce literature available on the coexistence of HLH and IRIS; therefore, medical management in the co-occurrence of these two conditions needs to be further investigated.
624	My jamais vu in post allogeneic hematopoietic cell transplant: a review on secondary hemophagocytosis in adults.	31611631	Post allogenic hematopoietic cell transplant (HCT) hemophagocytic lymphohistiocytosis (HLH) is an aggressive disease with unknown etiology. It has a poorly understood pathophysiology and poor outcome if untreated early. It's a state of hypercytokinemia. There are many proposed diagnostic criteria for Post HCT HLH. It usually occurs early in the first 2-6 weeks after allogeneic HCT but can present late. The incidence is highest among cord blood transplant compared with other sources of stem cells with a higher incidence in HLA mismatch donors. Post HCT HLH has a marked low survival rate, when compared with Non-HLH post HCT patients and specifically poor outcome is associated in patients with liver dysfunction, graft failure, and those with endothelial complications. Steroid is the mainstay treatment which can be followed up by cyclosporine and etoposide though an optimal therapy is not known. Intravenous immunoglobulin (IVIg) has been tried in virus associated HLH. Second bone marrow transplant is a rescue procedure in patient with HLH due to graft failure, though a very careful selection of individual patients is mandatory. It has been recently found that etoposide based conditioning regimen may reduce HLH post HCT. A prospective study on post HCT HLH are needed to evaluate this unrecognized condition.
625	A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.	31601675	Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
626	Comment on: CD163 as a valuable diagnostic and prognostic biomarker of sepsis-associated hemophagocytic lymphohistiocytosis in critically ill children. A call for HLA-DR in HLH.	315994783129848931584245	NaN
627	Reply to: Comment on: CD163 as a valuable diagnostic and prognostic biomarker of sepsis-associated hemophagocytic lymphohistiocytosis in critically ill children. A call for HLA-DR in HLH.	315842453129848931599478	NaN
628	Successful Modified Therapy in a Patient with Probable Infection-Associated Hemophagocytic Lymphohistiocytosis.	31583147	Hemophagocytic lymphohistiocytosis (HLH) is a rare, hyperinflammatory syndrome characterized by clinical signs and symptoms of extreme inflammation. In adults, HLH is typically a complication of infections, autoimmune diseases, and malignancies. While the disease is often fatal, classic management of HLH revolves around early diagnosis and initiation of protocolized therapy. We present a case of a previously healthy 56-year-old female who developed distributive shock requiring intubation, vasopressors, and continuous venovenous hemofiltration. In the setting of multiple infectious syndromes, severe cytopenias, and rising direct hyperbilirubinemia, her diagnosis of HLH was confirmed. Therapy was initiated with dexamethasone and two doses of reduced-intensity etoposide based on the patient's clinical course. Over the next few weeks, she continued to improve on dexamethasone monotherapy and has maintained remission up to the present with complete resolution of her cytopenias and return of baseline renal function. Our case highlights the variability in the management of probable infection-associated HLH (IHLH) with a good patient outcome. We demonstrate the potential to treat IHLH with partial protocols and minimal chemotherapeutics.
629	Extramacrochaetae promotes branch and bouton number via the sequestration of daughterless in the cytoplasm of neurons.	31581354	The Class I basic helix-loop-helix (bHLH) proteins are highly conserved transcription factors that are ubiquitously expressed. A wealth of literature on Class I bHLH proteins has shown that these proteins must homodimerize or heterodimerize with tissue-specific HLH proteins in order to bind DNA at E-box consensus sequences to control tissue-specific transcription. Due to its ubiquitous expression, Class I bHLH proteins are also extensively regulated posttranslationally, mostly through dimerization. Previously, we reported that in addition to its role in promoting neurogenesis, the Class I bHLH protein daughterless also functions in mature neurons to restrict axon branching and synapse number. Here, we show that part of the molecular logic that specifies how daughterless functions in neurogenesis is also conserved in neurons. We show that the Type V HLH protein extramacrochaetae (Emc) binds to and represses daughterless function by sequestering daughterless to the cytoplasm. This work provides initial insights into the mechanisms underlying the function of daughterless and Emc in neurons while providing a novel understanding of how Emc functions to restrict daughterless activity within the cell.
630	Influenza virus B-associated hemophagocytic lymphohistiocytosis.	31579142	This original report describes the diagnosis and management of a male with hemophagocytic lymphohistiocytosis (HLH) triggered by influenza B virus infection. The patient was diagnosed with HLH- 2004 clinical criteria and a bone marrow biopsy demonstrating hemophagocytes. Therapy consisted of etoposide and dexamethasone while monitoring hemoglobin and platelet levels. To enable early recognition and prompt treatment for this disease, physicians should be aware of this association.
631	A Small-Molecule Pan-Id Antagonist Inhibits Pathologic Ocular Neovascularization.	31577956	Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.
632	Hemophagocytic Lymphohistiocytosis: Prevalence, Risk Factors, Outcome, and Outcome-related Factors in Adult Idiopathic Inflammatory Myopathies.	3157570333004480	To clarify the prevalence, risk factors, outcome, and outcome-related factors of hemophagocytic lymphohistiocytosis (HLH) in patients with dermatomyositis (DM), polymyositis (PM), or clinically amyopathic dermatomyositis (CADM).Data of patients with DM, PM, or CADM who were admitted to the First Affiliated Hospital of Zhejiang University from February 2011 to February 2019 were retrospectively collected. Patients diagnosed with HLH constituted the case group. A 1:4 case-control study was performed to identify risk factors for HLH in patients with DM, PM, or CADM through comparison, univariate, and multivariate logistic regression analysis. Intragroup comparison was made among patients with HLH to identify factors influencing unfavorable short-term outcome.HLH was a rare (4.2%) but fatal (77.8%) complication in patients with DM, PM, or CADM. The retrospective case-control study revealed that higher on-admission disease activity (p = 0.008), acute exacerbation of interstitial lung disease (AE-ILD, p = 0.002), and infection (p = 0.002) were risk factors for complication of HLH in patients with DM, PM, or CADM. The following intragroup comparison showed that higher on-admission disease activity (p = 0.035) and diagnosis of CADM (p = 0.039) might influence the short-term outcome of patients with HLH. However, no risk factor was identified after false discovery rate correction.In this study, secondary HLH was a fatal complication, with higher on-admission disease activity, AE-ILD, and infection working as risk factors. The underlying role of infection and autoimmune abnormality in HLH in connective tissue disease was subsequently noted. Clinical factors influencing the short-term outcome of patients with secondary HLH require further study.
633	Ruxolitinib for the treatment of lymphoma-associated hemophagocytic lymphohistiocytosis: A cautionary tale.	31575356	Hemophagocytic lymphohistiocytosis is a hyperinflammatory syndrome characterized by fever, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, and pancytopenia. Three publications reported success with ruxolitinib, a Janus-associated kinase (JAK1/2) inhibitor. This therapy interrupts the production of cytokines associated with hemophagocytic lymphohistiocytosis, namely interferon-γ and interleukins 2, 6, and 10.We administered ruxolitinib to two patients with lymphoma-associated hemophagocytic lymphohistiocytosis who had failed standard treatment with dexamethasone and etoposide.Patient #1 was started on ruxolitinib 10 mg BID, and titrated to 15 mg BID. All but two of the hemophagocytic lymphohistiocytosis criteria resolved within two weeks, and she was able to restart therapy for lymphoma. During her ruxolitinib taper, she again presented with relapsed hemophagocytic lymphohistiocytosis. She was taking 2.5 mg a day at the time. Despite salvage treatment, she died from the disease. Patient #2 was a diffuse large B-cell lymphoma patient who presented with hemophagocytic lymphohistiocytosis and was treated with chemoimmunotherapy and achieved a complete response (CR). Hemophagocytic lymphohistiocytosis symptoms relapsed, and he was treated with ruxolitinib. He developed relapsed lymphoma and unfortunately died.While treating the underlying lymphoma is a clear priority, the cytopenias and other symptoms of hemophagocytic lymphohistiocytosis complicate the delivery of this therapy. Hence, the use of ruxolitinib as a bridge to definitive therapy was appealing. However, we are concerned about the progression of lymphoma while these patients were taking ruxolitinib. Ruxolitinib may be controlling cytokine storm associated with hemophagocytic lymphohistiocytosis, while other aspects of the condition are progressing. Therefore, we would advise caution in its use in lymphoma-associated-hemophagocytic lymphohistiocytosis until more data are available.
634	Coexistence of Myelolipoma and Primary Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome.	31572300	Introduction: Adrenal myelolipomas are usually isolated benign adrenal lesions, but can be adjacent to steroid-secreting adrenocortical tumors. We studied the aberrant regulation of cortisol secretion in a 61 year-old woman with combined bilateral myelolipomas and primary bilateral macronodular adrenal hyperplasia (BMAH) causing Cushing's syndrome. Materials and Methods: Cortisol response was measured during in vivo tests that transiently modulated the levels of ligands for potential aberrant receptors, including GIP. Response to medical therapies decreasing GIP was monitored. Expression of ACTH and of GIP receptors were examined in resected adrenal tissues by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Results: In vivo, cortisol increased in response to mixed meals (+353%), oral 75 g glucose (+71%), GIP infusion (+416%), and hLH IV (+243%). Suppression of GIP by pasireotide improved cortisol secretion but produced hyperglycemia. The left adrenal was predominantly composed of myelolipoma and strands of BMAH, while the right was mainly composed of BMAH with some foci of myelolipoma on pathology. No ACTH was detectable by immunohistochemistry in BMAH or myelolipomas tissue. Ectopic GIP receptor was confirmed by RT-PCR and immunohistochemistry in BMAH tissues but not in the myelolipomas. No germline mutations were identified in the ARMC5 gene of the patient's leucocyte DNA. Conclusion: This is the first report of interspersed myelolipoma and BMAH with GIP-dependent Cushing's syndrome. In contrast with the BMAH tissues, myelolipoma tissue did not express specific GIP receptors. The potential mechanisms responsible for the interspersed growth of those two lesions remain to be identified.
635	Awareness of Hemophagocytic Lymphohistiocytosis as an Unusual Cause of Liver Failure in the Neonatal Period.	31567788	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome that predominantly affects infants from birth to 18 months of age, characterized by fever and multiorgan failure. Liver injury has been rarely reported as a presenting sign in the neonatal period. This study reports a case with HLH in the neonatal period who presented with acute liver failure.Herein, a 3-day-old female newborn was admitted with cytopenia, increased liver enzymes, hypofibrinogenemia, and markedly elevated serum ferritin. Hemophagocytosis of bone marrow biopsy confirmed the diagnosis of HLH. The newborn was treated with HLH-2004 protocol, but she finally died from multiorgan failure.Growing awareness of HLH as a cause of liver failure in the neonatal period can be associated with early treatment and reduces mortality in this group of patients.
636	Application of blood and immunodeficiency gene detection in the diagnosis of hemophagocytic lymphohistiocytosis patients.	31562900	To investigate the value of genetic mutations in the pathogenesis and differential diagnosis of hemophagocytic lymphohistiocytosis (HLH), mutations related to blood and immune deficiency genes were analyzed in patients with HLH. Peripheral blood samples from 33 children diagnosed with HLH on the basis of the 2004 diagnostic criteria were collected, and 317 genes related to blood system diseases and 562 genes related to immunodeficiency were detected by second-generation targeted sequencing technology, bioinformatic analysis, and parental verification analysis. A total of 159 mutations related to blood system diseases and immunodeficiency were found in 33 patients, including 7 HLH-related gene mutations (UNC13D, XIAP, LYST, STX11, ITK, PRF1, and SRGN) in 12 patients. UNC13D was found in 6 patients, with the highest frequency. Two cases (6.1%, 2/33) were diagnosed as primary hemophagocytic lymphohistiocytosis (pHLH), and 6 cases (18.2%, 6/33) were diagnosed as primary immunodeficiency disease (PID) or hereditary hematopathy; the remainder were diagnosed as secondary hemophagocytic lymphohistiocytosis (sHLH). It is necessary to detect blood and immunodeficiency genes to exclude the possibility of pHLH, PID, or hereditary hematopathy associated with HLH for children.
637	Hemophagocytic Lymphohistiocytosis in a Patient of Scrub Typhus.	31559779	Hemophagocytic lymphohistiocytosis (HLH) is a rare but aggressive and potentially fatal condition characterized by excessive immune activation. It can occur as primary/ familial and secondary/sporadic/ acquired form. Infections can play a role as triggers in the secondary form of HLH. A case of Hemophagocytic lymphohistiocytosis (HLH) in a patient of Scrub typhus is being reported here. Such association of scrub typhus and HLH is rare.
638	Prognostic factors for survival of herpes simplex virus-associated hemophagocytic lymphohistiocytosis.	31549293	Hemophagocytic lymphohistiocytosis (HLH) occurs in neonates with disseminated infection of herpes simplex virus (HSV). Little has been reported on the control of rapid HLH progression. We studied the cytokine profile and genetic basis of two index cases with divergent outcomes after early treatment of type 2 HSV infection. One survivor had fever and elevated serum levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), interferon (IFN)-β, and IFN-γ at diagnosis. The other neonate had no fever or TNF-α production, but significant IL-6 or IFN responses during the treatment course, and died 19 days after birth. Among 16 reported cases of neonatal HSV-HLH including index cases, eight deceased neonates experienced significantly less fever at presentation (p = 0.028), lower platelet counts (p = 0.019), and lower ratios of soluble IL-2 receptor (sIL-2R) to ferritin levels (p = 0.044) than eight survivors. The 100-day overall survival rates were significantly higher in patients with fever (p = 0.004), > 100 × 109/L of platelet counts (p = 0.035) or > 20 of sIL-2R/ferritin ratio at diagnosis (p = 0.004). The first febrile and cytokine responses to HSV infection predict the early outcome of neonatal HSV-HLH.
639	TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP.	3154108231974356	The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap-/- macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap-/- myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1β, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components.
640	Evaluation of lymphocyte function by IFN-γ secretion capability assay in the diagnosis of lymphoma-associated hemophagocytic syndrome.	31540793	Lymphoma-associated hemophagocytic syndrome (LAHS) is a highly life-threatening disease characterized by an uncontrolled immune disorder. Both under-recognition and delayed diagnosis may contribute to aggressive diseases, and a poorer prognosis. Identification of laboratory features specific for LAHS patients may allow for early detection and intervention with improved outcomes. In the present study, 120 lymphoma patients at first diagnosis were recruited and the function of lymphocytes was evaluated by IFN-γ secretion assay at first diagnosis and follow up. During the surveillance period, 20 patients who complicated with hemophagocytic lymphohistiocytosis (HLH) were classified as LAHS group, and 30 patients without infectious diseases during the course of treatment were classified as lymphoma control group. In addition, 20 non-malignant associated HLH patients recruited as HLH control group and 50 healthy control (HC) subjects were also included. The IFN-γ secretion capability of lymphocytes was compared between first diagnosis of lymphoma patients who was complicate with HLH or not in the disease progression. Our results showed that only NK cell activity was decreased in lymphoma control group, but the activities of NK, CD4+ and CD8+ T cells were all significantly decreased at the time of lymphoma diagnosis in those who would progress with HLH. During the course of treatment, lymphocyte function was relatively stable in lymphoma patients but became further decreased when suffering from complication of LAHS. The IFN-γ secretion capability of lymphocytes in LAHS and non-malignant associated HLH patients were all significantly decreased compared with HCs. So the occurrence of HLH was the key factor leading to the impaired activity of lymphocytes. These data suggest that decreased lymphocyte function might be used as a predictor of LAHS, which has critical clinical significance in diagnosis and further understanding the pathogenesis of the disease.
641	Emapalumab for the treatment of relapsed/refractory hemophagocytic lymphohistiocytosis.	31537529	Emapalumab is a fully human immunoglobulin G1 monoclonal antibody directed against interferon-γ (IFN-γ), which in November 2018 received the first global approval for the treatment of pediatric and adult patients with primary hemophagocytic lymphohistiocytosis (HLH) with refractory, recurrent, or progressive disease or intolerance to HLH therapy. This review will highlight the pathophysiology of primary HLH, the therapeutic rationale for use of IFN-γ-targeting therapy, and potential limitations to its broader use in the treatment of HLH.
642	Ruxolitinib in adult patients with secondary haemophagocytic lymphohistiocytosis: an open-label, single-centre, pilot trial.	31537486	Hemophagocytic lymphohistiocytosis is a cytokine-driven inflammatory syndrome that is associated with substantial morbidity and mortality. Overall survival in adult patients with secondary haemophagocytic lymphohistiocytosis remains suboptimal, and novel therapeutic strategies are needed. The phosphorylation-dependent activation of the Janus family kinases JAK1 and JAK2 are hallmarks of the final common pathway in this disease. We therefore aimed to determine the activity and safety of ruxolitinib, a JAK inhibitor, in adults with secondary haemophagocytic lymphohistiocytosis.We performed an open-label, single-centre, pilot study of ruxolitinib in adults with secondary haemophagocytic lymphohistiocytosis at the University of Michigan Rogel Cancer Center (Ann Arbor, MI, USA). We included patients aged 18 years or more who fulfilled at least five of the eight HLH-2004 criteria for hemophagocytic lymphohistiocytosis. Discontinuation of corticosteroids was not required for enrolment in this study. Patients received oral ruxolitinib (15 mg twice a day) on a continuous 28-day cycle, or until disease progression or unacceptable toxicity. The primary endpoint was overall survival at 2 months from the first dose of ruxolitinib. Secondary endpoints included the assessment of adverse events, response (defined as the assessment of all quantifiable signs and laboratory abnormalities included in the diagnostic criteria for haemophagocytic lymphohistiocytosis), and pharmacodynamic biomarkers. Analyses were done in all treated patients with available data. This study is registered with ClinicalTrials.gov, number NCT02400463, and is still recruiting.As of Feb 7, 2019, five patients had been enrolled. The first patient was enrolled in February, 2016. No deaths were recorded, with a median follow-up of 490 days (IQR 190-1075). 2-month overall survival was 100% (95% CI 57-100). Regarding response, resolution of symptoms (either partial or complete) and disease-associated laboratory abnormalities was observed in all five patients. Cytopenias improved in all patients within the first week of treatment, leading to relatively rapid transfusion independence, discontinuation of corticosteroids, and hospital discharge. A single serious adverse event (ie, grade 4 febrile neutropenia) was reported. One patient discontinued treatment because of grade 2 extremity pain and no treatment-related deaths were observed. Improvements in inflammatory markers (eg, ferritin, soluble IL-2 receptor) and T cells and monocytes activation (ie, decreased STAT1 phosphorylation) were observed following treatment.These preliminary data suggest that ruxolitinib is active, well tolerated, and manageable in the outpatient setting in patients with secondary haemophagocytic lymphohistiocytosis. Given the paucity of effective, non-myelosuppressive therapies, these preliminary findings have important therapeutic implications for patients with haemophagocytic lymphohistiocytosis and other cytokine-release syndromes and warrant further investigation.National Cancer Institute, the University of Michigan Rogel Cancer Center, and Incyte Corporation.
643	An acute presentation of haemophagocytic lymphohistiocytosis due to visceral leishmaniasis in a British adult returning traveller.	31536056	A 62-year old British Caucasian woman normally resident in Spain presented with fever and pancytopaenia after returning to the UK. Her symptoms persisted despite broad-spectrum antibiotics, and she gradually became confused, hypotensive and progressively more pancytopaenic. Imaging demonstrated hepatosplenomegaly, and a bone marrow aspirate confirmed a diagnosis of haemophagocytic lymphohistiocytosis (HLH). Bone marrow polymerase chain reaction (PCR) and blood serology were both positive for Leishmania donovani, consistent with visceral leishmaniasis (VL). Following treatment with dexamethasone and amphotericin, she improved clinically and biochemically, and was able to return to Spain. Fever in the returning traveller is a common acute medical presentation. Although HLH and VL are rare diagnoses, both carry a very high mortality rate if undiagnosed and untreated.
644	Efficacy of plasma exchange with a high dose of acyclovir for disseminated varicella infection.	31520251	In individuals treated with immunosuppressive therapies, the varicella-zoster virus (VZV) infection can become disseminated and lead to a life-threatening condition. There is currently no established treatment strategy for this life-threatening condition. Here, we describe a case where plasma exchange (PE) with a high dose of acyclovir (ACV) ameliorated the severe effects, including VZV-hemophagocytic lymphohistiocytosis (VZV-HLH) and disseminated intravascular coagulation (DIC), in a 9-year-old girl with steroid-dependent nephrotic syndrome. This 9-year-old girl experienced frequent relapse steroid-dependent nephrotic syndrome. She had been treated with steroids, tacrolimus, mizoribine, and rituximab. She had not previously received a varicella vaccine. She was admitted with only one vesicular rash. At admission, a serum test revealed 1.6 × 106 copies/mL of VZV DNA. The patient rapidly developed VZV-HLH and DIC. A combination of a high dose of ACV, immunoglobulin, and steroid pulse therapy could not improve these severe complications. Therefore, PE was applied. PE with a high dose of ACV successfully reduced serum VZV DNA from 7.5 × 106 to 2.8 × 104 copies/mL. This reduction in the VZV DNA copy number suggested that the combination of PE and a high dose of ACV was effective in treating a disseminated VZV infection. To the best of our knowledge, this is the first report showing that PE with a high dose of ACV ameliorated the severe complications of disseminated VZV by reducing the VZV DNA copy number.
645	MUC1-C Activates the NuRD Complex to Drive Dedifferentiation of Triple-Negative Breast Cancer Cells.	31519689	The NuRD chromatin remodeling and deacetylation complex, which includes MTA1, MBD3, CHD4, and HDAC1 among other components, is of importance for development and cancer progression. The oncogenic mucin 1 (MUC1) C-terminal subunit (MUC1-C) protein activates EZH2 and BMI1 in the epigenetic reprogramming of triple-negative breast cancer (TNBC). However, there is no known link between MUC1-C and chromatin remodeling complexes. Here, we showed that MUC1-C binds directly to the MYC HLH-LZ domain and identified a previously unrecognized MUC1-C→MYC pathway that regulates the NuRD complex. MUC1-C/MYC complexes selectively activated the MTA1 and MBD3 genes and posttranscriptionally induced CHD4 expression in basal- but not luminal-type BC cells. In turn, MUC1-C formed complexes with these NuRD components on the ESR1 promoter. Downregulating MUC1-C decreased MTA1/MBD3/CHD4/HDAC1 occupancy and increased H3K27 acetylation on the ESR1 promoter, with induction of ESR1 expression and downstream estrogen response pathways. Targeting MUC1-C and these NuRD components also induced expression of FOXA1, GATA3, and other markers associated with the luminal phenotype. These findings support a model in which MUC1-C activates the NuRD complex to drive dedifferentiation and reprogramming of TNBC cells. SIGNIFICANCE: MUC1-C directly interacts with MYC to activate the NuRD complex, mediating regulation of the estrogen receptor in triple-negative breast cancer cells.
646	Benefit of Anakinra in Treating Pediatric Secondary Hemophagocytic Lymphohistiocytosis.	3151335331524337	To assess the benefit of the recombinant human interleukin-1 receptor antagonist anakinra in treating pediatric patients with secondary hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) associated with rheumatic and nonrheumatic conditions.A retrospective chart review of all anakinra-treated patients with secondary HLH/MAS was performed at Children's of Alabama from January 2008 through December 2016. Demographic, clinical, laboratory, and genetic characteristics, outcomes data, and information on concurrent treatments were collected from the records and analyzed using appropriate univariate statistical approaches to assess changes following treatment and associations between patient variables and outcomes.Forty-four patients with secondary HLH/MAS being treated with anakinra were identified in the electronic medical records. The median duration of hospitalization was 15 days. The mean pretreatment serum ferritin level was 33,316 ng/ml and dropped to 14,435 ng/ml (57% decrease) within 15 days of the start of anakinra treatment. The overall mortality rate in the cohort was 27%. Earlier initiation of anakinra (within 5 days of hospitalization) was associated with reduced mortality (P = 0.046), whereas thrombocytopenia (platelet count <100,000/μl) and STXBP2 mutations were both associated with increased mortality (P = 0.008 and P = 0.012, respectively). In considering patients according to their underlying diagnosis, those with systemic juvenile idiopathic arthritis (JIA) had the lowest mortality rate, with no deaths among the 13 systemic JIA patients included in the study (P = 0.006). In contrast, those with an underlying hematologic malignancy had the highest mortality rate, at 100% (n = 3).These findings suggest that anakinra appears to be effective in treating pediatric patients with non-malignancy-associated secondary HLH/MAS, especially when it is given early in the disease course and when administered to patients who have an underlying rheumatic disease.
647	Dilemmas in diagnosis and management of hemophagocytic lymphohistiocytosis in children.	31506890	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening entity which is characterized by severe hyperinflammation. Now the HLH-2004 protocol has been widely accepted and clinically used; however, many questions still remain in clinical practice. In this review, we discuss the dilemmas in the diagnosis and treatment of HLH in children.Original research for articles and literature reviews published in PubMed was carried out using the key term "hemophagocytic lymphohistiocytosis".As the gene sequencing technology progresses, the range of causal mutations and primary HLH has been redefined. The monoallelic variants may contribute to the pathogenesis of the disease. Many conditions without defective cytotoxicity of T or NK cells may lead to HLH, such as primary immunodeficiency (PID) and dysregulated immune activation or proliferation (DIAP). HLH shares overlapping clinical and laboratory characteristics with severe sepsis, but usually the single values are more pronounced in HLH than sepsis. H score is another approach to help the diagnosis of secondary HLH. Specific Th1/Th2 cytokine patterns are very helpful tools to differentiate HLH (reactivation of HLH) from sepsis. Moreover, it also has been used successfully to stratify the therapy intensity. The treatment of HLH should consider underlying diseases, triggers and severity. HLH-94 is recommended for patients who need etoposide-based therapy.Dramatic progress has been made during the past decades in understanding the pathophysiology of HLH. However, diagnosis and treatment of HLH remain with many dilemmas because of the heterogeneous nature of the disease. Better understanding new gene defects and more effective diagnostic approaches and salvage regimens are goals for the future.
648	An Unusual Presentation of Systemic Lupus Erythematosus as Hemophagocytic Lymphohistiocytosis in a Male.	31482047	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening hyper-inflammatory condition characterized by excessive activation of macrophages and T cells resulting in multi-organ dysfunction. HLH can be primary/familial or secondary to infections, malignancy, immunosuppression, and autoimmune conditions. Systemic lupus erythematosus (SLE) is an autoimmune condition that can predispose to HLH. SLE, as other immune conditions is more common in females than in males. However, the occurrence of SLE in males and subsequent predisposition to HLH is rare. We report the case of a 22-year-old gentleman who presented with fever for three months and one day of altered sensorium prior to admission. On evaluation, he fulfilled five out of the 17 diagnostic criteria for SLE. His bone marrow biopsy showed hemophagocytosis and met five out of the eight diagnostic criteria of HLH, and a diagnosis of HLH secondary to SLE was made. He was treated with pulse doses of intravenous methylprednisolone and azathioprine and showed dramatic improvement. A high index of suspicion is essential for the diagnosis of HLH and prompt initiation of treatment is of utmost importance for tackling such a rapidly progressive life-threatening condition.
649	A Novel BTK Gene Mutation in a Child With Atypical X-Linked Agammaglobulinemia and Recurrent Hemophagocytosis: A Case Report.	31481959	X-linked agammaglobulinemia (XLA), caused by a mutation in the Bruton's tyrosine kinase (BTK) gene, is rarely reported in patients with recurrent hemophagocytic lymphohistiocytosis (HLH). This mutation leads to significantly reduced numbers of circulatory B cells and serum immunoglobulins in patients. Therefore, they exhibit repetitive bacterial infections since infancy, and immunoglobulin (Ig) replacement therapy is the primary treatment. HLH is a life-threatening condition with manifestations of non-remitting fever, hepatosplenomegaly, cytopenias, coagulopathy, lipid disorder, and multiple organ failure. It is caused by the immune dysregulation between cytotoxic T cells, NK cells, and histiocytes. The treatment is based on HLH-2004 protocol including immunotherapy, chemotherapy, supportive therapy, and stem cell transplantation. However, as we know more about the classification and pathophysiology of HLH, the treatment is modified. T-cell-directed immunotherapy is effective in patients with primary HLH, and strong immunosuppression is contraindicated in patients with severe ongoing infections or some primary immunodeficiency diseases (PIDs). Here, we report the case of a 7-year-old boy who presented with ecthyma gangrenosum and several episodes of pyogenic infections during childhood. At the age of 5 years, he exhibited cyclic HLH every 2-3 months. The remission of HLH episodes finally achieved after he received monthly Ig replacement therapy (400 mg/kg) at the 4th HLH. However, transient elevation of IgM was incidentally discovered after 6 cycles of monthly Ig replacement therapy. IgM-secreting multiple myeloma, Waldenström's macroglobulinemia, and lymphoma were excluded. The IgM levels then declined and returned to the normal range within a year. The patient and his parents received whole-genome sequencing analysis. It revealed a novel hemizygous c.1632-1G>A mutation in the BTK gene and XLA was diagnosed. XLA exhibits a spectrum of clinical and immunological presentations in patients. The identification of the mutation in the BTK gene contribute to an accurate diagnosis. Ig replacement therapy is the primary treatment for HLH in patients with XLA.
650	Chediak-Higashi syndrome: Lessons from a single-centre case series.	31477396	Chediak-Higashi syndrome (CHS) is a rare and potentially fatal autosomal recessive disease characterized by frequent bacterial infections, bleeding tendency, oculocutaneous albinism, photosensitivity and progressive neurologic dysfunction. Owing to the rarity of this condition, the objective of this study was to describe patients with CHS.Retrospective evaluation of patients followed in a paediatric tertiary centre of Allergy and Immunology of São Paulo, Brazil, between 1986 and 2018 with a confirmed diagnosis of CHS. Data were obtained from medical records. Demographic aspects, family history, clinical findings, laboratory data, diagnosis, treatment and outcome were described.A total of 14 patients (five male) were included. Clinical manifestations were first recognized at a median age of two months (at birth-20 months). Median age at diagnosis was 1.7 years (0-5 years). All patients had recurrent infections. Albinism was present in 13 patients and silvery or light hair was present in 14. Seven patients developed hemophagocytic lymphohistiocytosis (HLH); the median age at the diagnosis of HLH was 5.7 years (2.6-6.7 years) and the median interval between the diagnosis of CHS and HLH was 3.3 years (0-5 years). Four of the most recently diagnosed patients underwent bone marrow transplantation (BMT). Nine patients are deceased, and one was lost to follow-up. The median age of death was 6.7 years (3.8-22 years). Five patients died of HLH, one of lymphoma, and three of infection. All the patients who had HLH before the year of 2000 died of HLH. The two most recently diagnosed patients with HLH were able to cure the HLH, although they died of other causes. Four patients are alive, three of them after successful BMT.Thirty years of follow up showed an improvement in the prognosis in patients with CHS. The better understanding of the underlying biological mechanisms of HLH allowed the standardization of management protocols, resulting in survival improvement. BMT is the only treatment that can change CHS prognosis, which emphasizes the need for early identification of the disease.
651	Risk factors of early death in adult patients with secondary hemophagocytic lymphohistiocytosis: a single-institution study of 171 Chinese patients.	31474196	Background: Adult secondary hemophagocytic lymphohistiocytosis (HLH) is a potentially life-threatening syndrome characterized by excessive activation of mononuclear-phagocytic system resulting in hyperinflammatory response. To date, the factors influencing early death of HLH are still not fully elucidated. Patients and Methods: We did a retrospective study of 171 adult patients with newly diagnosed HLH at our institution from January 2012 to April 2018. All patients' clinical features, laboratory findings, treatments and prognosis were reviewed. Results: The median age was 49 years (range, 18-88 years), and 110 (64.3%) were male. The major underlying trigger of HLH was malignancy (88/171, 51.5%), especially non-Hodgkin lymphoma. In a multivariate analysis, age ≥54 years (P = 0.002), platelet ≤39.5 × 109/L (P = 0.028), activated partial thromboplastin time (APTT) ≥54 sec (P = 0.048), triglyceride ≥3.23 mmol/L (P < 0.001), lactate dehydrogenase (LDH) ≥1300 U/L (P = 0.012) and malignancy (P = 0.001) were significantly associated with early death in HLH. Then, patients were classified into four groups according to the number of risk factors at the time of diagnosis: low risk (zero, one or two risk factors), low intermediate risk (three risk factors), high intermediate risk (four risk factors) and high risk (at least five risk factors), with the 30-day overall survival (OS) of 92.4%, 58.8%, 30.0% and 4.8%, respectively (P < 0.001). Conclusions: Patients with old age, thrombocytopenia, prolonged APTT, hypertriglyceridemia, elevated LDH and malignancy had inferior survival. It is important to identify those patients at risk of early death, which may guide treatment and reduce mortality.
652	Macrophage activation syndrome/haemophagocytic lymphohistiocytosis secondary to Burkholderia cepacia complex septicaemia in an elderly female carrier of X-linked chronic granulomatous disease with extreme lyonisation: 'cepacia syndrome' revisited.	31473638	X-linked carriers of chronic granulomatous disease (CGD) may become phenotypically affected if substantial skewing from lyonisation occurs. We describe a 73-year-old female carrier with an overt CGD phenotype due to skewed lyonisation, complicated by macrophage activation syndrome (MAS)/haemophagocytic lymphohistiocytosis (HLH) secondary to Burkholderiacepacia complex septicaemia that was successfully treated with a combination of three antibiotics, an antifungal, granulocyte colony stimulating factor, intravenous immune globulin (IVIG) and ciclosporin. Fully phenotypic immunodeficiency is possible in X-linked CGD carriers when skewed lyonisation occurs, rendering such patients to all the same sequelae of CGD such as MAS/HLH. MAS/HLH should be thoroughly excluded when evaluating 'cepacia syndrome' in non-CGD patients.
653	Molecular cloning and functional characterization of MYC transcription factor in pathogen-challenged Apostichopus japonicus.	31472172	Myelocytomatosis viral oncogene (MYC), a transcription factor in the MYC family, plays vital roles in vertebrate innate immunity by regulating related immune gene expressions. In this study, we cloned and characterized an MYC gene from sea cucumber Apostichopus japonicus via RNA-seq and RACE approaches (designated as AjMYC). A 2074 bp fragment representing the full-length cDNA of AjMYC was obtained. This gene includes an open reading frame (ORF) of 1296 bp encoding a polypeptide of 432 amino acid residues with the molecular weight of 48.85 kDa and theoretical pI of 7.22. SMART analysis indicated that AjMYC shares an MYC common HLH motif (354-406 aa) at the C-terminal. Spatial expression analysis revealed that AjMYC is constitutively expressed in all detected tissues with peak expression in the tentacle. Vibrio splendidus-challenged sea cucumber could significantly boost the expression of AjMYC transcripts by a 5.58-fold increase in the first stage. Similarly, 2.75- and 3.23-fold increases were detected in LPS-exposed coelomocytes at 1 and 24 h, respectively. In this condition, coelomocyte apoptotic rate increased from 11.98% to 56.23% at 1 h and to 59.08% at 24 h. MYC inhibitor treatment could not only inhibit the expression of AjMYC and Ajcaspase3, but also depress the coelomocyte apoptosis. Furthermore, AjMYC overexpression in EPC cells for 24 h also promoted the cell apoptosis rate from 21.31% to 45.85%. Collectively, all these results suggested that AjMYC is an important immune factor in coelomocyte apoptosis toward pathogen-challenged sea cucumber.
654	MDSC targeting with Gemtuzumab ozogamicin restores T cell immunity and immunotherapy against cancers.	31462392	Targeting of MDSCs is a major clinical challenge in the era of immunotherapy. Antibodies which deplete MDSCs in murine models can reactivate T cell responses. In humans such approaches have not developed due to difficulties in identifying targets amenable to clinical translation.RNA-sequencing of M-MDSCs and G-MDSCs from cancer patients was undertaken. Flow cytometry and immunohistochemistry of blood and tumours determined MDSC CD33 expression. MDSCs were treated with Gemtuzumab ozogamicin and internalisation kinetics, and cell death mechanisms determined by flow cytometry, confocal microscopy and electron microscopy. Effects on T cell proliferation and CAR-T cell anti-tumour cytotoxicity were identified in the presence of Gemtuzumab ozogamicin.RNA-sequencing of human M-MDSCs and G-MDSCs identified transcriptomic differences, but that CD33 is a common surface marker. Flow cytometry indicated CD33 expression is higher on M-MDSCs, and CD33+ MDSCs are found in the blood and tumours regardless of cancer subtype. Treatment of human MDSCs leads to Gemtuzumab ozogamicin internalisation, increased p-ATM, and cell death; restoring T cell proliferation. Anti-GD2-/mesothelin-/EGFRvIII-CAR-T cell activity is enhanced in combination with the anti-MDSC effects of Gemtuzumab ozogamicin.The study identifies that M-MDSCs and G-MDSCs are transcriptomically different but CD33 is a therapeutic target on peripheral and infiltrating MDSCs across cancer subtypes. The immunotoxin Gemtuzumab ozogamicin can deplete MDSCs providing a translational approach to reactivate T cell and CAR-T cell responses against multiple cancers. In the rare conditions of HLH/MAS gemtuzumab ozogamicin provides a novel anti-myeloid strategy. FUND: This work was supported by Cancer Research UK, CCLG, Treating Children with Cancer, and the alumni and donors to the University of Birmingham.
655	Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation.	31461843	Arginine-vasopressin (AVP) promotes muscle differentiation, hypertrophy, and regeneration through the combined activation of the calcineurin and Calcium/Calmodulin-dependent Protein Kinase (CaMK) pathways. The AVP system is impaired in several neuromuscular diseases, suggesting that AVP may act as a physiological factor in skeletal muscle. Since the Phosphoinositide 3-kinases/Protein Kinase B/mammalian Target Of Rapamycin (PI3K/Akt/mTOR) signaling plays a significant role in regulating muscle mass, we evaluated its role in the AVP myogenic effect. In L6 cells AKT1 expression was knocked down, and the AVP-dependent expression of mTOR and Forkhead box O3 (FoxO) was analyzed by Western blotting. The effect of the PI3K inhibitor LY294002 was evaluated by cellular and molecular techniques. Akt knockdown hampered the AVP-dependent mTOR expression while increased the levels of FoxO transcription factor. LY294002 treatment inhibited the AVP-dependent expression of Myocyte Enhancer Factor-2 (MEF2) and myogenin and prevented the nuclear translocation of MEF2. LY294002 also repressed the AVP-dependent nuclear export of histone deacetylase 4 (HDAC4) interfering with the formation of multifactorial complexes on the myogenin promoter. We demonstrate that the PI3K/Akt pathway is essential for the full myogenic effect of AVP and that, by targeting this pathway, one may highlight novel strategies to counteract muscle wasting in aging or neuromuscular disorders.
656	Haemophagocytic lymphohistiocytosis (HLH) following allogeneic haematopoietic stem cell transplantation (HSCT)-time to reappraise with modern diagnostic and treatment strategies?	31455895	NaN
657	Fatal familial hemophagocytic lymphohistiocytosis with perforin gene (PRF1) mutation and EBV-associated T-cell lymphoproliferative disorder of the thyroid.	31440481	Familial hemophagocytic lymphohistiocytosis (FHL) is a rare fatal autosomal recessive disorder of immune dysregulation. The disease presents most commonly in the first year of life; however, symptomatic presentation throughout childhood and adulthood has also been identified. Biallelic mutation in the perforin gene is present in 20%-50% of all cases of FHL. Secondary hemophagocytic lymphohistiocytosis (HLH) in association with hematological malignancies is known; however, whether mutations in HLH-associated genes can be associated with FHL and hematolymphoid neoplasms is not well documented. Also, Epstein-Barr-virus- (EBV) positive systemic T-cell lymphoproliferative disease (SE-LPD) in the setting of FHL is not clearly understood. Here, we present the case of a young boy who presented with typical features of childhood FHL harboring the perforin gene (PRF1) mutation, and had SE-LPD diagnosed on autopsy, along with evidence of recent EBV infection. The patient expired due to progressive disease. Five siblings died in the second or third decade of life with undiagnosed disease. Genetic counseling was provided to the two surviving siblings and parents, but they could not afford genetic testing. One surviving sibling has intermittent fever and is on close follow-up for possible bone marrow transplantation.
658	Macrophage Activation Syndrome Secondary to Underlying Sarcoidosis.	31431835	Hemophagocytic lymphohistiocytosis (HLH) due to an underlying rheumatologic condition is known as macrophage activation syndrome (MAS), a rare and serious complication that often has a delayed diagnosis. MAS can complicate any rheumatologic disease, although it is most prevalent in systemic juvenile idiopathic arthritis. MAS occurring as a sequela of sarcoidosis is seldom reported. Herein, we present an uncommon case of MAS occurring secondary to suspected extrapulmonary sarcoidosis and the associated diagnostic challenges. A 53-year-old White female presented with a 20-month history of constitutional symptoms of an unclear etiology. Her extensive workup included equivocal bone marrow and liver biopsies, suggestive of occasional hemophagocytosis. On admission, she met criteria for HLH based on the HLH-94 diagnostic guidelines. A repeat liver biopsy was performed revealing non-necrotizing granulomas in the parenchyma. Given the concern for an extrapulmonary sarcoidosis, she was started on pulse-dose steroids with subsequent symptomatic resolution. Two years later, she remains in complete remission. As a systemic disease, sarcoidosis can manifest in any organ and present in a variety of ways. While HLH and MAS have numerous etiologies, sarcoidosis should be considered as a potential underlying diagnosis, and prompt treatment initiation with steroids may reduce morbidity and mortality.
659	Metabolic regulation of lifespan from a C. elegans perspective.	31428207	Decline of cellular functions especially cognitive is a major deficit that arises with age in humans. Harnessing the strengths of small and genetic tractable model systems has revealed key conserved regulatory biochemical and signaling pathways that control aging. Here, we review some of the key signaling and biochemical pathways that coordinate aging processes with special emphasis on Caenorhabditis elegans as a model system and discuss how nutrients and metabolites can regulate lifespan by coordinating signaling and epigenetic programs. We focus on central nutrient-sensing pathways such as mTOR and insulin/insulin-like growth factor signaling and key transcription factors including the conserved basic helix-loop-helix transcription factor HLH-30/TFEB.
660	Hemophagocytic lymphohistiocytosis mimicking neonatal hemochromatosis.	31424295	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal illness characterized by impaired natural killer (NK) cell and cytotoxic T-cell function. Patients develop systemic inflammation, multisystem organ dysfunction, and if untreated, death. Patients who present in the neonatal period often have atypical presentations with evidence of liver dysfunction and cholestasis; this has a broad differential diagnosis including neonatal infection, congenital liver defects, or other causes of liver dysfunction, such as neonatal hemochromatosis. Here, we present an infant whose diagnosis of familial HLH was confounded by the history of a stillborn sibling with suspected neonatal hemochromatosis, ultimately delaying diagnosis and initiation of curative treatment. This highlights the need to maintain a low threshold for sending HLH work-up concurrently with evaluation of liver diseases in infants with liver dysfunction, to ensure timely diagnosis and initiation of treatment. Clinicians should maintain a high index of suspicion for HLH and be aware that HLH may mimic the findings on liver biopsy seen in neonatal hemochromatosis.
661	Secondary hemophagocytic syndrome after renal transplantation: two case-reports.	31419275	Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH) is an infrequent and underdiagnosed condition caused by an overactive immune response, resulting in blood cells phagocytosis. After kidney transplantation (KTx), HLH is usually secondary (or reactive) to infectious and neoplastic processes and has a high mortality rate. No effective treatment is available for this condition. Usual procedures include detecting and treating the pathology triggering the immune system dysregulation, other than administration of intravenous human immunoglobulin (IVIG) and high doses of steroids, and plasmapheresis. The best protocol for maintenance immunosuppressive therapy is also unknown. This article presents two cases of post-KTx reactive HLH that underwent adjuvant IVIG treatment and obtained good clinical results. Despite the high morbidity and mortality associated with reactive HLH after KTx, the early and precise diagnosis and the administration of IVIG therapy along with the treatment of the triggering disease, was an effective strategy to control HLH.
662	Signaling pathways involved in the T-cell-mediated immunity against Epstein-Barr virus: Lessons from genetic diseases.	31402499	Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein-Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non-malignant and malignant B-cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV - notably the central role of T-cell expansion and its regulation in the pathophysiology of EBV-associated diseases. On one hand, the defective expansion of EBV-specific CD8 T cells results from mutations in genes involved in T-cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co-stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4-1BB)) leads to impaired elimination of proliferating EBV-infected B cells and the occurrence of lymphoma. On the other hand, protracted T-cell expansion and activation after the defective killing of EBV-infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T- and NK cell-cytotoxicity. In this setting, the persistence of EBV-infected cells results in HLH, a condition characterized by unleashed T-cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co-receptor molecules (CD27, CD137, and SLAM-R) selectively involved in immune responses against infected B cells via specific T-B cell interactions.
663	HLH-2/E2A Expression Links Stochastic and Deterministic Elements of a Cell Fate Decision during C. elegans Gonadogenesis.	31402303	Stochastic mechanisms diversify cell fate in organisms ranging from bacteria to humans [1-4]. In the anchor cell/ventral uterine precursor cell (AC/VU) fate decision during C. elegans gonadogenesis, two "α cells," each with equal potential to be an AC or a VU, interact via LIN-12/Notch and its ligand LAG-2/DSL [5, 6]. This LIN-12/Notch-mediated interaction engages feedback mechanisms that amplify a stochastic initial difference between the two α cells, ensuring that the cell with higher lin-12 activity becomes the VU while the other becomes the AC [7-9]. The initial difference between the α cells was originally envisaged as a random imbalance from "noise" in lin-12 expression/activity [6]. However, subsequent evidence that the relative birth order of the α cells biases their fates suggested other factors may be operating [7]. Here, we investigate the nature of the initial difference using high-throughput lineage analysis [10]; GFP-tagged endogenous LIN-12, LAG-2, and HLH-2, a conserved transcription factor that orchestrates AC/VU development [7, 11]; and tissue-specific hlh-2 null alleles. We identify two stochastic elements: relative birth order, which largely originates at the beginning of the somatic gonad lineage three generations earlier, and onset of HLH-2 expression, such that the α cell whose parent expressed HLH-2 first is biased toward the VU fate. We find that these elements are interrelated, because initiation of HLH-2 expression is linked to the birth of the parent cell. Finally, we provide a potential deterministic mechanism for the HLH-2 expression bias by showing that hlh-2 is required for LIN-12 expression in the α cells.
664	Hemophagocytic lymphohistiocytosis: a rare association with pulmonary cryptococcosis.	31401574	Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory syndrome characterised by unregulated macrophage and T-lymphocyte activation, resulting in cytokine overproduction and subsequent histiocytic phagocytosis. Here we report a case of pulmonary cryptococcosis, in a 59-year-old diabetic patient, with no other risk factors whose clinical course was complicated by secondary hemophagocytosis. Even after addressing the primary underlying illness (pulmonary cryptococcosis), his clinical condition continued to worsen. After excluding the other causes of HLH and possible reasons of his clinical worsening, glucocorticoids were added following which the patient experienced a remarkable improvement in his clinical and laboratory parameters. To our knowledge, this is the first case report of HLH being caused by pulmonary cryptococcosis and only second case report of cryptococcosis being complicated with HLH (previous report being associated with meningoencephalitic cryptococcosis).
665	Genetic Deficiency of Interferon-γ Reveals Interferon-γ-Independent Manifestations of Murine Hemophagocytic Lymphohistiocytosis.	31400073	Familial hemophagocytic lymphohistiocytosis (FHLH) is a complex cytokine storm syndrome caused by genetic abnormalities rendering CD8+ T cells and natural killer cells incapable of cytolytic killing. In murine models of FHLH, interferon-γ (IFNγ) produced by CD8+ T cells has been identified as a critical mediator of disease, and an IFNγ-blocking antibody (emapalumab) has recently been approved by the Food and Drug Administration. However, development of hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) in patients who are genetically unresponsive to IFNγ questions the absolute necessity of IFNγ in driving disease. This study was undertaken to determine the necessity of IFNγ in driving HLH.IFNγ-/- Prf1-/- mice were infected with lymphocytic choriomeningitis virus (LCMV), and HLH immunopathologic features, including survival, weight loss, cytopenias, cytokine profiles, and immune cell phenotypes, were assessed. Mixed bone marrow chimeras were created to determine the immune cell-intrinsic role of IFNγ receptor signaling. CD8+ T cell depletion and interleukin-33 (IL-33)/ST2 blockade were performed using monoclonal antibodies.LCMV infection of IFNγ-/- Prf1-/- mice resulted in severe HLH-like disease. CD8+ T cells and the IL-33/ST2 axis remained essential mediators of disease; however, IFNγ-independent HLH immunopathology correlated with a 10-15-fold increase in neutrophilia (P < 0.001) and an altered cytokine milieu dominated by IL-6, IL-1β, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05). Furthermore, IFNγ regulated CD8+ T cell expression of GM-CSF and neutrophil survival.IFNγ is not necessary for the development of fulminant HLH, requiring physicians to consider case-by-case treatment strategies. Use of therapies that target upstream activators of CD8+ T cells, such as IL-33/ST2 signaling, may be more universally applicable treatment options that ameliorate both IFNγ-dependent and -independent manifestations of HLH/MAS.
666	Age- and stress-associated C. elegans granulins impair lysosomal function and induce a compensatory HLH-30/TFEB transcriptional response.	31398187	The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases are key contributors to the maintenance of protein homeostasis with age. We previously reported that expression of granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, enhance the accumulation and toxicity of TAR DNA binding protein 43 (TDP-43) in Caenorhabditis elegans (C. elegans). In this study we show that C. elegans granulins are produced in an age- and stress-dependent manner. Granulins localize to the endolysosomal compartment where they impair lysosomal protease expression and activity. Consequently, protein homeostasis is disrupted, promoting the nuclear translocation of the lysosomal transcription factor HLH-30/TFEB, and prompting cells to activate a compensatory transcriptional program. The three C. elegans granulin peptides exhibited distinct but overlapping functional effects in our assays, which may be due to amino acid composition that results in distinct electrostatic and hydrophobicity profiles. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease.
667	[Hemophagocytic lymphohistiocytosis in unspecific virus infection].	31396675	Hemophagocytic lymphohistiocytosis (HLH), also known as hemophagocytic syndrome or macrophage activation syndrome within a pre-existing rheumatological disease, remains undiagnosed in over 70% of all cases in intensive care units (ICU) due to the sepsis-like clinical presentation. This report describes the case of a 30-year-old previously healthy male patient who was admitted to the normal infectiology ward of the Charité - Universitätsmedizin Berlin with unclear fever after a 3‑month journey around Asian and South America. The patient was transferred to the ICU after 3 days because of respiratory failure. Due to the immediate diagnostics of HLH and initiation of specific immunosuppressive treatment with dexamethasone, immunoglobulins and anakinra, the patient completely recovered and could finally be discharged after a 2‑week stay in hospital. Furthermore, the current diagnostic and therapeutic options are discussed. Ferritin is a decisive diagnostic marker that should be determined in every patient with unclear organ failure.
668	Current Flow Cytometric Assays for the Screening and Diagnosis of Primary HLH.	31396234	Advances in flow cytometry have led to greatly improved primary immunodeficiency (PID) diagnostics. This is due to the fact that patient blood cells in suspension do not require further processing for analysis by flow cytometry, and many PIDs lead to alterations in leukocyte numbers, phenotype, and function. A large portion of current PID assays can be classified as "phenotyping" assays, where absolute numbers, frequencies, and markers are investigated using specific antibodies. Inherent drawbacks of antibody technology are the main limitation to this type of testing. On the other hand, "functional" assays measure cellular responses to certain stimuli. While these latter assays are powerful tools that can be used to detect defects in entire pathways and distinguish variants of significance, it requires samples with robust viability and also skilled processing. In this review, we concentrate on hemophagocytic lymphohistiocytosis (HLH), describing the principles and accuracies of flow cytometric assays that have been proven to assist in the screening diagnosis of primary HLH.
669	Genetic characterization of pediatric primary hemophagocytic lymphohistiocytosis in China: a single-center study.	31388699	To study the genetic characteristics of primary hemophagocytic lymphohistiocytosis (pHLH) in China, we investigated the genetic data and clinical features of Chinese HLH patients. We retrospectively reviewed the genetic and clinical data of patients with HLH from November 2015 to June 2018. As a result, 26 patients were diagnosed with pHLH. The median age at diagnosis was 2.8 years (range 0.1-13.7 years). The probable overall survival at 12 and 24 months was 87.6% and 62.6%, respectively. Mutations in PRF1 (38.4%) and UNC13D (26.9%) were the most common genetic abnormalities. Furthermore, we identified 19 novel mutations that had not been previously reported and were predicted to likely be pathogenic. In addition to HLH-associated genes, there were 27 other genes identified. Genotype-phenotype analysis showed that patients with disruptive mutations were significantly younger at diagnosis than those with other mutation types (2.9 years vs. 6.4 years, P = 0.036). Familial HLH patients were more prone to central nervous system involvement and seizures compared with other patients (83.3% vs. 37.5%, P = 0.019; 55.6% vs. 12.5%, P = 0.04, respectively). In summary, numerous new mutations in HLH-related genes and other genes were identified in Chinese children with pHLH. Significantly, disruptive mutation types were more likely to be found in younger patients, and familial HLH patients tended to exhibit central nervous system involvement and seizures.
670	Population pharmacokinetics and initial dosing regimen optimization of cyclosporin in pediatric hemophagocytic lymphohistiocytosis patients.	31382792	Hemophagocytic lymphohistiocytosis (HLH) is induced by various triggers, including genetic factors, infections, autoimmune diseases, lymphoma or other malignancies. Cyclosporin is one of the clinical treatments for HLH. However, cyclosporin has considerable inter- and intra-individual variabilities in pharmacokinetics and also displays a narrow therapeutic window, making it difficult to define an optimal dose for HLH treatment. This study is aimed to establish cyclosporin population pharmacokinetic (PPK) model of pediatric HLH patients and formulate an initial dose regimen for personalized medicine.Pediatric HLH patients between June 2014 and March 2019 from Children's Hospital of Fudan University were analyzed using NONMEM. Dose recommended was investigated using Monte Carlo simulations.The final cyclosporin PPK model was: CL/F = 91×(WT/70)0.75×(1+ Piperacillin-Tazobactam × θP-T); V/F = 4250×(WT/70), where WT, and θP-T were weight, and the coefficient of the Piperacillin-Tazobactam, respectively. Based on the simulation results of our model, new initial dosage suggestions were recommended. In conclusion, the first cyclosporin PPK model in pediatric HLH patients was established and the model could be used to predict individualized initial dosing regimens in children with HLH.
671	Regulation of Notch output dynamics via specific E(spl)-HLH factors during bristle patterning in Drosophila.	31375669	The stereotyped arrangement of sensory bristles on the adult fly thorax arises from a self-organized process, in which inhibitory Notch signaling both delimits proneural stripes and singles out sensory organ precursor cells (SOPs). A dynamic balance between proneural factors and Enhancer of split-HLH (E(spl)-HLH) Notch targets underlies patterning, but how this is regulated is unclear. Here, were identify two classes of E(spl)-HLH factors, whose expression both precedes and delimits proneural activity, and is dependent on proneural activity and required for proper SOP spacing within the stripes, respectively. These two classes are partially redundant, since a member of the second class, that is normally cross-repressed by members of the first class, can functionally compensate for their absence. The regulation of specific E(spl)-HLH genes by proneural factors amplifies the response to Notch as SOPs are being selected, contributing to patterning dynamics in the notum, and likely operates in other developmental contexts.
672	Evolutions of gas temperature inside fire compartment and external facade flame height with a casement window.	31374375	Gas temperature of a fire compartment and external facade flame height pose serious hazard upon urban environment and safety, where the window (ventilation) condition has an essential impact. This paper investigates their evolutions with a casement window of various opening angles, which has not been quantified previously although is commonly used practically. Experiments are conducted employing a model-scale fire compartment under different heat release rates with a casement window of various dimensions and opening angles. It is found that the upper-part gas temperature inside the compartment increases, while the critical heat release rate for flame ejection as well as the external facade flame height decreases, with increasing of the window opening angle as it is less than 60 degrees (θ < 60°). Then, all these quantities change little as the window opening angle is further increased (θ ≥ 60°). New non-dimensional models are proposed to describe the evolutions of these quantities based on the derived new length scale according to window dimensions and opening angle. This paper provides for the first time both essential data and new correlations for the gas temperature inside fire compartment and external facade flame height with a casement window.
673	A Rare Case of Hemophagocytic Lymphohistiocytosis in an Adult with Diffuse Large B-Cell Lymphoma.	31360558	A 71-year-old Indian female presented with a 3-month history of weight loss and fatigue. Further review confirmed a histological diagnosis of diffuse large B-cell lymphoma. Although bone marrow analysis did not reveal hemophagocytosis, she had some clinical and laboratory pointers to hemophagocytic lymphohistiocytosis (HLH). Her clinical state deteriorated rapidly with development of acute respiratory distress syndrome, diffuse alveolar hemorrhage, and subsequently death.
674	Editorial: Epstein-Barr Virus-Associated T/NK-Cell Lymphoproliferative Diseases.	31355168	NaN
675	Severe Dengue and Associated Hemophagocytic Lymphohistiocytosis in PICU.	31353429	To study the clinico-laboratory profile and outcome of children with severe dengue and dengue-associated hemophagocytic lymphohistiocytosis (HLH).In this retrospective study, 22 children with laboratory confirmed severe dengue admitted to pediatric intensive care unit (PICU) were enrolled. Clinical features, laboratory parameters, and outcome were noted and compared between cases fulfilling HLH-2004 criteria and those without HLH.Median (IQR) age was 8 (5-10.3) y. Fever was present for mean (SD) duration of 5.3 (2.1) d. Vomiting, respiratory distress, pain abdomen and hepatomegaly were other clinical features. Thrombocytopenia, anemia and elevated serum transaminases were noted in 91%, 41% and 30% respectively; coagulopathy and hypoalbuminemia were seen in 36% each. Half (n = 11, 50%) had dengue shock syndrome. Acute respiratory distress syndrome (ARDS) (n = 7, 32%) and acute kidney injury (AKI) (n = 6, 28%) were other major organ dysfunctions. Mean (SD) duration of PICU stay was 3.6 (1.5) d with 13.6% mortality. HLH was noted in 7 (32%) cases at a median (IQR) hospital stay of 5 (2-8) d. Children with HLH had significantly higher Pediatric Index of Mortality 2 (PIM 2) score at admission and higher frequency of pain abdomen, anemia, hypoalbuminemia, elevated alanine aminotransferase (ALT) and ARDS. Length of PICU stay (5.1 vs. 2.9 d) and mortality (28.6% vs. 6.7%) were higher in HLH group, however the difference was not statistically significant. Steroids were used in 4 cases with HLH and all survived, whereas among 3 who did not receive steroids, 2 died (p = 0.23).Severe dengue presents with life-threatening organ dysfunctions. HLH is increasingly recognized in dengue infection and maybe considered as a differential diagnosis in children with lower hemoglobin, hypoalbuminemia, elevated ALT and severe organ dysfunction.
676	A bHLH transcription factor TsMYC2 is associated with the blue grain character in triticale (Triticum × Secale).	31352584	RNA-Seq was employed to compare the transcriptome differences between the triticale lines and to identify the key gene responsible for the blue aleurone trait. The accumulation of anthocyanins in the aleurone of triticale results in the formation of the blue-grained trait, but the identity of the genes associated with anthocyanin biosynthesis in the aleurone has not yet been reported. In this manuscript, RNA-Seq was employed to compare the transcriptome differences between the triticale lines HM13 (blue aleurone) and HM5 (white aleurone), and to identify the key genes responsible for the blue aleurone trait. There were 32,406 differentially expressed genes between HM13 and HM5. Seventy-three unigenes were homologous to the structural genes related to anthocyanin biosynthesis, and the average transcript level of the structural genes was higher in HM13 than in HM5, so that quantitative differences between the two lines in transcription rates could be the cause of the blue aleurone. The MYB and bHLH transcription factors had two homologous unigenes, but contained only one differentially expressed unigene each. The relative transcript level of bHLH Unigene5672_All (TsMYC2) in HM13 was 42.71 times that in HM5, while the relative transcript level of the MYB transcription factor Unigene12228_All in HM13 was 2.20 times that in HM5. qPCR experiments determined the relative transcript level of TsMYC2 in developing grain, with the expression of TsMYC2 in grain being the highest compared with that in root, stem or leaf tissue. TsMYC2 was homologous to the bHLH transcription factor regulating anthocyanin biosynthesis and contained three entire functional domains: bHLH-MYC_N, HLH and ACT-like, which were important for exercising regulation of anthocyanin biosynthesis as a bHLH transcription factor. Transient expression of ZmC1 and TsMYC2 could induce anthocyanin biosynthesis in white wheat coleoptile cells, demonstrating that TsMYC2 was a functional bHLH transcription factor. These results indicated that TsMYC2 was associated with the blue aleurone trait and could prove to be a valuable gene with which to breed new triticale cultivars with the blue aleurone trait.
677	GATA2 mutation underlies hemophagocytic lymphohistiocytosis in an adult with primary cytomegalovirus infection.	31350183	We report a case of a 27-year old woman with persistent fever and pancytopenia who had multiple episodes of a hemophagocytic lymphohistiocytosis (HLH) like condition. The criterion for HLH was satisfied; primary cytomegalovirus (CMV) infection was identified as the cause. Further examination revealed a GATA binding protein 2 mutation. Reports of GATAs deficiency presenting with HLH after primary CMV infection is very limited. As early recognition and diagnosis will improve patients' outcomes, internists and infectious disease specialists should be aware of this disease.
678	The cellular regulators PTEN and BMI1 help mediate NEUROGENIN-3-induced cell cycle arrest.	31341016	Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice. However, the human NEUROG3 loss-of-function phenotype differs subtly from that in mice, but the reason for this difference remains poorly understood. Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines. NEUROG3 overexpression induced a reversible cell cycle exit, whereas expression of a neuronal lineage homolog, NEUROG1, had no such effect. In endocrine lineage cells, the cellular quiescence induced by short-term NEUROG3 expression required cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21CIP1 expression. Expression of endocrine differentiation markers required sustained NEUROG3 expression in the quiescent, but not in the senescent, state. Inhibition of the phosphatase and tensin homolog (PTEN) pathway reversed quiescence by inducing cyclin-dependent kinase 2 (CDK2) and reducing p21CIP1 and NEUROG3 protein levels in BON4 cells and human enteroids. We discovered that NEUROG3 expression stimulates expression of CDKN2a/p16INK4a and BMI1 proto-oncogene polycomb ring finger (BMI1), with the latter limiting expression of the former, delaying the onset of CDKN2a/p16INK4a -driven cellular senescence. Furthermore, NEUROG3 bound to the promoters of both CDKN1a/p21CIP1 and BMI1 genes, and BMI1 attenuated NEUROG3 binding to the CDKN1a/p21CIP1 promoter. Our findings reveal how human NEUROG3 integrates inputs from multiple signaling pathways and thereby mediates cell cycle exit at the onset of differentiation.
679	[Clinical analysis of secondary hemophagocytic lymphohistiocytosis complicated with capillary leak syndrome].	31340624	Objective: To investigate the clinical characteristics of secondary hemophagocytic lymphohistiocytosis (sHLH) complicated with capillary leak syndrome (CLS) . Methods: The clinical and laboratory data of 87 sHLH patients, who were treated in our hospital between January 2015 and December 2017, were retrospectively analyzed. Depending on whether they were complicated with CLS, 21 sHLH patients were classified as the CLS-sHLH group, while 66 were classified as the non-CLS-sHLH group. The differences of clinical manifestations, laboratory tests, treatment and prognosis between the two groups were compared. Results: There was no significant difference in the etiology of sHLH between the CLS-sHLH group and the non-CLS-sHLH group (P>0.05) . The neutrophil, fibrinogen and albumin levels in the CLS-sHLH group were lower than those in the non-CLS-sHLH group, while the triacylglycerol levels were higher than those in the non-CLS-sHLH group (P<0.05) . Varying degrees of edema, weight gain, hypotension, hypoproteinemia, oliguria and multiple serous effusions were observed in the CLS-sHLH group. Among them, there were 15 patients that CLS get improved, and the medial time of improvement was 7 (5-14) days. The other 6 patients did not get remission, while they died within 6-30 days. The median overall survival of the CLS-sHLH group was lower than that of the non-CLS-sHLH group (75 days vs not reached, P=0.031) . Conclusions: There may be no correlation between the cause of sHLH and the occurrence of CLS. Severity of neutropenia, fibrinogen and albumin levels, and triglyceride levels may be accompanied for sHLH patients complicated with CLS. Patients with sHLH who complicated with CLS have a poor prognosis. Active treatment of HLH and its primary disease, reasonable fluid replacement and oxygen supply are crucial, which can effectively control disease progression.目的： 探讨继发性噬血细胞性淋巴组织细胞增多症（sHLH）合并毛细血管渗漏综合征（CLS）的临床特征。 方法： 回顾性分析2015年1月至2017年12月江苏省人民医院收治的87例sHLH患者的临床及实验室资料，其中21例合并CLS（CLS-sHLH组），66例未合并CLS（非CLS-sHLH组）。比较两组的临床表现、实验室检查、治疗及预后差异。 结果： 比较CLS-sHLH组和非CLS-sHLH组患者引起sHLH的病因，差异无统计学意义（P>0.05）。CLS-sHLH组患者ANC、血清纤维蛋白原、白蛋白较非CLS-sHLH组降低，而甘油三酯较非CLS-sHLH组升高（P值均<0.05）。CLS-sHLH组患者均有不同程度水肿、体重增加、低血压、低蛋白血症、少尿及多浆膜腔积液。其中15例患者CLS症状消失，CLS中位好转时间为7（5~14）d。6例患者CLS症状未消失，并于15（6~30）d死亡。CLS-sHLH组中位OS时间低于非CLS-sHLH组（75 d对未达到，P=0.031）。 结论： 在sHLH患者中，CLS的发生可能与sHLH的原发病无明显相关。严重的ANC、纤维蛋白原及白蛋白减低，甘油三酯升高，可能是sHLH发生CLS的伴随症状。sHLH合并CLS时预后差。.
680	Challenges in the diagnosis of hemophagocytic lymphohistiocytosis: Recommendations from the North American Consortium for Histiocytosis (NACHO).	31339233	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of pathologic immune activation, often associated with genetic defects of lymphocyte cytotoxicity. Though a distinctive constellation of features has been described for HLH, diagnosis remains challenging as patients have diverse presentations associated with a variety of triggers. We propose two concepts to clarify how HLH is diagnosed and treated: within the broader syndrome of HLH, "HLH disease" should be distinguished from "HLH disease mimics" and HLH subtypes should be categorized by specific etiologic associations, not the ambiguous dichotomy of "primary" and "secondary." We provide expert-based advice regarding the diagnosis and initiation of treatment for patients with HLH, rooted in improved understanding of its pathophysiology.
681	Hemophagocytic Lymphohistiocytosis Secondary to Acute Hepatitis E Infection: A Rare Association.	31325278	Viral infections are commonest cause of secondar y hemophagoc ytic lymphohistiocytosis (HLH) and Ebstein Bar Virus is associated with majority of cases. We report a rare case of HLH associated with acute hepatitis E virus infection.
682	Hemophagocytic Lymphohistiocytosis: A Rare Complication of an Ultrarare Lysosomal Storage Disease.	31318819	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening hyperinflammatory condition that may be triggered by infections, autoimmune and immunologic disorders, malignancies, and metabolic diseases. Early and accurate diagnosis of HLH and its underlying cause is of paramount importance for proper management and prognosis. We report the case of a Tunisian 21-month-old girl who initially presented clinical features of HLH related to a lysosomal acid lipase deficiency. The genetic sequence analysis of the LIPA gene revealed a never described homozygous mutation c.966G>C (p.Gln322His). The parents were heterozygous for this mutation. Enzyme replacement therapy was not provided for the patient. She received etoposide, corticosteroids, and cyclosporine for the HLH. She is waiting for hematopoietic stem cell transplantation. To the best of our knowledge, this is the second Tunisian case of secondary HLH complicating lysosomal acid lipase deficiency related to a new homozygous mutation: c.966G>C (p.Gln322His).
683	How to Treat Cytomegalovirus-induced Hemophagocytic Lymphohistiocytosis in a Child With Leukemia.	31306340	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by a hyperinflammatory state due to an aberrant activation of the immune cells. It can be familial or secondary to malignancy, autoimmune or metabolic diseases. Most HLH cases are triggered by infection. Histiocyte society suggested HLH-2004 protocol for diagnosis and treatment of both forms. Here, we present a three-year-old girl with B-cell acute lymphoblastic leukemia who developed HLH secondary to cytomegalovirus infection during maintenance therapy. She was successfully treated without needing full HLH protocol therapy. We discuss modified therapy for this specific group of HLH, summarizing 5 other similar cases in the literature.
684	CD163 as a valuable diagnostic and prognostic biomarker of sepsis-associated hemophagocytic lymphohistiocytosis in critically ill children.	312984893158424531599478	To investigate CD163 as an effective biomarker for identifying and predicting the outcomes of sepsis-associated hemophagocytic lymphohistiocytosis (SAHS) in children.We prospectively enrolled presumed sepsis patients who had developed prolonged fever (>7 days), hepatosplenomegaly, cytopenias, and hyperferritinemia (>500 ng/mL) despite antibiotic therapy. Blood samples were collected within 24 hours after enrolment. A nested case-control study was performed. The number of patients who fulfilled the HLH-2004 criteria, 28-day mortality outcomes, and 90-day mortality outcomes were recorded.Sixty-nine patients were enrolled in the study. Significant increases in the levels of ferritin and soluble CD163 (sCD163) and the percentage of CD163-positive peripheral blood mononuclear cells (mCD163) and decreases in fibrinogen levels and the percentage of natural killer cells (NK %) were observed in patients with SAHS (n = 23) compared with those of patients with sepsis (n = 46). The area under the ROC curve (AUC) for ferritin combined with sCD163 was superior to the AUC for either ferritin or sCD163 for distinguishing SAHS from sepsis. Moreover, sCD163 was a prognostic factor for 28-day mortality (0.857 [0.659-1.000]).sCD163 is a valuable biomarker for the differential diagnosis of SAHS from sepsis and effectively predicts 28-day mortality in children with SAHS.
685	Haemophagocytic lymphohistiocytosis complicating visceral leishmaniasis in the UK: a case for detailed travel history, a high index of suspicion and timely diagnostics.	31296633	A 4-month-old male infant presented acutely unwell with fever. He was initially treated for sepsis but failed to improve with IV broad spectrum antibiotics. Haemophagocytic lymphohistiocytosis (HLH) was diagnosed due to his fever, pancytopenia, splenomegaly, hypertriglyceridaemia, hypofibrinogenaemia and significant hyperferritinaemia. An array of differentials for HLH including both immunological and infectious causes were considered and excluded. He had travelled to Madrid, and hence visceral leishmaniasis (VL) was suspected, but was not confirmed on the initial bone marrow aspirate (BMA) microscopy or culture. He improved with empirical treatment with dexamethasone and liposomal amphotericin B. VL was later confirmed on BMA PCR. He made a good recovery and remained well at 12 month follow-up. Non-endemic countries need rapid and sensitive VL diagnostics. A thorough travel history and high clinical index of suspicion are necessary to avoid the pitfall of treatment with intense immunosuppression recommended in treatment guidelines for HLH.
686	Calming the storm in HLH.	3129654131015190	NaN
687	Disseminated CMV infection and HLH in a patient with well-controlled HIV and ulcerative colitis.	31296498	We present a case of haemophagocytic lymphohistiocytosis (HLH) in the context of disseminated cytomegalovirus (CMV) viraemia in a 50-year-old man with well-controlled HIV infection and ulcerative colitis (UC), for which he was receiving azathioprine. Peak CMV viral load was 371 000 copies/ml with evidence of end-organ CMV in the lungs and colon. A bone marrow biopsy showed evidence of haemophagocytosis of platelets, neutrophils and erythrocytes. The azathioprine was stopped, and he received intravenous ganciclovir and corticosteroids with suppression of the CMV viral load and resolution of the HLH.
688	Insulin-like peptides and the mTOR-TFEB pathway protect Caenorhabditis elegans hermaphrodites from mating-induced death.	3128286231418689	Lifespan is shortened by mating, but these deleterious effects must be delayed long enough for successful reproduction. Susceptibility to brief mating-induced death is caused by the loss of protection upon self-sperm depletion. Self-sperm maintains the expression of a DAF-2 insulin-like antagonist, INS-37, which promotes the nuclear localization of intestinal HLH-30/TFEB, a key pro-longevity regulator. Mating induces the agonist INS-8, promoting HLH-30 nuclear exit and subsequent death. In opposition to the protective role of HLH-30 and DAF-16/FOXO, TOR/LET-363 and the IIS-regulated Zn-finger transcription factor PQM-1 promote seminal-fluid-induced killing. Self-sperm maintenance of nuclear HLH-30/TFEB allows hermaphrodites to resist mating-induced death until self-sperm are exhausted, increasing the chances that mothers will survive through reproduction. Mothers combat males' hijacking of their IIS pathway by expressing an insulin antagonist that keeps her healthy through the activity of pro-longevity factors, as long as she has her own sperm to utilize.
689	Adjuvant recombinant thrombomodulin therapy for hepatopathy induced by vincristine, actinomycin D, and cyclophosphamide in pediatric rhabdomyosarcoma: A case report.	31281657	Hepatopathy induced by vincristine, actinomycin D and cyclophosphamide (VAC) is a potentially lethal complication of VAC chemotherapy for pediatric malignancy, which is managed by conventional anticoagulation and liver-supporting treatment alone. We report a case of VAC-induced hepatopathy with coagulopathy and severe inflammation. A 15-year-old male with rhabdomyosarcoma receiving adjuvant chemotherapy presented with refractory thrombocytopenia, followed by abdominal tenderness and non-neutropenic fever. Hepatic dysfunction and coagulopathy subsequently emerged with persistent fever. This condition indicated disseminated intravascular coagulation. A diagnosis of 'very severe' sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) was established in accordance with the European Society for Blood and Marrow Transplantation diagnostic criteria for hepatic SOS/VOD in children. Early administration of recombinant thrombomodulin (rTM) (380 U/kg/day) and prednisolone (1.8 mg/kg/day) successfully controlled the condition. Serum concentrations of pro-inflammatory cytokines increased with hepatopathy development but immediately decreased after drug initiation. rTM administration may be promising for the control of inflammatory VAC-induced hepatopathy.
690	An Elusive Diagnosis: Case Reports of Secondary Hemophagocytic Lymphohistiocytosis and Review of Current Literature.	31275772	Hemophagocytic lymphohistiocytosis (HLH) is a rare and serious hematologic disorder characterized by severe immune system dysregulation with a cytokine storm and histologic evidence of hemophagocytosis. It can be inherited or develop secondary to other diseases. We present three cases of secondary HLH in patients with distinct backgrounds. Our objective is to characterize the unique features of the disease, its underlying associations, treatment, and potential prognostic variables. The first case was a 20-year-old male with a history of intravenous (IV) drug abuse who presented with multi-organ failure and septic shock. A diagnosis of HLH was suspected after finding a ferritin of >100,000 ng/mL and confirmed with bone marrow biopsy. Furthermore, the patient was found to have significant Epstein-Barr virus (EBV) viremia. He responded well to the HLH-94 protocol with the addition of rituximab and ganciclovir. The second case was a 50-year-old female with a history of human immunodeficiency virus (HIV) who presented with multi-organ failure and severe anemia. Ferritin was also significantly elevated and a bone marrow biopsy confirmed the diagnosis of HLH. She was started on HLH-94 protocol. Despite treatment, the patient expired due to worsening renal failure and shock. Her autopsy report also showed evidence of Hodgkin's lymphoma. The third case was a 57-year-old male with a history of Crohn's disease treated with infliximab and adalimumab, who presented with multi-organ failure and pancytopenia. A diagnosis of HLH was made based on clinical findings and later confirmed on bone marrow biopsy. He responded to HLH-94 protocol but experienced fatal gastrointestinal bleeding. Patients presenting with HLH are often critically ill and deteriorate rapidly. The diagnosis is often challenging to establish due to its variable presentation and association with other pathologies. A moderate index of suspicion should be present for patients who have febrile illness with pancytopenia, multi-organ failure, high ferritin, and low fibrinogen levels. We discuss associations with viral infections, hematologic malignancies and immunosuppressive therapy. Treatment is directed at suppressing the immune response and for secondary HLH, addressing the underlying conditions, such as use of rituximab for EBV viremia and treatment of lymphoma.
691	Miliary Tuberculosis-Related Acute Respiratory Distress Syndrome Complicated with Hemophagocytic Lymphohistiocytosis Syndrome.	31275676	Acute respiratory distress syndrome (ARDS) and hemophagocytic lymphohistiocytosis (HLH) are accompanied with poor outcome and high mortality when miliary tuberculosis is a causative pathogen for both of them. A patient complicated with ARDS and HLH is unusual in critical care, and few case reports are present in PudMed. Besides, the relationship between HLH and ARDS is still unknown and has not been reviewed in the literature. In this report, we present the case of a 74-year-old Taiwanese woman suffering from pulmonary tuberculosis and miliary tuberculosis, and she developed ARDS and HLH on the 3rd day after admission. We arranged serial laboratory examination, various serum markers, bone marrow aspiration, and bronchoscopy with alveolar lavage for survey; we prescribed empirical antibiotics and antituberculosis medication soon after alveolar lavage showing positive acid-fast stain. She was extubated on hospital day 31 and discharged on hospital day 73. In conclusion, early diagnosis and intervention for underlying disease and intensive bundle care for multiorgan failure are crucial for both ARDS and HLH.
692	Hemophagocytic lymphohistiocytosis associated with Epstein-Barr virus infection: case report and literature review.	31275547	Hemophagocytic lymphohistiocytosis (HLH) is rare and life threatening syndrome. There are only a few reported cases of HLH with GI symptoms. We describe the case of an 18 months old boy who presented with a history of fever for 40 days, abdominal distention and hepatosplenomegaly. Abdominal x-ray showed a pneumoperitoneum. Urgent laparotomy was done which revealed an isolated cecal perforation. The histopathological findings in the subsequent resected bowel was HLH with evidence of positive EBV Barr infection.
693	Neonatal Hemophagocytic Lymphohistiocytosis.	31261095	Hemophagocytic lymphohistiocytosis (HLH) is extremely rare in the neonatal period. The incidence of neonatal HLH is not confirmed and may range from 1 in 50,000 to 150,000. The incidence varies based on ethnicity, particularly in populations in which consanguinity is common. HLH is associated with a high fatality rate and poor prognosis, making it important to recognize and diagnose it early. This review will concentrate primarily on the diagnosis and management of neonatal HLH.
694	Effects of dietary supplementation of three strains of Lactococcus lactis on HIFs genes family expression of the common carp following Aeromonas hydrophila infection.	31252044	HIFs (Hypoxia inducible factors) are the main regulators of the expression change of oxygen-dependent genes, in addition, they also play important roles in immune regulation. HIFs participate in infectious diseases and inflammatory responses, providing us a new therapeutic target for the treatment of diseases. In this study, 16 HIFs were identified in common carp genome database. Comparative genomics analysis showed large expansion of HIF gene family and approved the four round whole genome duplication (WGD) event in common carp. To further understand the function of HIFs, the domain architectures were predicted. All HIF proteins had the conserved HLH-PAS domain, which were essential for them to form dimer and bind to the downstream targets. The differences in domain of HIFα and HIFβ might result in their different functions. Phylogenetic analysis revealed that all HIFs were divided into two subfamilies and the HIFs in common carp were clustered with their teleost counterparts indicating they are highly conservative during evolution. In addition, the tissue distribution was examined by RT-PCR showed that most of HIF genes had a wide range of tissue distribution but exhibited tissue-specific expression patterns. The expression divergences were observed between the copy genes, for example, HIF1A-1, HIF2A-1, ARNT-2 had wide tissue distribution while their copies had limited tissue distribution, proving the function divergence of copies post the WGD event. In order to find an effective activation of HIFs and apply to treatment of aquatic diseases, we investigate the dietary supplementation effects of different strains of Lactococcus lactis on the expression of HIFα subfamily members in kidney of common carp infected with A. hydrophila. In addition, all of the HIF genes have a high expression in the early stages of infection, and decreased in the treatment time point of 48 h in common carp. This phenomenon confirms that as a switch, the main function of HIFs is to regulate the production of immune response factors in early infection. So activation of the switch may be an effective method for infectious disease treatment. As expected, the treatment groups improved the expression of HIFs compared with the control group, and the effects of the three strains are different. The strain1 of L. lactis had a stronger induction on HIF genes than strain2 and strain3, and it might be applied as a potential activation of HIF genes for disease treatment. So, adding befitting L. lactis maybe a well method to activate the HIF genes to protect them from mycobacterial infection.
695	Identification and functional analysis of three new anthocyanin R2R3-MYB genes in Petunia.	31245756	We identified three novel members of the R2R3-MYB clade of anthocyanin regulators in the genome of the purple flowering Petunia inflata S6 wild accession, and we called them ANTHOCYANIN SYNTHESIS REGULATOR (ASR). Two of these genes, ASR1 and ASR2, are inactivated by two different single base mutations in their coding sequence. All three of these genes are absent in the white flowering species P. axillaris N and P. parodii, in the red flowering P. exserta, and in several Petunia hybrida lines, including R27 and W115. P. violacea and other P. hybrida lines (M1, V30, and W59) instead harbor functional copies of the ASR genes. Comparative, functional and phylogenic analysis of anthocyanin R2R3-MYB genes strongly suggest that the ASR genes cluster is a duplication of the genomic fragment containing the other three R2R3-MYB genes with roles in pigmentation that were previously defined, the ANTHOCYANIN4-DEEP PURPLE-PURPLE HAZE (AN4-DPL-PHZ) cluster. An investigation of the genomic fragments containing anthocyanin MYBs in different Petunia accessions reveals that massive rearrangements have taken place, resulting in large differences in the regions surrounding these genes, even in closely related species. Yeast two-hybrid assays showed that the ASR proteins can participate in the WMBW (WRKY, MYB, B-HLH, and WDR) anthocyanin regulatory complex by interacting with the transcription factors AN1 and AN11. All three ASRs can induce anthocyanin synthesis when ectopically expressed in P. hybrida lines, but ASR1 appeared to be the most effective. The expression patterns of ASR1 and ASR2 cover several different petunia tissues with higher expression at early stages of bud development. In contrast, ASR3 is only weakly expressed in the stigma, ovary, and anther filaments. The characterization of these novel ASR MYB genes completes the picture of the MYB members of the petunia anthocyanin regulatory MBW complex and suggests possible mechanisms of the diversification of pigmentation patterns during plant evolution.
696	Application of Flow Cytometry in Primary Immunodeficiencies: Experience From India.	31244832	Primary immunodeficiency diseases (PID) are a clinically and immunologically heterogeneous group of disorders of immune system. Diagnosis of these disorders is often challenging and requires identification of underlying genetic defects, complemented by a comprehensive evaluation of immune system. Flow cytometry, with its advances in the last few decades, has emerged as an indispensable tool for enumeration as well as characterization of immune cells. Flow cytometric evaluation of the immune system not only provides clues to underlying genetic defects in certain PIDs and helps in functional validation of novel genetic defects, but is also useful in monitoring immune responses following specific therapies. India has witnessed significant progress in the field of flow cytometry as well as PID over last one decade. Currently, there are seven Federation of Primary Immunodeficiency Diseases (FPID) recognized centers across India, including two Indian Council of Medical research (ICMR) funded centers of excellence for diagnosis, and management of PIDs. These centers offer comprehensive care for PIDs including flow cytometry based evaluation. The key question which always remains is how one selects from the wide array of flow cytometry based tests available, and whether all these tests should be performed before or after the identification of genetic defects. This becomes crucial, especially when resources are limited and patients have to pay for the investigations. In this review, we will share some of our experiences based on evaluation of a large cohort of hemophagocytic lymphohistiocytosis, severe combined immunodeficiency, and chronic granulomatous disease, and the lessons learned for optimum use of this powerful technology for diagnosis of these disorders.
697	A Case of Hemophagocytic Lymphohistiocytosis Secondary to Ralstonia Solanacearum Infection.	31232034	Hemophagocytic lymphohistiocytosis (HLH), as known as Hemophagocytic syndrome (HPS), is deemed to a severe clinical syndrome caused by excessive human being immune system activation. Bacteria of Ralstonia genus is a non-fermentative, gram-negative bacillus and also in the category of human opportunistic pathogenic bacteria. In this article, authors report a rare case of Hemophagocytic lymophohistiocytosis which probably was triggered by Ralstonia solanacearum (R. solanacearum) infection.Hematologic investigation, biochemical examination, high throughput genetic test for infectious agents and bone marrow puncture.The patient achieved complete remission and no signs of relapse have as yet been found.The bacteria of Ralstonia genus merely infect humans, and there were no reports about the infection of R. solanacearum in humans and secondary HLH. The prognosis of the patient in this case was very good. This result, we think shows that the relationship between HLH and R. solanacearum infection should be taken into the diagnosis process. Recognition of this will promote the correct diagnosis in clinical work.
698	Systemic Epstein-Barr Virus-Positive T/NK Lymphoproliferative Diseases With SH2D1A/XIAP Hypomorphic Gene Variants.	31231620	X-linked lymphoproliferative disease (XLP) is one of the X-linked primary immunodeficiency diseases (PIDs) with defective immune response to Epstein-Barr virus (EBV) infection. Chronic active EBV infection (CAEBV) and EBV-hemophagocytic lymphohistiocytosis (HLH) are recognized as systemic EBV-positive T-cell and natural killer (NK)-cell lymphoproliferative diseases (LPDs) arising from the clonal proliferations of EBV-infected T cells and NK cells. A high incidence of CAEBV in East Asia implies the unknown genetic predisposition. In patients with XLP, EBV-infected cells are generally B cells. No mutation of SH2D1A/XIAP genes has ever been identified in patients with systemic EBV-positive T-cell and NK-cell LPD. We report herewith a male case of NK-cell type CAEBV with SH2D1A hypomorphic mutation (c.7G > T, p.Ala3Ser), two male cases of CAEBV/EBV-HLH with XIAP hypomorphic variant (c.1045_1047delGAG, p.Glu349del), and another female case of CD4+CAEBV with the same XIAP variant. The female underwent bone marrow transplantation from an HLA-matched sister with the XIAP variant and obtained a complete donor chimerism and a cure of laryngeal LPD lesion, but then suffered from donor-derived CD4+ T cell EBV-LPD. These observations demonstrated that SH2D1A and XIAP genes are critical for the complete regulation of EBV-positive T/NK cell LPD. X-linked lymphoproliferative disease (XLP) is one of the X-linked primary immunodeficiency diseases (PIDs) reported to have a defective immune response to Epstein-Barr virus (EBV) infection. Mutations in SH2D1A and XIAP genes cause XLP. Systemic EBV-positive T-cell and natural killer (NK)-cell lymphoproliferative diseases (LPDs) consist of three major types: EBV-positive hemophagocytic lymphohistiocytosis (HLH), chronic active EBV infection (CAEBV), and EBV-positive T-cell/NK-cell lymphoma. CAEBV is recognized as a poor prognostic disease of EBV-associated T-cell and NK-cell LPD arising from the clonal proliferation of EBV-infected T cells (CD4+, CD8+, and TCRγδ+) and/or NK cells. The majority of cases with CAEBV were reported from East Asia and South America. In Caucasian patients with CAEBV disease, the target of infection is exclusively B cells. These imply a genetic predisposition to EBV-positive T/NK cell LPD according to ethnicity. In reported cases with XLP, EBV-infected cells are B cells. On the other hand, no mutation of SH2D1A/XIAP genes have been determined in patients with T/NK-cell-type (Asian type) CAEBV. We here describe, for the first time, four case series of CAEBV/EBV-HLH patients who carried the hypomorphic variants of XLP-related genes. These cases included a male patient with CAEBV carrying SH2D1A hypomorphic mutation (c.7G > T, p.Ala3Ser) and two male patients with CAEBV/EBV-HLH carrying the XIAP hypomorphic variant (c.1045_1047delGAG, p.Glu349del), along with another female patient with CAEBV carrying the same XIAP variant. The female case underwent bone marrow transplantation from a healthy HLA-matched sister having the same XIAP variant. Although a complete donor chimerism was achieved with the resolution of laryngeal LPD lesions, systemic donor-derived CD4+ T-cell EBV-LPD developed during the control phase of intractable graft- vs. -host-disease. These observations demonstrated that SH2D1A and XIAP genes are critical for the complete regulation of systemic EBV-positive T/NK-cell LPD.
699	Haemophagocytic lymphohistiocytosis in patients with human immunodeficiency virus infection: to treat or not to treat.	31223395	Haemophagocytic lymphohistiocytosis (HLH) in Human Immunodeficiency Virus (HIV) infected individuals can either be due to the disease itself or due to associated infections/malignancies. The treatment for HLH requires immunosuppressive therapy but administering immunosuppressive therapy to an already immunosuppressed patient (HIV infection) is complex. We present two such cases of HLH in patients infected with HIV. In the first case, no alternate cause for HLH was found even after extensive investigations and it was attributed to the uncontrolled HIV replication. Patient was started on dexamethasone for the same but succumbed to hospital acquired pneumonia. The second patient was diagnosed with Hodgkin's lymphoma but he succumbed to his illness before initiating immunosuppressive therapy for HLH. We report these cases to highlight the dilemma and a need for further research in this direction.
700	To Transplant or Not to Transplant? Late-Onset Primary HLH in a Patient: A Case Report and Review of Literature.	31219909	Primary, or familial, hemophagocytic lymphohistiocytosis (P-HLH) is a rare inherited autosomal-recessive immune deficiency which generally manifests during infancy or early childhood. Recent literature suggests an increased number of reports of late-onset P-HLH, especially in association with infection and underlying malignancy. The authors describe a case of subcutaneous T-cell lymphoma in a 8-year-old child that was complicated by primary, perforin-deficient HLH. In contrast, we examined retrospective data for 19 cases of late-onset P-HLH with available treatment data and compared the results of conservative medical therapy with hematopoietic stem cell transplant (HSCT) postremission therapy. Our patient displayed compound heterozygous mutations in PRF1 that have not been described in the literature previously: allele 1 [c.786_801del(p.Gln263fs)] and allele 2 [c.886T>C(p.Tyr296His)]. Of the 19 cases analyzed, 14 achieved remission. Postremission, 7 of 14 (50%) received HSCT and were reported alive at a median time of 24 months, 5 of 14 (36%) received medical therapy and were reported alive at a median time of 24 months, and 2 of 14 (14%) received medical therapy and died at a median of 73 months postremission. Our retrospective literature review suggests that some patients can survive late-onset, perforin-deficient, P-HLH without the potential lifelong risks of HSCT when in the first remission.
701	Tocilizumab for the treatment of chimeric antigen receptor T cell-induced cytokine release syndrome.	31219357	Introduction: Cancer-directed immunotherapies are transforming the landscape in oncology as new and exciting therapies move from the laboratory to the bedside. Chimeric antigen receptor T (CAR-T) cells are one of these novel therapies, demonstrating impressive efficacy against B-cell malignancies. With the development of new therapies, it is not uncommon to identify new and unanticipated toxicities. CAR-T cells cause unique toxicities not typically found with traditional cytotoxic chemotherapy or small molecule inhibitors. Areas covered: CAR-T cell associated toxicities include cytokine release syndrome (CRS) and CAR-T cell-related encephalopathy syndrome (CRES), alternatively known as immune effector cell-associated neurotoxicity syndrome (ICANS). Prompt identification and management of CRS and CRES are imperative for the prevention of life-threatening complications of these innovative therapies. This literature review describes the seminal trials of CD19-directed immunotherapy and the pathophysiology and management of the toxicities found with CAR-T cells. In addition, the use of the interleukin-6 receptor antibody tocilizumab for CRS is reviewed. Expert opinion: This review describes the recommended management of CRS and CRES and examines the current limitations in management. Alternative therapies for the treatment of CAR-T cell related toxicities are also explored. Furthermore, the review proposes future directions for research.
702	Hemophagocytic lymphohistiocytosis in pregnancy: a case report and review of the literature.	31215279	Hemophagocytic lymphohistiocytosis (HLH) during pregnancy is rare and seldom reported in literature. Here we report a case of pregnancy-associated HLH. A patient was admitted at 26 weeks and 2 days pregnancy complaining of cough and fever was admitted. She was found having bi-cytopenia, elevated ferritin and hypertriglyceridaemia. HLH was not confirmed because of the negative results from the first bone marrow biopsy. As the situation worsened, a timely termination of pregnancy was carried out. The second bone marrow biopsy did reveal hemophagocytosis and then pregnant-related HLH was confirmed. Prompt treatment of dexamethasone and etoposide have the puerpernat in a better situation. From this case, we learned HLH can be likely ignored and not be diagnosed promptly in a pregnant patient. In order to confirm this disease, a bone marrow biopsy should be performed promptly or repeatly for suspicious HLH. The time of termination and treatment is both essential for the foetus and the puerperant. Given the high morbidity and mortality of this disease in recent years, an expert consensus or clinical guideline should be developed.
703	Estrogenic Compounds or Adiponectin Inhibit Cyclic AMP Response to Human Luteinizing Hormone in Mouse Leydig Tumor Cells.	31212720	Mouse Leydig Tumor cells (mLTC), transiently expressing cAMP-dependent luciferase, were used to study the influence of sexual steroids and of adiponectin (ADPN) on the cAMP response to luteinizing hormones (LH). While testosterone and progesterone had no significant effect, several molecules with estrogenic activity (17β-estradiol, ethynylestradiol, and bisphenol A) provoked a decrease in intracellular cyclic AMP accumulation under 0.7 nM human LH stimulation. Adiponectin exhibited a bimodal dose-effect on LH response: synergistic between 2-125 ng/mL and inhibitory between 0.5-5 µg/mL. In brief, our data indicate that estrogens and ADPN separately exert rapid (<1 h) inhibitory and/or synergistic effects on cAMP response to LH in mLTC-1 cells. As the inhibitory effect of each estrogenic molecule was observed after only 1-h preincubation, it might be mediated through the G protein-coupled estrogen receptor (GPER) membrane receptor, but this remains to be demonstrated. The synergistic effect with low concentrations of ADPN with human Luteinizing Hormone (hLH) was observed with both fresh and frozen/thawed ADPN. In contrast, the inhibitory effect with high concentrations of ADPN was lost with frozen/thawed ADPN, suggesting deterioration of its polymeric structure.
704	An Effective Approach for the Multiobjective Regional Low-Carbon Location-Routing Problem.	31212710	In this paper, we consider a variant of the location-routing problem (LRP), namely the the multiobjective regional low-carbon LRP (MORLCLRP). The MORLCLRP seeks to minimize service duration, client waiting time, and total costs, which includes carbon emission costs and total depot, vehicle, and travelling costs with respect to fuel consumption, and considers three practical constraints: simultaneous pickup and delivery, heterogeneous fleet, and hard time windows. We formulated a multiobjective mixed integer programming formulations for the problem under study. Due to the complexity of the proposed problem, a general framework, named the multiobjective hyper-heuristic approach (MOHH), was applied for obtaining Pareto-optimal solutions. Aiming at improving the performance of the proposed approach, four selection strategies and three acceptance criteria were developed as the high-level heuristic (HLH), and three multiobjective evolutionary algorithms (MOEAs) were designed as the low-level heuristics (LLHs). The performance of the proposed approach was tested for a set of different instances and comparative analyses were also conducted against eight domain-tailored MOEAs. The results showed that the proposed algorithm produced a high-quality Pareto set for most instances. Additionally, extensive analyses were also carried out to empirically assess the effects of domain-specific parameters (i.e., fleet composition, client and depot distribution, and zones area) on key performance indicators (i.e., hypervolume, inverted generated distance, and ratio of nondominated individuals). Several management insights are provided by analyzing the Pareto solutions.
705	[Clinical diagnosis and treatment of perinatal stage hemophagocytic lymphohistiocytosis].	31207702	Objective: To analyze the clinical features of patients with hemophagocytic lymphohistiocytosis (HLH) during perinatal stage. Methods: From January 2011 to October 2016, the obstetric materials, clinical features, associated disease/factors and therapeutic outcomes were studied in 11 patients with HLH in perinatal period. Results: In these 11 patients, 9 were primipara. As for the onset time of HLH, 5 were before delivery and 6 were after delivery. Six were complicated with other associated disease/factors, and infection was the commonest (5/6) . Treatment included glucocorticoid + fludarabine in 1 case, HLH-94/04 protocols in 9 cases, and monotherapy corticosteroids in 1 case. Two patients died eventually. Conclusions: Perinatal HLH is commonly observed in the pregnancy/postpartum related HLH (28th week of pregnancy to one week after delivery) . Infection is still the commonest associated factors. HLH-94/04 protocols after the cessation of pregnancy may be effective, but the cessation of pregnancy itself may not be enough for the perinatal stage related-HLH. The perinatal stage related-HLH still has a better outcome than the other subtype of secondary-HLH.目的： 分析围产期相关噬血细胞综合征（hemophagocytic lymphohistiocytosis，HLH）的临床特征。 方法： 对2011年1月至2016年10月诊治的11例围产期HLH患者基本信息、临床特点、相关疾病/因素和治疗效果进行观察。 结果： 11例HLH患者中，初产妇9例。妊娠期发病5例，产后发病6例。6例患者存在相关疾病/因素，感染最为常见（6例）。治疗方案包括糖皮质激素+氟达拉滨1例，HLH-94/04方案9例，单药激素1例。最终2例患者死亡。 结论： 围产期（妊娠28周至产后7 d）HLH在妊娠/产后相关HLH噬血中很常见。感染是最常见的相关因素。终止妊娠后进行治疗是有效的，而仅仅终止妊娠可能还不足以控制围产期相关HLH。围产期相关HLH的转归优于其他继发性HLH亚型。.
706	A case series of endemic infections associated hemophagocytic lymphohistiocytosis (HLH) mimicking severe sepsis syndrome.	31193414	NaN
707	Dengue Infection Complicated by Hemophagocytic Lymphohistiocytosis: Experiences From 180 Patients With Severe Dengue.	31188423	Globally, ~500 000 people with severe dengue (SD) require hospitalization yearly; ~12 500 (2.5%) die. Secondary hemophagocytic lymphohistiocytosis (sHLH) is a potentially fatal hyperinflammatory condition for which HLH-directed therapy (as etoposide and dexamethasone) can be life-saving. Prompted by the high mortality in SD and the increasing awareness that patients with SD may develop sHLH, our objectives were to (1) determine the frequency of dengue-HLH in SD, (2) describe clinical features of dengue-HLH, (3) assess mortality rate in SD and dengue-HLH, and (4) identify mortality-associated risk factors in SD.A 5-year retrospective single-center study in all adult patients with SD admitted to a tertiary intensive care unit in Malaysia.Thirty-nine of 180 (22%) patients with SD died. Twenty-one of 180 (12%) had HLH defined as an HLH probability ≥70% according to histo score (HScore); 9 (43%) died. Similarly, 12 of 31 (39%) fulfilling ≥4 and 7 of 9 (78%) fulfilling ≥5 HLH-2004 diagnostic criteria died. Peak values of aspartate aminotransferase (AST), alanine aminotransferase, lactate dehydrogenase, and creatinine correlated to fatality (odds ratios [ORs], 2.9, 3.4, 5.8, and 31.9; all P < .0001), as did peak ferritin (OR, 2.5; P = .0028), nadir platelets (OR, 1.9; P = .00068), hepatomegaly (OR, 2.9; P = .012), and increasing age (OR, 1.2; P = .0043). Multivariable logistic regression revealed peak AST (OR, 2.8; P = .0019), peak creatinine (OR, 7.3; P = .0065), and SOFA (Sequential Organ Failure Assessment) score (OR, 1.4; P = .0051) as independent risk factors of death.Be observant of dengue-HLH due to its high mortality. A prospective study is suggested on prompt HLH-directed therapy in SD patients with hyperinflammation and evolving multiorgan failure at risk of developing dengue-HLH.
708	A Rare Case of HHV-8 Associated Hemophagocytic Lymphohistiocytosis in a Stable HIV Patient.	31183227	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition associated with viral infections including HIV. Cases have been reported mainly in advanced HIV/AIDS. This is a rare case that reports HLH associated with human herpes virus-8 (HHV-8) associated multicentric Castleman disease in a stable HIV patient.A 70-year-old Asian male patient with history of stable HIV on medications with CD 4 cell count above 200 presented with cough and fever and was initially treated for pneumonia as an outpatient. Persisting symptoms prompted presentation to the hospital. The patient was found to have anemia which persisted despite repeated transfusion of packed red cells. A bone marrow biopsy to investigate anemia revealed hemophagocytosis. A CT scan revealed multiple enlarged lymph nodes and hepatosplenomegaly. An excisional lymph node biopsy revealed HHV-8 associated multicentric Castleman disease. The patient deteriorated despite initiation of treatment.HLH can occur at any stage of HIV, rapid diagnosis to identify possible underlying reactive infectious etiology and prompt initiation of treatment is crucial to survival.
709	Disseminated Herpes Simplex Masquerading as Hemophagocytic Lymphohistiocytosis: A Case Report.	31167703	Marked elevation in serum ferritin levels may be seen in disseminated infection or severe organ failure states, but it is also present in hemophagocytic lymphohistiocytosis (HLH). Herpes simplex virus (HSV) hepatitis has a high mortality rate, even in immunocompetent individuals, in whom it is rarely reported. We present a case of hyperferritinemia with features initially suggestive of a diagnosis of HLH but that ultimately proved to be fulminant HSV hepatitis.A 56-year-old man with an indolent undiagnosed brain mass presented with progressive neurologic deficits and was found to have fevers, cytopenias, transaminitis, and hyperferritinemia. Initially, HLH was suspected; however, the ultimate diagnosis was HSV hepatitis with dissemination. Although the patient was treated with intravenous acyclovir, multiorgan failure developed, and he died.This case highlights the importance of considering alternative causes for a rise in ferritin levels when HLH is on the differential. Additionally, we discuss the diagnostic and therapeutic implications of HSV hepatitis, and we review the literature for cases presenting in immunocompetent hosts.
710	Hemorrhagic fever with renal syndrome with secondary hemophagocytic lymphohistiocytosis in West China: a case report.	31164087	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease characterized by an excessive systemic inflammatory response, which can be classified as primary HLH (pHLH) and secondary HLH (sHLH). Viruses are the primary pathogens causing sHLH. Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease caused by hantaviruses. Its main characteristics include fever, circulatory collapse with hypotension, hemorrhage, and acute kidney injury. The case of HFRS presented with sHLH is very rare in clinic. We reported the HFRS inducing by Hantaan virus (HTNV) presented with sHLH as the first case in Shaanxi province of west China.A case of HFRS in 69-year-old Chinese woman, which had persistent fever, cytopenia, coagulopathy, ferritin significantly increased, hepatosplenomegaly and superficial lymphadenopathy. The hemophagocytosis was found in bone marrow, which was consistent with the characteristics of the HLH. The patient recovered completely after timely comprehensive treatments.HTNV should be considered as one of the underlying viruses resulting in hemophagocytosis, and if occurs, the early diagnosis and rapid therapeutic intervention are very important to the prognosis of sHLH.
711	Clinical characteristics of liver failure with hemophagocytic lymphohistiocytosis.	31148551	Liver failure with hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome with high mortality. The aim of this study was to decipher clinical and laboratory characteristics of hemophagocytic lymphohistiocytosis after definite diagnosis of liver failure and to provide clues for early diagnosis and treatment of HLH in patients with liver failure. Eleven patients diagnosed with liver failure and HLH were retrospectively investigated in this study. All patients presented with jaundice, persistent high-grade fever, pancytopenia, splenomegaly, evidence of hemophagocytes in the bone marrow and laboratory abnormalities indicating HLH. The average interval from the earliest diagnosis of liver failure to a definitive diagnosis of HLH was 17.27 days. Six (54.55%) patients died during follow-up. For patients with liver failure after admission and subsequently definitively diagnosed with HLH, bilirubin and INR were significantly decreased. HLH is definitely diagnosed at an intermediate or late stage when patients have already suffered from liver failure. The initial dose of glucocorticoid (methylprednisolone) was decreased to 1-1.5 mg/kg/d and gradually reduced thereafter. In conclusion, for patients with liver failure, HLH should be screened as early as possible upon persistent fever, splenomegaly and unexplained pancytopenia. For patients with liver failure and HLH, the dosage of glucocorticoid should be reduced to avoid serious side effects.
712	Hemophagocytic lymphohistiocytosis as a diagnostic consideration of fever of unknown origin with pancytopenia and chronic liver disease.	31143751	Hemophagocytic lymphohistiocytosis (HLH) is a severe disorder of systemic immune dysregulation which can be primary or secondary to autoimmune disorders, malignancy, or infections. We hereby describe a case of a 23-year-old male with severe hepatitis along with pancytopenia and prolonged fever of unknown origin that developed HLH triggered by staphylococcal urinary tract infection. This is a discussion of this unusual disease and its presentation and the diagnostic difficulties which may be encountered in general clinical practice.
713	Surviving starvation simply without TFEB.	3113656731086360	Starvation is among the most ancient of selection pressures, driving evolution of a robust arsenal of starvation survival defenses. In order to survive starvation stress, organisms must be able to curtail anabolic processes during starvation and judiciously activate catabolic pathways. Although the activation of metabolic defenses in response to nutrient deprivation is an obvious component of starvation survival, less appreciated is the importance of the ability to recover from starvation upon re-exposure to nutrients. In order for organisms to successfully recover from starvation, cells must be kept in a state of ready so that upon the return of nutrients, activities such as growth and reproduction can be resumed. Critical to this state of ready is the lysosome, an organelle that provides essential signals of nutrient sufficiency to cell growth-activating pathways in the fed state. In this issue, Murphy and colleagues provide evidence that exposure of Caenorhabditis elegans roundworms to 2 simple nutrients, glucose and the polyunsaturated fatty acid linoleate, is able to render lysosomal function competent to activate key downstream starvation recovery pathways, bypassing the need for a master transcriptional regulator of lysosomes. These findings provide a quantum leap forward in our understanding of the cellular determinants that permit organisms to survive cycles of feast and famine.
714	Higher Fatality for Severe Fever with Thrombocytopenia Syndrome Complicated by Hemophagocytic Lymphohistiocytosis.	31124344	Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious zoonosis caused by the SFTS virus. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome associated with excessive immune activation. Cytokine storms are often seen in both SFTS and HLH, resulting in rapid disease progression and poor prognosis. The aim of this study was to identify whether SFTS cases complicated by HLH are related to higher rates of mortality. Descriptive analysis of the frequency of clinical and laboratory data, complications, treatment outcomes, and HLH-2004 criteria was performed. Cases presenting with five or more clinical or laboratory findings corresponding to the HLH-2004 diagnostic criteria were defined as SFTS cases complicated by HLH. Eighteen cases of SFTS were identified during a 2-year study period, with a case-fatality proportion of 22.2% (4 among 18 cases, 95% confidence interval 9%-45.2%). SFTS cases complicated by HLH were identified in 33.3% (6 among 18 cases). A mortality rate of 75% (3 among 4 cases) was recorded among SFTS cases complicated by HLH. Although there were no statistically significant differences in outcomes, fatal cases exhibited more frequent correlation with HLH-2004 criteria than non-fatal cases [3/14 (21.4%) vs. 3/4 (75%), p=0.083]. In conclusion, the present study suggests the possibility that SFTS cases complicated by HLH are at higher risk of poor prognosis.
715	An unusual presentation of purine nucleoside phosphorylase deficiency mimicking systemic juvenile idiopathic arthritis complicated by macrophage activation syndrome.	31118063	Systemic juvenile idiopathic arthritis (sJIA) is an inflammatory condition that presents with fever, rash and arthritis. At onset systemic features are predominant and the diagnosis may be a challenge. Secondary hemophagocytic lymphohistiocytosis (sHLH) forms may be associated with different disorders, including rheumatic diseases, and this form is called macrophage activation syndrome (MAS). CXCL9 levels, a chemokine induced by IFNγ, are significantly elevated in patients with sHLH or MAS and are correlated with laboratory features of disease activity. High levels of IL-18 have been reported in patients with MAS during sJIA, as well as in some patients with sHLH and IL-18 is indeed known to induce IFNγ production.We report a patient with a clinical presentation highly suggestive for systemic juvenile idiopathic arthritis (sJIA) onset complicated by MAS, and was later diagnosed with purine nucleoside phosphorylase (PNP)-deficiency with HLH. Some unusual features appeared when HLH was controlled and further investigations provided the correct diagnosis. Serum CXCL9 and IL-18 levels were found markedly elevated at disease onset, during the active phase of MAS and decreased progressively during the course.The reported case underlines the potential difficulties in discriminating sJIA from other causes of systemic inflammation. Furthermore, this supports the notion that especially in young children with a sJIA-like disease other mimicking conditions should be actively sought for. CXCL9 and IL-18 levels suggested that patients with PNP-deficiency may have a subclinical activation of the IFNγ pathway and indeed they are predisposed to develop sHLH.
716	Requirement for etoposide in the initial treatment of Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis.	31115044	Haemophagocytic lymphohistiocytosis (HLH) is a severe, even fatal, inflammatory condition. Epstein-Barr virus (EBV) infection-associated HLH (EBV-HLH) is one of the most common types of secondary HLH. Etoposide is a key drug in HLH-94/04 regimen. We sought to identify the importance of etoposide in the initial treatment of EBV-HLH. Ninety-three patients with EBV-HLH of all ages admitted to our centre in 2017 were divided into two groups according to whether the initial treatment contained etoposide or not. The survival of the group whose initial therapy included etoposide (Group 1, 52 patients; 6-month survival rate 0·769) was significantly better than that of the group whose initial therapy did not include etoposide (Group 2, 41 patients; 6-month survival rate 0·269) (P < 0·001). In patients aged <18 years old, the survival of Group 1 patients was not significantly better than that of Group 2 patients (P = 0·447), in contrast to patients aged ≥18 years, where the survival of Group1 patients was significantly better than that of Group 2 patients (P < 0·001). We concluded that including etoposide in the initial treatment of EBV-HLH patients can improve their prognosis, especially adult patients. This may be because that adult patients are recognized as "higher risk" patients.
717	Progressive and disseminated histoplasma infection and hemophagocytic lymphohistiocytosis in an immunocompetent adult.	31110713	Hemophagocytic lymphohistiocytosis (HLH) in adults is a life-threatening underdiagnosed disorder that complicates different infectious syndromes and overlaps with sepsis. No guidelines are available for the management of HLH in adults. A high index of suspicion is required in patients with disseminated histoplasmosis.
718	Disturbances in NK Cells in Various Types of Hemophagocytic Lymphohistiocytosis in a Population of Polish Children.	31107368	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease associated with immune system hyperactivation and the appearance of serious systemic disturbances. The purpose of this study was an assessment of natural killer (NK) cell disturbances in a group of children with clinical signs of HLH. A total of 43 children with HLH and 17 healthy children were enrolled in the study. NK phenotyping, intracellular perforin staining, and cytotoxicity tests were performed by using the flow cytometry method. HLH patients were divided into 6 HLH types: 9% infection-related HLH; 7% malignancy-related HLH; 21% macrophage activating syndrome; 12% familial hemophagocytic lymphohistiocytosis; 2% X-linked lymphoproliferative syndrome; and 49% as HLH of unknown background. A positive correlation was observed between cytotoxicity and NK cells in children with HLH (P=0.01). In all HLH groups, the percentage of NK cells was significantly lower than in the control population. The spontaneous cytotoxicity was significantly lower in HLH patients. The results presented in this study indicate the importance of impaired function and the number of NK cells in the pathogenesis of HLH. Nonetheless, the background of disturbances seems to be different in various cases.
719	Enterovirus-Associated HLH: Addition of Anakinra to IVIG and Corticosteroids.	31104275	NaN
720	[Hemophagocytic lymphohistiocytosis with granulomatosis and diffuse T-cell infiltration associated with disseminated Nocardiosis and pulmonary infection due to Streptomyces spp].	31103241	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome frequently secondary to infectious disease, especially in immuno-compromised patients. We report a HLH secondary to disseminated nocardiosis and Streptomyces spp pulmonary infection.A 69-years-old women had recent subcutaneous nodules of the forearms and loins associated with peripheral neuropathy and pulmonary nodule of the right upper lobe. Cutaneous biopsy revealed granuloma. Cutaneous lesions worsened and the patient developed a HLH with probable cardiac and neurological involvement, associated with cutaneous granulomatosis and diffuse polyclonal lymphocyte proliferation. Nocardia PCR was positive in cutaneous biopsy. Pulmonary samples revealed Streptomyces in culture and Nocardia in PCR. The evolution under antibiotic treatment was favorable.Recent diagnosis of HLH without obvious etiology should lead to etiological investigation, including the search for infections with slow-growing bacteria such as Nocardia or Streptomyces spp.
721	A clinicopathologic study of the spectrum of systemic forms of EBV-associated T-cell lymphoproliferative disorders of childhood: A single tertiary care pediatric institution experience in North America.	31099136	Systemic forms of EBV-associated T-cell lymphoproliferative disorders of childhood (S-EBV-T-LPD) comprise three major forms: EBV-positive hemophagocytic lymphohistiocytosis (EBV-HLH), systemic EBV-positive T-cell lymphoma (S-EBV-TCL), and systemic chronic active EBV infection (S-CAEBV). These disorders occur rarely in children in Western countries. Here, we described eight children of such entities.Eight cases (six clinical and two autopsy) with S-EBV-T-LPD of childhood were retrospectively identified from 1990 to 2015. Clinicopathologic parameters including histomorphology, immunophenotype, EBV studies, and T-cell receptor gene rearrangement studies were recorded.Patients include five females and three males of Hispanic, Asian, and Caucasian origins with an age range of 14 months to 9 years. Fever, hepatosplenomegaly, cytopenias, abnormal EBV serologies, and very high EBV viral loads were common findings. Histologic findings showed EBV+ T-cell infiltrates with variable degrees of architectural distortion and cytologic atypia ranging from no to mild cytologic atypia to overt lymphoma and tissue hemophagocytosis. All showed aberrant CD4+ or CD8+ T cells with dim to absent CD5, CD7, and CD3, and bright CD2 and CD45 by flow cytometry or loss of CD5 by immunohistochemistry. TCR gene rearrangement studies showed monoclonal rearrangements in all clinical cases (6/6). Outcomes were poor with treatment consisting of chemotherapy per the HLH-94 or HLH-2004 protocols with or without bone marrow transplant.In this large pediatric clinicopathologic study of S-EBV-T-LPD of childhood in the United States, EBV-HLH, S-EBV-TCL, and S-CAEBV show many overlapping features. Diagnosis is challenging, and overall outcome is poor using current HLH-directed therapies.
722	Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival.	3108636031136567	Lysosomes are ubiquitous acidified organelles that degrade intracellular and extracellular material trafficked via multiple pathways. Lysosomes also sense cellular nutrient levels to regulate target of rapamycin (TOR) kinase, a signaling enzyme that drives growth and suppresses activity of the MiT/TFE family of transcription factors that control biogenesis of lysosomes. In this study, we subjected worms lacking basic helix-loop-helix transcription factor 30 (hlh-30), the Caenorhabditis elegans MiT/TFE ortholog, to starvation followed by refeeding to understand how this pathway regulates survival with variable nutrient supply. Loss of HLH-30 markedly impaired survival in starved larval worms and recovery upon refeeding bacteria. Remarkably, provision of simple nutrients in a completely defined medium (C. elegans maintenance medium [CeMM]), specifically glucose and linoleic acid, restored lysosomal acidification, TOR activation, and survival with refeeding despite the absence of HLH-30. Worms deficient in lysosomal lipase 2 (lipl-2), a lysosomal enzyme that is transcriptionally up-regulated in starvation in an HLH-30-dependent manner, also demonstrated increased mortality with starvation-refeeding that was partially rescued with glucose, suggesting a critical role for LIPL-2 in lipid metabolism under starvation. CeMM induced transcription of vacuolar proton pump subunits in hlh-30 mutant worms, and knockdown of vacuolar H+-ATPase 12 (vha-12) and its upstream regulator, nuclear hormone receptor 31 (nhr-31), abolished the rescue with CeMM. Loss of Ras-related GTP binding protein C homolog 1 RAGC-1, the ortholog for mammalian RagC/D GTPases, conferred starvation-refeeding lethality, and RAGC-1 overexpression was sufficient to rescue starved hlh-30 mutant worms, demonstrating a critical need for TOR activation with refeeding. These results show that HLH-30 activation is critical for sustaining survival during starvation-refeeding stress via regulating TOR. Glucose and linoleic acid bypass the requirement for HLH-30 in coupling lysosome nutrient sensing to survival.
723	Role of Damage-Associated Molecular Patterns and Uncontrolled Inflammation in Pediatric Sepsis-Induced Multiple Organ Dysfunction Syndrome.	31073505	The incidence of multiple organ dysfunction syndrome (MODS) in sepsis varies from 17 to 73% and furthermore, increases the risk of death by 60% when controlled for the number of dysfunctional organs. Several MODS phenotypes exist, each unique in presentation and pathophysiology. Common to the phenotypes is the stimulation of the immune response by pathogen-associated molecular patterns (PAMPs), or danger-associated molecular patterns (DAMPs) causing an unremitting inflammation. Two of the MODS phenotypes are discussed in detail, thrombocytopenia-associated multiple organ failure (TAMOF) and the hyperinflammatory phenotype-macrophage activating syndrome (MAS) and hemophagocytic lymphohistiocytosis (HLH). In the end, we will briefly review the role of mitochondrial dysfunction as a significant contributor to the pathogenesis of MODS.
724	Nontuberculous Mycobacterium infection complicated with Haemophagocytic syndrome: a case report and literature review.	31072325	Non-tuberculous mycobacterial (NTM) infection is usually observed in patients with immunosuppressive conditions. It may also cause unregulated immune responses. While there have been increasing numbers of reported tuberculosis-related HPS (haemophagocytic syndrome), HPS caused by NTM infection is still very rarely reported.We report a previously healthy 21-year-old Chinese female with fever, night sweats and fatigue, in whom HPS was diagnosed according to the HLH-2004 criteria. Mycobacterium intracellulare was cultured from her peripheral blood. After treatment with corticosteroid, clarithromycin, rifampicin, ethambutol and amikacin, the patient finally recovered. We also reviewed relevant publications on NTM infection complicated with HPS and found 11 cases, including ours. Clinical presentations, diagnoses and prognoses were analysed and summarized to deepen our understanding of this rare condition.Most reported NTM-related cases were caused by disseminated infection. The lack of localized symptoms might add to the difficulty involved in making the right diagnosis. While it usually takes time to obtain tissue or blood culture results, granuloma in a bone marrow biopsy might be an early indicator of possible mycobacterial infection. Although treatment varied, the overall prognosis of NTM-related HPS was promising.
725	[Fatal exacerbations of chronic active Epstein-Barr virus infection subsequent to cytotoxic chemotherapy].	31068557	Chronic active Epstein-Barr virus infection (CAEBV) is critical owing to lethal complications such as hemophagocytic lymphohistiocytosis (HLH), multiple organ failure, and malignant lymphoma. Here we present two cases of CAEBV who developed rapid and life-threatening disease progression after cytotoxic chemotherapy. Case 1: In a 34-year-old male, CAEBV recurred after 4-month remission obtained by initial therapy with etoposide, cyclosporine, and prednisolone. Accordingly, cord blood transplantation was planned. A day after administering high-dose melphalan as the conditioning, he developed respiratory failure, pancytopenia, and hyperferritinemia. He died 3 days later. Case 2: A 53-year-old female attained remission after initial therapy for CAEBV. After 1 month, she relapsed, and high-dose cytarabine (HDAC) was administered. A day after HDAC administration, she suddenly developed respiratory failure, which was followed by multiple organ failure. She died 3 days later. Thus, planned strategy for prompt allogeneic hematopoietic stem cell transplantation is necessary to prevent disease progression and control cytokinemia before cytotoxic chemotherapy for CAEBV.
726	A key gene bHLH115 in iron homeostasis: comprehensive bioinformatics analyses in Arabidopsis, tomato, rice, and maize.	31062267	bHLH115 transcription factor (TF) is a positive regulator of the Fe-deficiency and plays essential roles in the stress-related regulation network. In this study, orthologous bHLH115 genes in Arabidopsis, tomato, rice, and maize were analyzed using in silico methods. All bHLH115 proteins contained PF00010 (HLH: Helix-loop-helix DNA-binding domain) domain structure and their sub-cellular localizations were predicted as nucleus. The bHLH115 orthologues in monocots and dicots clearly diverged from each other. The expression analyses revealed that orthologous genes of bHLH115 in queried species were highly expressed in seed parts, leaf, stem, and flower parts. The bHLH115 genes were co-expressed with genes in plant defense system, and with genes involving in biotic and abiotic stress responses. In terms of protein structures, OsbHLH115 and ZmbHLH115, and AtbHLH115 and SlbHLH115 had the highest protein structure similarities. In addition, bHLH115 proteins have bZIP, bHLH and MYB transcription factor binding sites strengthens their engagement in various metabolic ways. Molecular docking analyses showed the different binding sites based on plant species, suggesting functional flexibilities of bHLH115 gene.
727	Hemophagocytic Lymphohistiocytosis Associated with Natural T-cell Leukemia.	31058001	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening syndrome of excessive immune activation. It can be triggered by a variety of events that disrupt immune homeostasis, infection being the most common of them. We report a case of a 36-year-old male diagnosed with HLH associated with natural T-cell leukemia. The purpose of this report is to call attention to the clinical presentation, diagnosis, and treatment of HLH.
728	[Analysis of PRF1gene variant in a child with late-onset familial hemophagocytic lymphohistiocytosis type 2 and severe central nervous system disease].	31055813	To detect genetic mutations in a child with late-onset hemophagocytic lymphohistiocytosis (HLH).Clinical data of an 8-year-5-month-old girl with recurrent HLH and severe central nervous system disease was analyzed. Next-generation sequencing was used to detect mutation in the exons and adjacent introns of 17 genes associated with HLH. Suspected mutations were confirmed by Sanger sequencing. Influence of mutations on protein function was predicted with SIFT and PolyPhen-2 software.The child was found to carry compound heterozygous mutations of the PRF1 gene. Among these, the c.1349C>T (p.Thr450Met) mutation, with a SIFT predictive value of -4.921 (Deleterious variant) and a PolyPhen-2 predictive value of 1.000 (Probably damaging), was inherited from her father and known to be pathogenic. The c.1273dupT (p.Trp425fsX457) mutation was inherited from her mother and previously unreported, which resulted in the deletion of almost the entire C2 domain (amino acid residues 413 to 540) and carboxyl terminal of perforin, which seriously affected the function of the protein.The c.1349C>T (p.Thr450Met) and c.1273 dupT (p.Trp425fsX457) compound heterozygous mutations of the PRF1 gene probably underlie the disease in this patient.
729	Haemophagocytic lymphohistiocytosis in patients treated with immune checkpoint inhibitors: analysis of WHO global database of individual case safety reports.	31046841	Immune checkpoint inhibitor (ICI) use in clinical practice has unravelled a spectrum of immune-related adverse events (irAEs) due to immune system hyper-activation. ICI-related haemophagocytic lymphohistiocytosis (HLH) has been recently outlined in single case reports, raising a concern about the need of increasing our knowledge on this rare yet life threatening ICI haematological toxicity.To determine ICI-related HLH clinical, haematological, and coagulation features, its timing and outcome, concurrent irAEs and concomitant infections, we performed a retrospective observational cross-sectional study and queried VigiBase, the WHO global database of suspected adverse drug reactions (ADRs), on September 30th, 2018. We retrieved the individual case safety reports reporting HLH in association with ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab or durvalumab, gathered in the database starting from the ICIs' approval dates by the US Food and Drug Administration. The main outcome measures were co-suspected drugs, concurrent irAEs, HLH clinical, haematological and coagulation features, concomitant infections, HLH median time to onset and outcome.Among 49'883 ICI-related ADRs collated in VigiBase as of September 30th, 2018, HLH was reported in 38 cases of which 34 (90%) mentioned ICIs as the solely suspected drugs. ICI-related HLH showed clinical, haematological and coagulation features similar to those of HLH with different etiology. Concurrent irAEs occurred in 5 (13%) patients and 6 (16%) reported concomitant viral infections. 31 (82%) cases defined ICI-related HLH outcome, which resolved in 19 (61%) cases. HLH developed a median of 6.7 weeks after initiation of ICI treatment (IQR 2.9-15.4, n = 18, 47%).By evaluating the largest cohort of ICI-related HLH cases, we observed that ICI-related HLH arises with a delayed timing with respect to initiation of ICI treatment, and usually presents without other irAEs and concomitant infections. Keeping in mind these findings, clinicians should consider ICIs' involvement in the onset of HLH whenever they diagnose a disease of this group of syndromes in cancer patients treated with ICIs.
730	Flow Cytometric Analysis of T Cells in Hemophagocytic Lymphohistiocytosis.	31037861	T cell immunophenotypes in patients with hemophagocytic lymphohistiocytosis (HLH) have been described. Downregulation of CD5 or CD7 on T cells has been reported in patients with Epstein-Barr virus (EBV)-positive HLH. As the utility of T cell immunophenotypes as an adjunctive diagnostic or a prognostic marker for HLH has not been evaluated, we analyzed T cell immunophenotypes in HLH patients for this purpose.We classified 45 HLH patients into three subgroups: EBV-positive HLH (N=27), EBV-negative secondary HLH (N=15), and familial HLH (N=3). We retrospectively characterized downregulation patterns of CD5 or CD7 on activated T cells, using flow cytometry. Overall survival was estimated using Kaplan-Meier curves and compared using the log-rank test.An aberrant immunophenotype, including CD5 and/or CD7 downregulation on T cells, was observed in 55.6% (15/27) of the EBV-positive HLH patients and 100% of the familial HLH (3/3). Only one (1/15, 6.7%) patient with EBV-negative secondary HLH showed an aberrant loss of CD7 antigen on CD8+ T cells. The presence of an aberrant T cell immunophenotype was not related to overall survival in EBV-positive HLH and EBV-negative secondary HLH patients.An aberrant T cell immunophenotype may assist in discriminating EBV-negative secondary HLH and EBV-positive HLH. However, it may not be useful as a prognostic marker.
731	Significance of serum ferritin level in hemophagocytic lymphohistiocytosis diagnosis.	31022329	The threshold of serum ferritin (SF) level listed in diagnostic guidelines for hemophagocytic lymphohistiocytosis (HLH) of HLH-2004 has a low specificity. The goal of this research was to evaluate the role of admission SF in HLH diagnostic procedure and to find an appropriate threshold for a HLH suspected cohort with fever.All patients admitted to Beijing Children's Hospital during the period of September 1, 2015 and July 31, 2016 with fever and SF tested at admission were included in this study. The significance of SF in HLH diagnosis and its relationships with HLH-relevant clinical characteristics were studied.Among 357 patients, 39 HLH cases were diagnosed (24 EBV-related HLH, 13 unknown etiologies triggered HLH, and two familial HLH). The best cutoff value of admission SF was 934 ng/mL, with sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) being 87.2%, 88.4%, 47.9%, and 98.3%, respectively. Compared to 500 ng/mL, specificity and PPV of the new SF standard in HLH diagnose increased by 11.7% and 14.0%, which indicated improvements in diagnostic ability of "non-HLH" and in veracity of "HLH" identification. Among four HLH patients whose admission SF was between 500 ng/mL and 934 ng/mL, HLH diagnosis was guaranteed by other laboratory results in two patients; however, possible misdiagnosis was made in the rest two patients.Elevated cutoff value of admission SF level seems to be more appropriate for distinguishing HLH in patients with fever. The exact cutoff value of SF level at diagnosis needs to be determined.
732	UNC13D Deficiency Associated With Epileptic Seizures and Antibody Deficiency: The First Case from the Iranian National Registry.	31017122	NaN
733	Takotsubo cardiomyopathy secondary to haemophagocytic lymphohistiocytosis in HIV patients: a comprehensive review.	31015233	Haemophagocytic lymphohistiocytosis (HLH) is an immune dysregulation disorder with variable presentations and non-specific features making it extremely difficult to diagnose early in the clinical course. Here, we are presenting a case of a young man who presented in cardiogenic shock with findings of anterolateral wall ischaemia on ECG. Echocardiography findings were consistent with takotsubo cardiomyopathy (TCM). Cardiac catheterisation showed clean coronary arteries and pulmonary artery pressure measurements showed high output cardiac failure. After extensive workup, the patient was diagnosed with HLH. In spite of aggressive supportive and definitive therapy, he eventually died due to a complicated clinical course. We did a comprehensive literature review and found that this is the first reported case of HLH presenting as TCM as the initial clinical manifestation.
734	Mechanisms of action of ruxolitinib in murine models of hemophagocytic lymphohistiocytosis.	3101519031296541	Hemophagocytic lymphohistiocytosis (HLH) is an often-fatal disorder characterized by the overactivation of T cells and macrophages that excessively produce proinflammatory cytokines, including interferon-γ (IFN-γ). Previously, we reported that the JAK inhibitor ruxolitinib dampens T-cell activation and lessens inflammation in a model of HLH in which perforin-deficient (Prf1 -/-) mice are infected with lymphocytic choriomeningitis virus (LCMV). Ruxolitinib inhibits signaling downstream of IFN-γ, as well as several other JAK-dependent cytokines. As a consequence, it remained unclear whether ruxolitinib was exerting its beneficial effects in HLH by inhibiting IFN-γ signaling or by targeting signaling initiated by other proinflammatory cytokines. To address this question, we compared the effects of ruxolitinib with those obtained using an IFN-γ-neutralizing antibody (αIFN-γ) in 2 murine HLH models. In both models, ruxolitinib and αIFN-γ reduced inflammation-associated anemia, indicating that ruxolitinib operates in an IFN-γ-dependent manner to reverse this HLH manifestation. In contrast, the number and activation status of T cells and neutrophils, as well as their infiltration into tissues, were significantly reduced following treatment with ruxolitinib, but they remained unchanged or were increased following treatment with αIFN-γ. Notably, despite discontinuation of ruxolitinib, LCMV-infected Prf1 -/- mice exhibited enhanced survival compared with mice in which αIFN-γ was discontinued. This protective effect could be mimicked by transient treatment with αIFN-γ and a neutrophil-depleting antibody. Thus, ruxolitinib operates through IFN-γ-dependent and -independent mechanisms to dampen HLH by targeting the deleterious effects of T cells and neutrophils, with the latter representing an unappreciated and understudied cell type that contributes to HLH pathogenesis.
735	Pneumococcal Bacteremia Complicated by Hemophagocytic Lymphohistiocytosis.	31011972	From pneumonia to pericarditis, from sepsis to splenic abscess, Streptococcus pneumoniae is the causative agent of a diverse array of pyogenic disease. With the introduction of vaccines and effective antibiotic treatments, the incidence of complicated streptococcal infection has declined. We report a case of S. pneumoniae bacteremia, in the setting of occult sinusitis, complicated by hemophagocytic lymphohistiocytosis (HLH), disseminated intravascular coagulation (DIC), and recurrent pneumococcal infection. Although severe streptococcal infection has been associated with immunodeficiency or splenectomy, no such predisposition was identified in our patient. We discuss the association of streptococcal infection with HLH and DIC and review occult sinusitis as a source of pneumococcal bacteremia, with the goal of enhancing the "illness scripts" of general medical practitioners to include such entities.
736	Prevalence and Outcome of Secondary Hemophagocytic Lymphohistiocytosis Among SIRS Patients: Results from a Prospective Cohort Study.	31010216	Secondary hemophagocytic lymphohistiocytosis (sHLH) is a life-threatening condition clinically presenting as SIRS (Systemic Inflammatory Response Syndrome). However, there is no comprehensive data concerning diagnostic algorithms, prevalence, outcome and biomarker performance in SIRS patients. We conducted a prospective observational cohort study on 451 consecutive patients fulfilling ≥2 SIRS criteria. The Hscore and the HLH-2004 criteria were used to determine the presence of sHLH, and the correlation of the screening-biomarkers ferritin, sCD25, and sCD163 with both scores was assessed. Out of 451 standard-care SIRS patients, five patients had high Hscores (≥169), suggesting incipient or HLH-like disease, and these patients were in urgent need for intensified therapy. However, none of these patients fulfilled five HLH-2004 criteria required for formal diagnosis. From the studied biomarkers, ferritin correlated strongest to both the HLH-2004 criteria and the Hscore (rs = 0.72, 0.41, respectively), and was the best predictor of 30-day survival (HR:1.012 per 100 μg/L, 95% CI: 1.004-1.021), when adjusted for patient's age, sex, bacteremia and malignant underlying-disease. Also, the HLH-2004 (HR per point increase: 1.435, 95% CI: 1.1012-2.086) and the Hscore (HR per point increase:1.011, 95% CI: 1.002-1.020) were independent predictors of 30-day-survival. The Hscore detected patients in hyperinflammatory states requiring urgent therapy escalation. Degrees of hyperinflammation, as assessed by ferritin and both HLH scores, are associated with worse outcomes.
737	Hydroa vacciniforme: a distinctive form of Epstein-Barr virus-associated T-cell lymphoproliferative disorders.	30998212	Hydroa vacciniforme (HV) is a cutaneous subset of Epstein-Barr virus (EBV)-associated T/NK lymphoproliferative disorders (LPDs). Our previous case series study clearly showed a clinical spectrum of EBV-associated T/NK LPDs including HV, hypersensitivity to mosquito bites (HMB), chronic active EBV infection (CAEBV), and hemophagocytic lymphohistiocytosis (HLH). Patients with HV are divided into two groups: a benign subtype designated "classic HV" (cHV) and more serious systemic HV (sHV), also called "HV-like LPD" in the 2017 World Health Organization (WHO) classification. Patients with cHV usually have an increased number of EBV-infected γδT cells and patients with sHV without HMB are further classified into two groups: γδT-cell- and αβT-cell-dominant types. Patients with HMB, with or without HV-like eruptions, have an increased number of EBV-infected NK cells in the blood. Patients with cHV and γδT-cell-dominant sHV show a favourable prognosis, but the other subtypes such as αβT-cell-dominant sHV and HMB have a poor prognosis with mortality rates of 11.5 and 3.51 per 100 person-years, respectively. In addition to the clinical subtypes and the dominant lymphocyte subsets, the poor prognostic indicators include onset age over nine years and expression of the reactivation marker, BZLF1 mRNA. No prognostic correlation has been reported for anti-EBV antibody titres or EBV DNA load. The clinical subtypes and their prognostic factors should be considered for therapeutic interventions.
738	Hemophagocytic lymphohistiocytosis associated with the use of lamotrigine.	3099605930996062	To describe adverse event reports of hemophagocytic lymphohistiocytosis (HLH) reported in association with lamotrigine.The Food and Drug Administration Adverse Event Reporting System database of spontaneous adverse event reports and medical literature databases were searched for cases of HLH reported in association with lamotrigine. Cases were included if they met the case definition of suspected or confirmed HLH and if causal association was assessed as robust or supportive.Eight cases met the case definition for HLH and were deemed causally associated with lamotrigine. These 8 cases of HLH had a plausible temporal relationship because they occurred within a 24-day interval from lamotrigine initiation. The doses ranged from 25 mg every other day to 250 mg once daily in the 6 cases that reported this information. Seven patients improved with drug discontinuation and one patient died after drug discontinuation and receiving an unspecified chemotherapy.Lamotrigine is associated with immune-related adverse reactions including HLH. HLH is a potentially fatal event; prompt recognition and early therapeutic intervention to mitigate the event is important in improving patient outcomes.
739	Recommendations for the management of hemophagocytic lymphohistiocytosis in adults.	3099226532321563	Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome induced by aberrantly activated macrophages and cytotoxic T cells. The primary (genetic) form, caused by mutations affecting lymphocyte cytotoxicity and immune regulation, is most common in children, whereas the secondary (acquired) form is most frequent in adults. Secondary HLH is commonly triggered by infections or malignancies but may also be induced by autoinflammatory/autoimmune disorders, in which case it is called macrophage activation syndrome (MAS; or MAS-HLH). Most information on the diagnosis and treatment of HLH comes from the pediatric literature. Although helpful in some adult cases, this raises several challenges. For example, the HLH-2004 diagnostic criteria developed for children are commonly applied but are not validated for adults. Another challenge in HLH diagnosis is that patients may present with a phenotype indistinguishable from sepsis or multiple organ dysfunction syndrome. Treatment algorithms targeting hyperinflammation are frequently based on pediatric protocols, such as HLH-94 and HLH-2004, which may result in overtreatment and unnecessary toxicity in adults. Therefore, dose reductions, individualized tailoring of treatment duration, and an age-dependent modified diagnostic approach are to be considered. Here, we present expert opinions derived from an interdisciplinary working group on adult HLH, sponsored by the Histiocyte Society, to facilitate knowledge transfer between physicians caring for pediatric and adult patients with HLH, with the aim to improve the outcome for adult patients affected by HLH.
740	Hemophagocytic lymphohistiocytosis due to Streptococcus suis in a 12-year-old girl: A case report.	30985681	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome that can be caused by bacterial infection. Streptococcus suis (S. suis) is a zoonotic pathogen that can cause severe disease in both pigs and humans. We report the first-ever documented case of HLH secondary to S. suis infection.A 12-year-old girl presented with fever, rash, hepatosplenomegaly, pancytopenia, and elevated levels of ferritin and soluble CD25. Bone marrow examination revealed hemophagocytosis. Blood culture was positive for S. suis.A diagnosis of hemophagocytic syndrome due to S. suis was established.We treated the patient with intravenous immunoglobulin, intravenous imipenem, and supportive care.The patient eventually showed complete recovery.Inflammatory response plays an important role in S. suis infection. Aberrant inflammatory response to S. suis infection may induce HLH. This case report illustrates that early definitive diagnosis and prompt treatment is a key imperative in patients with suspected S. suis infection.
741	Case Series of Hemophagocytic Lymphohistiocytosis from a Tertiary Care Centre: An Underdiagnosed Entity.	30977862	Hemophagocytic Lymphohistiocytosis (HLH) is an uncommon, life-threatening hyperinflammatory syndrome, caused by severe hypercytokinemia, due to an overstimulated but ineffective immune process. The presenting features of HLH are non-specific, mimicking many other diseases, and hence its early recognition still remains a challenge. It requires a high index of suspicion and detailed analysis of clinical and laboratory findings to arrive at a conclusive diagnosis. The objective of this study is to present detailed clinical and laboratory features of a series of HLH cases.A retrospective study was conducted wherein all the bone marrow aspirate smears diagnosed as HLH between January 2015 to June 2018 were reviewed. Detailed correlation of clinical and laboratory criteria was done with the bone marrow findings.A total of twelve cases were diagnosed as HLH from January 2015 to June 2018. Ten patients fulfilled 5 out of 8 clinical and lab diagnostic criteria of HLH (2004). After correlating clinical and laboratory criteria along with bone marrow findings the diagnosis of HLH was suggested.We present a series of twelve cases of Hemophagocytic Lymphohistiocytosis from a tertiary care hospital in New Delhi which will add not only to the understanding of this rare life threatening disease but also to the early diagnosis and intervention.
742	Correction to: Factors predicting the recurrence of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children after treatment using the HLH-2004 protocol.	3097710630788725	The correct name of the first author should be ''Ryu Yanagisawa'', and not ''Ryu Yanagaisawa'' as given in the original publication of the article.
743	Immune dysregulation: EBV+ DLBCL and HLH in a patient with T-LGL.	30975643	NaN
744	Hematopoietic stem cell transplantation in children with Griscelli Syndrome type 2: Experience and outcomes.	30971555	Griscelli syndrome is a rare autosomal recessive inherited disorder characterized by hypopigmentation, silver colored hair, and associated immunological deficiency, which proves fatal in the absence of timely intervention. Our patients diagnosed with Griscelli syndrome-2 presented with fever, hepatosplenomegaly, and deranged hematological and biochemical parameters. Both cases underwent detailed investigations comprising of hair mount microscopic examination, degranulation assay, and mutational studies. Our cases showed defective degranulation activity by NK cells and gene mutation analysis revealed RAB27A mutation that causes defect of cytotoxic granule exocytosis from natural killer (NK) and T-cells, manifesting clinically as hemophagocytic lymphohistiocytosis (HLH). Hematopoietic stem cell transplantation in one of the patients resulted in stable chimerism; however, the second case relapsed within a month after SCT. Stem cell transplantation is the only curative therapeutic option for GS2; thus, improvement in posttransplantation management may reduce mortality and posttransplant complications. Hence, any child who presents with partial albinism and clinical features suggestive of HLH, a peripheral blood, hair shaft mount examination along with basic immunological NK and T-cell cytotoxicity assay by flow cytometry will help clinch the diagnosis early. It can subsequently be confirmed by molecular study. Timely therapeutic intervention can prevent relapses and severe infection and improve outcome in these cases.
745	Lifespan Extension in C. elegans Caused by Bacterial Colonization of the Intestine and Subsequent Activation of an Innate Immune Response.	3096503330965036	Mechanisms that control aging are important yet poorly defined. To discover longevity control genes, we performed a forward genetic screen for delayed reproductive aging in C. elegans. Here, we show that am117 is a nonsense mutation in the phm-2 gene, which encodes a protein homologous to human scaffold attachment factor B. phm-2(lf) mutant worms have an abnormal pharynx grinder, which allows live bacteria to accumulate in the intestine. This defect shortens lifespan on highly pathogenic bacteria but extends lifespan and health span on the standard E. coli diet by activating innate immunity pathways that lead to bacterial avoidance behavior and dietary restriction. eat-2(lf) mutants displayed a similar phenotype, indicating accumulation of live bacteria also triggers extended longevity in this mutant. The analysis of phm-2 elucidates connections between pathogen response and aging by defining a mechanism of longevity extension in C. elegans-bacterial colonization, innate immune activation, and bacterial avoidance behavior.
746	Treatment of refractory secondary hemophagocytic lymphohistiocytosis with umbilical cord mesenchymal stem cells.	30961409	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome of excessive immune activation. Mesenchymal stem cells (MSCs) generate an immunosuppressive microenvironment by secreting cytokines and have been used to treat autoimmune diseases. We report the first case of refractory secondary HLH treated with umbilical cord MSCs. A 52-year-old Chinese female patient with a history of type 2 diabetes was diagnosed with refractory secondary HLH based upon the HLH-2004 protocol and was treated by infusion of third-party umbilical cord MSCs (1.4 × 106 cells/kg of body weight, 70 × 106 cells in total) from the stem cell bank of Hainan Province. Body temperature recovered to normal on the sixth day after infusion with umbilical cord MSCs, and the levels of inflammatory factors macrophage inflammatory protein (MIP)-1α, interleukin (IL)-12p70, stromal cell-derived factor (SDF)-1α, and IL-7 decreased significantly. Blood glucose levels were significantly lower than before treatment, and the amount of insulin needed was significantly reduced. Umbilical cord MSCs can relieve the symptoms of refractory secondary HLH and have a therapeutic effect on insulin resistance in type 2 diabetes mellitus.
747	An unusual case of hemophagocytic lymphohistiocytosis diagnosed by spinal nerve root biopsy.	30952113	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease process characterized by aberrant immune system activation and an exaggerated inflammatory response. Establishing the diagnosis may be challenging and is achieved by satisfying a number of clinical criteria, in addition to demonstrating tissue hemophagocytosis. This syndrome is rapidly fatal if prompt diagnosis and treatment are not achieved. The authors present the case of a 17-year-old male patient with CNS HLH involving both the brain and spinal cord, highlighting the variable CNS manifestations in pediatric patients with HLH and the challenges that accompany establishing diagnosis.
748	A case of pulmonary tuberculosis diagnosed in a patient with manifestations of haemophagocytic lymphohistiocytosis.	30949351	An 80-year-old woman was admitted with continuous fever, hepatic dysfunction and cytopaenia. The presence of hepatosplenomegaly, hyperferritinaemia, hypofibrinogenaemia and phagocytosis by macrophages in the bone marrow was consistent with a diagnosis of haemophagocytic lymphohistiocytosis (HLH). We suspected that HLH was induced by pre-existing tuberculosis, and antitubercular agents were started. Positive nucleic acid amplification and sputum culture for Mycobacterium tuberculosis resulted in a diagnosis of pulmonary tuberculosis. The patient improved with three months of treatment. In this patient, manifestations of HLH preceded those of pulmonary tuberculosis. A diagnosis of HLH should increase suspicion of disseminated tuberculosis and thus contribute to early detection.
749	Natural Killer Cell Degranulation Defect: A Cause for Impaired NK-Cell Cytotoxicity and Hyperinflammation in Fanconi Anemia Patients.	30949167	Fanconi anemia (FA) is a rare inherited syndrome characterized by progressive bone marrow failure (BMF), abnormal skin pigmentation, short stature, and increased cancer risk. BMF in FA is multifactorial and largely results from the death of hematopoietic stem cells due to genomic instability. Also, inflammatory pathology in FA has been previously reported, however the mechanism is still not clear. In literature, decreased NK-cell count and/or impaired NK-cell activity, along with other immunological abnormalities have been described in FA-patients (1). However, to the best of our knowledge, this is the first report showing a defective degranulation mechanism leading to abnormal NK-cell cytotoxicity in FA-patients, which may explain the development of a hyperinflammatory response in these patients. This may predispose some patients to develop Hemophagocytic lymphohistiocytosis (HLH) which manifests with prolonged fever, progressive cytopenias and organomegaly. Early diagnosis and initiation of immunosuppressive therapy in these patients will help to better manage these patients. We also propose FA genes to be listed as a cause of familial HLH.
750	Severe cytokine release syndrome resulting in purpura fulminans despite successful response to nivolumab therapy in a patient with pleomorphic carcinoma of the lung: a case report.	30944043	Immune checkpoint inhibitors (ICIs) have provided more options in the treatment of lung cancer. However, ICIs can cause several unfavorable reactions generally referred to as immune-related adverse effects.In this report, we present the case of a 52-year-old woman with successful regression of pleomorphic carcinoma of the lung following nivolumab therapy. She developed purpura fulminans (PF) ultimately resulting in amputation of both lower extremities. Blood tests revealed thrombocytopenia with increased serum soluble IL-2 receptor, ferritin, and triglyceride levels suggesting hemophagocytic lymphohistiocytosis (HLH). In addition, serum A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 activity was decreased, suggesting thrombotic thrombocytopenic purpura (TTP). Further detailed analysis revealed severe hypercytokinemia including increased levels of IL-1β, IL-6, IL-10, TNFα, IFNγ, and G-CSF.The severe systemic inflammatory reaction and impaired peripheral circulation in this patient was attributed to excessive immunological effect induced by nivolumab resulting in cytokine release syndrome (CRS). This is the first report of a patient with multiple pathological conditions including HLH, TTP-like condition, and PF presumably arising from ICI-induced CRS. Further accumulating thoroughly investigated cases would lead to better understanding of the disease and development of reliable cancer immunotherapy.
751	Hemophagocytic Lymphohistiocytosis (HLH) with Unusual Trigger.	30935194	NaN
752	Infections and HLH - Experience from a Tertiary Care Centre.	30935175	NaN
753	Epstein-Barr Virus-Associated T and NK-Cell Lymphoproliferative Diseases.	30931288	EBV-associated T and NK-cell lymphoproliferative diseases (EBV-T/NK LPDs) are characterized by the transformation and proliferation of EBV-infected T or NK cells. The 2016 revised World Health Organization classification recognizes the following EBV-positive lymphoproliferative disorders (LPD): chronic active EBV infection (CAEBV) of T- and NK-cell type (cutaneous and systemic forms), systemic EBV-positive T-cell lymphoma of childhood, aggressive NK-cell leukemia, extranodal NK/T-cell lymphoma, nasal type, and the new provisional entity primary EBV-positive nodal T/NK-cell lymphoma. EBV-associated hemophagocytic lymphohistiocytosis (HLH), although not included in the WHO classification because it is a reactive, inflammatory disease, is included in this review because it can be life-threatening and may have overlapping features with other EBV+ T/NK LPDs. EBV+ T/NK LPDs are rare diseases difficult to diagnose and manage properly, because some LPDs have unusual presentations, and discrepancies between clinical and histological findings might be encountered. Furthermore, EBV+ T/NK disorders share some clinico-pathological features, and may evolve into other categories during the clinical course, including malignant transformation of CAEBV. Here, we review the EBV+ T/NK LPDs in terms of their definitions, clinical features, histology, immunophenotype, molecular findings, and pathogenesis. This review aims to increase our understanding and awareness of the differential diagnosis among the different EBV+ T/NK LPDs. New insights into the genetic characteristics of these disorders will also be discussed.
754	Refractory macrophage activation syndrome in the setting of adult-onset Still disease with hemophagocytic lymphohistiocytosis detected on skin biopsy treated with canakinumab and tacrolimus.	30927277	A 19-year-old Caucasian female with adult-onset Still disease (AOSD) presented for evaluation of an acute clinical decompensation and atypical annular papules and plaques with purpura on the lower extremities. A punch biopsy demonstrated histiocytes with engulfed degenerated erythrocytes and lymphocytes, consistent with hemophagocytic lymphohistiocytosis (HLH). HLH, clinically referred to as macrophage activation syndrome, is a rare complication of AOSD and is life-threatening. Relevant clinical, laboratory, and histologic features of this diagnosis are reviewed.
755	The Transcription Factors TFEB and TFE3 Link the FLCN-AMPK Signaling Axis to Innate Immune Response and Pathogen Resistance.	30917316	TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK confers pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, whereas ablation of total AMPK activity abolishes this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages is observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status with innate immunity.
756	The inhibitory receptors on NK cells and CTLs are upregulated in adult and adolescent patients with secondary hemophagocytic lymphohistiocytosis.	30914280	Hemophagocytic lymphohistiocytosis (HLH) includes primary HLH (pHLH) and secondary HLH (sHLH). Mutations that cause abnormal functions in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) are frequently identified in pHLH. However, why NK cells and CTLs exhibit abnormal functions in sHLH remains unclear. Here, we demonstrated that NK cells in sHLH exhibited a high expression of inhibitory receptor NKG2A and a low expression of activating receptor NKG2D. Besides, the expression of HLA-E on lymphocyte, the adaptor of NKG2A on NK cells, was elevated in sHLH. Moreover, CTLs in sHLH patients expressed a higher level of functional exhaustion markers PD-1, TIM-3 and LAG-3 as well as a lower secretion of IFN-γ and CD107a upon stimulation. In addition, the expression of MHC-I on lymphocytes was decreased. Taken together, our study indicates a potentially pathological mechanism of sHLH and may open up new avenues for the development of immunotherapies against sHLH.
757	Human ehrlichiosis at a tertiary-care academic medical center: Clinical associations and outcomes of transplant patients and patients with hemophagocytic lymphohistiocytosis.	30913447	Ehrlichiosis is an acute febrile tick-borne disease which can rarely be a trigger for secondary hemophagocytic lymphohistiocytosis (HLH).We reviewed our experience with Ehrlichia infections at a tertiary-care academic medical center.Over 10 years, 157 cases of ehrlichiosis were identified. Ten patients (6.4%) had infection with E. ewingii, 7(4.5%) of whom were transplant patients as compared to 3(1.9%) non-transplant patients (p = .035). Transplant patients were more likely to have leukopenia and elevated creatinine compared to immunocompetent patients; length of hospital stay and early mortality were not different between the two groups. Ten patients met the HLH-2004 diagnosis criteria, which could be an underestimation of HLH occurrence as most patients were not completely evaluated for these criteria. We calculated the H-Score to find the probability of HLH; 25 patients scored high making the occurrence rate of HLH at least 16%. Ehrlichia-induced HLH patients (N = 25) had more anemia, thrombocytopenia, elevated creatinine and AST. Moreover, they had a significantly longer hospital stay (median 9 days) compared to patients without HLH (median 4 days) (p = .006).Ehrlichia-induced HLH is a potential serious complication with relatively high occurrence rate; patients manifest severe disease with end-organ damage requiring longer hospital stay.
758	Western blot analysis of the autophagosomal membrane protein LGG-1/LC3 in Caenorhabditis elegans.	30910027	Autophagy is being studied intensively in Caenorhabditis elegans in the context of protein homeostasis and aging. However, in contrast to the yeast and mammalian autophagosomal membrane proteins Atg8 and LC3, lipidation of the C. elegans ortholog LGG-1 with phosphatidylethanolamine has rarely been investigated by western blotting. We attribute this shortcoming to technical problems with separating the nonlipidated from the lipidated LGG-1 protein by gel electrophoresis. Our new protocol for Western blot analysis is applicable for both the detection of transgenic and endogenous LGG-1 proteins and provides a quantifiable method to assess autophagic flux. As a proof of principle, we use this assay to analyze the role of the transcriptional master regulator HLH-30/TFEB in starvation-induced autophagy.
759	Pathogenic Gene Mutations or Variants Identified by Targeted Gene Sequencing in Adults With Hemophagocytic Lymphohistiocytosis.	30899265	Hemophagocytic lymphohistiocytosis (HLH) can be classified into primary HLH and secondary HLH. Primary HLH usually occurs in infants and children with an underlying genetic defect, and there are also teens and occasional adults with primary HLH. Most cases with secondary HLH are adult patients with secondary triggers including infections, malignancies, and autoimmune diseases. The distinction between primary HLH and secondary HLH seems to be less straightforward, as patients with secondary HLH may also have genetic defects while primary HLH can be triggered by secondary causes. In this study, using amplicon-based targeted gene sequencing (TGS), we sequenced eighteen HLH-related genes in 112 adult HLH cases, which were mostly secondary HLH. Mutations or rare variants were identified in 48 cases (42.9%). All the variants except one were missense variants, and biallelic gene mutations were identified in 3 cases in which only one case harbored homogenous missense mutation. Recurrent variants including UNC13D p.G863D and AP3B1 p.T359A are much more prevalent in our cohort than in normal East Asian population, and in silico analysis predicted pathogenicity of these variants. In conclusion, according to our study, genetic defects may also contribute to the development of adult HLH cases or secondary HLH cases.
760	Effect of Serum Deprivation Stress on Signal Induction Regulatory Protein-Alpha (SIRP-Alpha)-Mediated Erythrophagocytosis by Macrophages.	30894504	BACKGROUND Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome that involves loss of macrophages' self-cells recognition resulting in auto-phagocytosis of erythrocytes, leukocytes, and platelets and leading to multi-system effects. The pathogenesis of HLH is unclear but can be explained by malfunction of the physiologic inhibitory pathway through interaction between macrophage SIRP-alpha and erythrocyte CD 47. The goal of the present study was to evaluate if erythrocytes phagocytosis occurs as a result of altered macrophage SIRP-alpha expression during inflammatory/stressful conditions as seen in HLH. MATERIAL AND METHODS RAW264.7 macrophages were cultured in serum-free media (SFM) and complete media (CM) to simulate stressful and physiologic conditions, respectively. CD47+ mouse erythrocytes were used to test interactions with macrophages at different stages. SIRP-alpha expressions and phagocytosis assays were measured and analyzed at different steps. The study was in vitro and used murine cells to simulate in vivo human interactions. RESULTS SIRP-alpha expressions and phagocytosis rates were higher in SFM compared to CM. Interestingly, after adding SIRP-alpha blocking antibodies (Ab), phagocytosis rates significantly decreased. CONCLUSIONS Serum deprivation and LPS/INF-Gamma induction resulted in increased SIRP-alpha expression and erythrophagocytosis. Using SIRP-alpha Ab during this condition decreased the rate of erythrophagocytosis, which indicates that SIRP-alpha receptor can have pro-phagocytic activity.
761	Cross-regulation of defective endolysosome trafficking and enhanced autophagy through TFEB in UNC13D deficiency.	30892133	Several lines of evidence support the occurrence of cross-regulation between the endocytic pathway and autophagy, but the molecular mechanisms regulating this process are not well-understood. Here, we show that the calcium sensor UNC13D regulates the molecular mechanism of late endosomal trafficking and endosomal maturation, and defects in UNC13D lead to macroautophagy upregulation. unc13d-null cells showed impaired endosomal trafficking and defective endocytic flux. The defective phenotypes were rescued by the expression of UNC13D but not by its STX7-binding-deficient mutant. This defective endosomal function in UNC13D-deficient cells resulted in increased autophagic flux, increased long-lived protein degradation, decreased SQSTM1/p62 protein levels and increased autolysosome formation as determined by biochemical, microscopy and structural methods. The autophagic phenotype was not associated with increased recruitment of the UNC13D-binding proteins and autophagy regulators, RAB11 or VAMP8, but was caused, at least in part, by TFEB-mediated upregulation of a subset of autophagic and lysosomal genes, including Atg9b. Downregulation of TFEB decreased Atg9b levels and decreased macroautophagy in unc13d-null cells. UNC13D upregulation corrected the defects in endolysosomal trafficking and decreased the number of accumulated autophagosomes in a cellular model of the lysosomal-storage disorder cystinosis, under both fed and starvation conditions, identifying UNC13D as an important new regulatory molecule of autophagy regulation in cells with lysosomal disorders. Abbreviations ACTB: actin, beta; CTSB: cathepsin B; EEA1: early endosome antigen 1; ESCRT: endosomal sorting complex required for transport; FHL3: familial hemophagocytic; lymphohistiocytosis type 3; HEX: hexosaminidase; HLH: hemophagocytic lymphohistiocytosis; LSD: lysosomal storage disorder; MEF: mouse embryonic fibroblast; SEM: standard errors of the mean; SNARE: soluble n-ethylmaleimide-sensitive-factor attachment receptor; STX: syntaxin; SYT7: synaptotagmin VII; TFE3: transcription factor E3; TFEB: transcription factor EB; TIRF: total internal reflection fluorescence ULK1: unc-51 like kinase 1; UNC13D: unc-13 homolog d; VAMP: vesicle-associate membrane protein; WT: wild-type.
762	Diagnostic Dilemma of Disseminated Histoplasmosis Mimicking Hemophagocytosis Lymphohistiocytosis in Patient with Rheumatoid Arthritis on Anti-TNF Therapy: Case Report and Review of the Literature.	30891325	Tumor necrosis factor inhibitors (TNFi) have become the cornerstone for the treatment of rheumatoid arthritis and other systemic autoimmune conditions. However, these biologic DMARDs can lead to various opportunistic infections such as viral infection, tuberculosis, and histoplasmosis. Furthermore, these biologics can also cause severe systemic inflammatory reactions known as hemophagocytosis lymphohistiocytosis (HLH) that can lead to multiorgan failure and high mortality. Due to overlapping clinical features and time-intensive microbiological culture methods, distinguishing between HLH and opportunistic infections can be challenging early in the disease course. We present a similar situation with our patient where the patient met the diagnostic criteria for HLH however was found to have disseminated histoplasmosis. This case uniquely evaluates the utility of the HLH diagnostic criteria and hemophagocytosis for accurate diagnosis of HLH.
763	Hemophagocytic Lymphohistiocytosis in Pregnancy: A Case Series and Review of the Current Literature.	30891322	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease that can be fatal in pregnancy. We report two cases of severe HLH that highlight etoposide use in pregnancy.28-year-old G2P1 with lupus presented at 18 weeks with acute hypoxic respiratory failure, hepatic dysfunction, leukopenia, thrombocytopenia, and elevated ferritin. Bone marrow biopsy confirmed HLH. Etoposide and corticosteroid treatment was initiated per HLH protocol; however clinical status declined rapidly. Fetal demise occurred at 21 weeks and she subsequently suffered a massive cerebral vascular accident. She was transitioned to comfort measures and the patient deceased.37-year-old G4P3 presented at 25 weeks with fever, acute liver failure, thrombocytopenia, and elevated ferritin. HLH treatment was initiated, including etoposide, and diagnosis confirmed with liver biopsy. Fetal growth restriction was diagnosed at 27 weeks. Delivery occurred at 37 weeks. The neonate was found to be CMV positive despite negative maternal serology.The addition of etoposide to corticosteroid use is a key component in HLH treatment of nonpregnant individuals. While this is usually avoided in pregnancy, the benefit to the mother may outweigh the potential harm to the fetus in severe cases and it should be strongly considered.
764	Severe skin disease in lupus associated with hemophagocytic lymphohistiocytosis: case reports and review of the literature.	30886995	Hemophagocytic lymphohistiocytosis (HLH) is a severe clinical entity associated with high mortality in the adult population. HLH has been associated with infections, malignancy and autoimmune conditions such as Systemic Lupus Erythematosus (SLE), however this is often in the context of a disease flare. Currently, there are limited reports of inaugural SLE manifesting as HLH with a lack of consensus on treatment and management of these patients.Here, we present two rare case reports of severe cutaneous manifestation of lupus associated with HLH. Both patients presented with sinister clinical courses with primarily rheumatologic complaints including malaise, arthralgia, and myalgia with biochemical abnormalities. Both patients were diagnosed with HLH as a result of first presentation from cutaneous lupus. A comprehensive literature review using the PubMed database with cases comprising keywords of HLH and SLE up to September 2017 was conducted, with an emphasis on inaugural cutaneous SLE cases.Ultimately, we highlight that a keen clinical acumen is required as misdiagnosis may lead to insufficient treatment with adverse clinical outcomes with the unique presentation of HLH from inaugural cases of SLE.
765	Hepatitis A infection related haemophagocytic syndrome: a case report and systematic review.	30871416	Haemophagocytic lymphohistiocytosis (HLH) is a clinical syndrome of excessive inflammation and tissue destruction owing to abnormal immune activation. We report an unusual case of haemophagocytosis associated with hepatitis A virus (HAV) infection in a 21-year-old man. This was further complicated by haemolysis secondary to G-6-PD deficiency and fungal sepsis. Our patient was treated successfully with intravenous immunoglobulin (IVIg) and supportive care. A systematic review of all reported cases of HAV associated haemophagocytosis is presented.
766	Secondary Hemophagocytic Lymphohistiocytosis: A Challenging Diagnosis in a Patient with Autoimmune Hepatitis.	30863647	We describe a case of secondary Hemophagocytic Lymphohistiocytosis (HLH) from autoimmune hepatitis mimicking severe sepsis in a man admitted to the intensive care unit.A 34-year-old Pakistani male with a prior history of biopsy-proven autoimmune hepatitis presented to a regional hospital with severe fever, cytopenias, hyperferritinemia, hypertriglyceridemia, splenomegaly, and a bone marrow biopsy showing hemophagocytosis. After ruling out mimicking conditions, a diagnosis of HLH was made using the HLH-2004 diagnostic criteria. He was treated with dexamethasone and etoposide, without bone marrow transplantation (BMT) due to poor functional status. At one-year after follow-up, he had returned to his baseline functional status without recurrence.We describe a rare case of secondary HLH in the setting of autoimmune hepatitis. Broadly, this case report educates clinicians to consider this potentially missed diagnosis. This case also informs clinicians that treatment of secondary HLH with BMT may not be necessary for the management of secondary HLH due to autoimmune hepatitis. Finally, it provides a detailed description of the natural history of a single patient with secondary HLH due to autoimmune hepatitis.
767	The Performance of Diagnostic Criteria for Hemophagocytic Lymphohistiocytosis in Critically Ill Patients.	30862243	The diagnostic criteria for secondary hemophagocytic lymphohistiocytosis (HLH) have not been validated in the critically ill adult population. We set out to evaluate the performance of diagnostic criteria and determine the ferritin cutoff in critically ill adults.A retrospective single-center study.Patients admitted to intensive care unit between 2008 and March 2010. Data were collected on consecutive patients who had ferritin measured. Charts were reviewed for the diagnostic criteria of HLH and components of Hscore.A total of 445 patients had a ferritin level measured during the study period. A diagnosis of HLH was made for 10 patients. Having 5 of 6 criteria had a specificity of 97% and a sensitivity of 70%. Hemophagocytosis was found in 41 (47.1%) of 87 bone marrow biopsies. Two hundred thirty-one patients had a ferritin level above 500 ng/dL. When determining the odds of HLH being clinically diagnosed, the optimal cut point for ferritin was 1197 ng/dL. When determining the odds of HLH based on the Hscore, the best cutoff was 143.5 (sensitivity of 90% and specificity of 90%) and patients who had HLH in our study population had an Hscore of 203.8 ± 64.9.In this cohort of critically ill patients, the HLH criteria are specific for HLH but not sensitive. Critically ill patients can have a higher incidence of hemophagocytosis without HLH. A higher ferritin cutoff in combination with 5 other clinical criteria is comparable to the Hscore for the recognition of HLH in the critically ill population.
768	Expression analysis of Inhibitor Of DNA Binding 1 (ID-1) gene in breast cancer.	30856107	ID-1 gene codes for a helix-loop-helix (HLH) protein that inhibits the DNA binding and transcriptional activation function of these proteins.We analyzed ID-1 expression in microarray and RNA Sequencing databases as well as 61 breast cancer tissues compared with adjacent non-cancerous tissues (ANCTs).Expression analysis of ID-1 gene in two microarray datasets and RNA sequencing data showed down-regulation of ID-1 in tumoral tissues compared with normal tissues. However, ID-1 expression analysis in tumoral tissues and ANCTs obtained from 61 patients revealed its over-expression in tumoral tissues. A negative association was detected between ID-1 expression levels and ER status.ID-1 expression may be implicated in the pathogenesis of breast cancer especially in patient with ER negative status.
769	Hemophagocytic lymphohistiocytosis as a harbinger of aggressive lymphoma: a case series.	30850926	Hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome, which can manifest either secondary to a variety of underlying causes, or due to a primary genetic defect. Malignancy is the most common underlying disease in adults with HLH, with lymphomas being the most common malignancy. Lymphoma-associated hemophagocytic syndrome (LAHS) typically follows a rapidly progressive clinical course and is associated with poor prognosis. We herein present four patients with HLH associated with aggressive lymphoma. At initial presentation, the underlying etiology of the HLH was unclear. Two patients were eventually diagnosed with anaplastic large cell lymphoma, while the other two had diffuse large B cell lymphoma. Two of the patients experienced rapid clinical deterioration, one at diagnosis and the other at relapse, and both died prior to diagnosis of lymphoma despite HLH-directed therapy. These cases highlight the need for intensive management in adults with HLH without a clear etiology, especially in cases when lymphoma-associated HLH is suspected. We describe the current pitfalls in diagnosis and treatment of LAHS and discuss possible ways to improve patient management.
770	Rare cause of Hemophagocytic Lymphohistiocytosis due to mutation in PRF1 and SH2D1A genes in two children - a case report with a review.	30849948	Hemophagocytic Lymphohistiocytosis (HLH) is a rare, complex, life-threatening hyper-inflammatory condition due to over activation of lymphocytes mediated secretory cytokines in the body. It occurs as a primary HLH due to genetic defect that mostly occurs in the childhood and associated with early neonatal death. Secondary HLH is triggered by secondary to infection and can occur at any age.The current report presents two cases of HLH. Case 1, three-months-old boy born to second degree consanguineous parents was clinically suspected with HLH. A pathogenic variant in exon 2 of PRF1 gene [c.386G > C (p.Trp129Ser); FLH-type2] was detected. The parents and the fetus under investigation were shown to be heterozygous carriers, while Case-1 was homozygous for the said variant. Case 2, a one and half-year old male child referred for work-up was born to non-consanguineous young parents. His HLH suspicion was in accordance with HLH-2004 Revised diagnostic guidelines (fulfilling 5/8 criteria). Molecular study revealed hemizygous likely pathogenic variant c.138-3C > G in intron 1 of SH2D1A gene. Both the mother and younger sister were confirmed to be the carrier of the same variant.This study has represented two rare cases of HLH carrying missense variant in PRF1 and splice site variant in SH2D1A gene. Detailed molecular analysis has helped the families with precise genetic counselling and prenatal diagnosis during subsequent pregnancy. It is advocated that male patients presenting with EBV-associated HLH may be screened for XLP that may lead to early diagnosis and therapeutic implication if any.
771	[Cytomegalovirus meningoencephalitis in a diffuse large B-cell lymphoma patient undergoing salvage chemotherapy].	30842379	A 63-year-old woman was admitted to our hospital to receive a fourth course of modified rituximab-ESHAP chemotherapy for relapsed primary breast diffuse large B-cell lymphoma. She developed hemophagocytic lymphohistiocytosis (HLH) 20 days after admission. Polymerase chain reaction (PCR) detected cytomegalovirus (CMV) DNA in her peripheral blood; therefore, she was diagnosed with CMV-associated HLH and consequently treated with foscarnet (FCN). Her general condition and pancytopenia soon improved, and the antiviral drug was stopped for 1 week. However, she suddenly became disoriented 10 days later, and this condition rapidly worsened. Cerebrospinal fluid (CSF) examination revealed an elevated white blood cell count with lymphocytic predominance and a high CMV DNA load, prompting a final diagnosis of CMV meningoencephalitis. We began intravenous combination therapy with FCN and ganciclovir (GCV), and her conscious state gradually improved. CMV DNA sequencing did not reveal drug resistance associated with mutations, and intravenous GCV was stopped for 1 week. FCN treatment was then continued until CMV DNA was no longer detected in her CSF samples via PCR. CMV meningoencephalitis is a rare neurological infection complicated with hematological malignancy in non-transplant patients and can be serious and life-threatening with a high mortality rate. This infection requires a differential diagnosis of consciousness impairment that develops in a patient with lymphoid malignancy during chemotherapy.
772	A Case of Congenital Anaplastic Large Cell Lymphoma in a Very Preterm Low-Birth Weight Neonate.	30830032	A premature infant male was born at 30 weeks' gestation with severe coagulopathy and thrombocytopenia. Over the first days of his life, the patient developed evidence of immune hyperactivation with adenopathy, hepatosplenomegaly, and elevated ferritin. Although the patient met diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH), flow cytometric based assays were not consistent with primary HLH. A lymph node and bone marrow biopsy eventually revealed the presence of anaplastic lymphoma kinase+anaplastic large cell lymphoma. To our knowledge, this is the earliest presentation of a lymphoma, and expands the known timeframe of lymphomagenesis.
773	Critically Severe Case of Neonatal Herpes with High Viral Load and Hemophagocytic Syndrome.	30828029	Neonatal disseminated herpes simplex virus (HSV) infection is a severe disease with high mortality and morbidity; yet, the pathophysiology remains unclear. Here, we report a male infant with disseminated HSV type 1 (HSV-1) infection, complicated by hemophagocytic lymphohistiocytosis (HLH) and multiple organ failure. The infant, born at 39 weeks of gestation by normal delivery, developed fever (38.5˚C) with the high serum C-reactive protein levels on the 1st day of life, and exhibited tachypnea on the 3rd day. On the 5th day of life, the patient received mechanical ventilation and was transferred to our neonatal ICU. Real-time PCR for HSV-1 DNA revealed an extremely high serum concentration (1.0 × 109 copies/µL), and he was diagnosed with HSV-1 infection. Acyclovir (ACV) and corticosteroid pulse therapies with methylprednisolone were started. Continuous hemodiafiltration (CHDF) using cytokine-absorbing hemofilters was also initiated because of renal failure. These therapies, however, failed to control the disease, and the patient died on the 41st day of life. The dose of ACV on CHDF might not be adequate, although we could not measure the serum ACV concentrations. After the patient's death, we measured his serum cytokine concentrations taken four times during the clinical course. Serum concentrations of interleukin (IL)-6, IL-10, IL-1β, and interferon (IFN)-γ were elevated at the time of admission and were remarkably decreased by 10 days after treatment. In particular, the concentrations of IL-1β and IFN-γ were lower than the measurable ranges. It is therefore important to measure serum cytokine concentrations in real time to prevent excessive immune suppression.
774	Secondary hemophagocytic lymphohistiocytosis in pediatric patients: a single center experience and factors that influenced patient prognosis.	30821552	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of excessive immune activation. Secondary HLH syndrome develops as a complication of infection, drugs, rheumatologic conditions, or malignancy. The main objectives of this work were to identify the etiology of secondary HLH and prognostic factors associated with mortality. Patients diagnosed with secondary HLH, between January 2011 and December 2016, were retrospectively included in this study. We analyzed clinical and laboratory findings as well as prognostic factors from 24 pediatric patients diagnosed with secondary HLH. The mean age of patients at the time of diagnosis was 79.9 ± 68.7 months (range: 2-202) and 54.2% of the patients were male. The most frequent HLH-2004 criterion was fever (100%). Underlying triggers of HLH were as follows: 13 (54.1%) infections, juvenile idiopathic arthritis in 5 patients (20.8%), drugs in 3 patients (12.5%), malignancies in 2 (0.8%), Kawasaki disease in 1 (0.4%) patient, and 1 (0.4%) with unknown triggers. The median time of diagnosis was 3 days (1-67 days). Overall, the mortality rate was 20.8%. In our logistic regression model, factors associated with mortality were decreased albumin levels (OR1 = 2.3[1.48-3.43]) and etoposide usage (OR2 = 1.22 [1.14-1.89]). The patient's 30-day survival was inferior among patients whose albumin level was 2 g/dL or less compared to those over 2 g/dL. Increased awareness of the underlying condition is critical in HLH patients. Our study emphasizes the prognostic significance of albumin level.
775	Posttransplant Hemophagocytic Lymphohistiocytosis Driven by Myeloid Cytokines and Vicious Cycles of T-Cell and Macrophage Activation in Humanized Mice.	30814997	Hemophagocytic lymphohistiocytosis (HLH) has recently been increasingly reported as an important complication after stem cell transplantation, in line with the increase in the number of HLA-mismatched transplantation. Although previous clinical studies have shown an elevation of inflammatory cytokines in patients with HLH after hematopoietic stem cell transplantation, as well as those after viral infection or autoimmune disease, the disease pathogenesis remains poorly understood. Here we explored this issue in humanized mice with functional human lymphohematopoietic systems, which were constructed by transplantation of human CD34+ cells alone, or along with human fetal thymus into NOD/SCID/γc-/- (NSG) or NSG mice carrying human SCF/GM-CSF/IL-3 transgenes (SGM3). In comparison with humanized NSG (huNSG) mice, huSGM3 mice had higher human myeloid reconstitution and aggressive expansion of human CD4+ memory T cells, particularly in the absence of human thymus. Although all huNSG mice appeared healthy throughout the observation period of over 20 weeks, huSGM3 mice developed fatal disease characterized by severe human T cell and macrophage infiltrations to systemic organs. HuSGM3 mice also showed severe anemia and thrombocytopenia with hypoplastic bone marrow, but increased reticulocyte counts in blood. In addition, huSGM3 mice showed a significant elevation in human inflammatory cytokines including IL-6, IL-18, IFN-α, and TNF-γ, faithfully reproducing HLH in clinical situations. Our study suggests that posttransplant HLH is triggered by alloresponses (or xenoresponses in our model), driven by myeloid cytokines, and exacerbated by vicious cycles of T-cell and macrophage activation.
776	Hyperacidification of Citrus fruits by a vacuolar proton-pumping P-ATPase complex.	30808865	The sour taste of Citrus fruits is due to the extreme acidification of vacuoles in juice vesicle cells via a mechanism that remained elusive. Genetic analysis in petunia identified two vacuolar P-ATPases, PH1 and PH5, which determine flower color by hyperacidifying petal cell vacuoles. Here we show that Citrus homologs, CitPH1 and CitPH5, are expressed in sour lemon, orange, pummelo and rangpur lime fruits, while their expression is strongly reduced in sweet-tasting "acidless" varieties. Down-regulation of CitPH1 and CitPH5 is associated with mutations that disrupt expression of MYB, HLH and/or WRKY transcription factors homologous to those activating PH1 and PH5 in petunia. These findings address a long-standing enigma in cell biology and provide targets to engineer or select for taste in Citrus and other fruits.
777	OsHLH61-OsbHLH96 influences rice defense to brown planthopper through regulating the pathogen-related genes.	30796564	In plants, basic helix-loop-helix (bHLH) proteins form the largest transcription factor (TF) family. Among them, HLH proteins are a small group of atypical members that lack the basic domain, and form dimers with bHLH proteins. Although bHLH proteins have been proved to play important roles in plant development and physiology, the function of HLH proteins is rarely studied, not to mention in plant biotic resistance. Brown planthopper (BPH) is a kind of rice-specific insect that causes devastating yield losses each year.In this study, we identified OsHLH61 gene that encodes HLH protein. OsHLH61 gene could be highly induced by BPH infestation. Furthermore, Methyl Jasmonic acid (Me-JA) and cis-12-oxo- phytodienoic acid (OPDA) induced expression of OsHLH61, while SA repressed it. We knocked down expression of OsHLH61 by RNA interference (RNAi), the transgenic plants were susceptible to BPH infestation. RNA-seq analysis revealed that some pathogen-related (PR) genes in the Salicylic acid (SA) signaling pathway that mediate plant immunity were obviously down-regulated in the OsHLH61 RNAi plants. Meanwhile, yeast two-hybrid assay and bimolecular luciferase complementation (BiLC) analysis identified bHLH096 to be an interacting factor of OsHLH61. Also, some PR genes were down-regulated in the OsbHLH96 over expressing lines. Expression of OsbHLH96 was inhibited. Besides, OsbHLH96 might interact with Jasmonate Zim-Domain3 (OsJAZ3).Altogether, we identified an OsHLH61-OsbHLH96 complex that might mediate defense to BPH through regulating PR genes. And OsHLH61-OsbHLH96 might be important in mediating SA and JA signaling crosstalk.
778	Specific and Fuzzy Interactions Cooperate in Modulating Protein Half-Life.	30790629	Protein degradation is critical for maintaining cellular homeostasis. The 20S proteasome is selective for unfolded, extended polypeptide chains without ubiquitin tags. Sequestration of such segments by protein partners, however, may provide a regulatory mechanism. Here we used the AP-1 complex to study how c-Fos turnover is controlled by interactions with c-Jun. We show that heterodimerization with c-Jun increases c-Fos half-life. Mutations affecting specific contact sites (L165V, L172V) or charge separation (E175D, E189D, K190R) with c-Jun both modulate c-Fos turnover, proportionally to their impact on binding affinity. The fuzzy tail beyond the structured b-HLH/ZIP domain (~165 residues) also contributes to the stabilization of the AP-1 complex, removal of which decreases c-Fos half-life. Thus, protein turnover by 20S proteasome is fine-tuned by both specific and fuzzy interactions, consistently with the previously proposed "nanny" model.
779	Factors predicting the recurrence of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children after treatment using the HLH-2004 protocol.	3078872530977106	Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) is highly prevalent in Japan. To date, no standard treatment for EBV-HLH has been established owing to the diversity in treatment response and the difficulty in assessing prognostic factors. The present prospective study recruited 27 children with EBV-HLH who were also part of the HLH-2004 study. EBV load in the peripheral blood was monitored at diagnosis and 2, 4, and 8 weeks after treatment initiation. Additionally, T-cell receptor (TCR) clonality and other laboratory data were evaluated. TCR clonality was positive in 14 patients at diagnosis. Seven of 27 patients experienced recurrences after treatment. No correlation was noted among any clinical data at diagnosis of patients with and without recurrence. However, the recurrence rate was significantly higher in patients aged < 2 years and/or those with a high plasma EBV load of > 103 copies/mL 2 weeks after treatment than that in patients without these factors. These findings suggest that a younger age or a high EBV load in plasma at the early phase of treatment is a factor predicting a recurrence and helps guide the intensity of subsequent treatment phases for children with EBV-HLH.
780	Hemophagocytic Lymphohistiocytosis (HLH) Triggered by Wasp Venom Immunotherapy Is Rare but Noteworthy.	30785100	NaN
781	IFN-g:IL-10 Ratio: a Putative Predictive Biomarker to Discriminate HLH From Severe Viral Infections.	30783923	NaN
782	Novel mutations of STXBP2 and LYST associated with adult haemophagocytic lymphohistiocytosis with Epstein-Barr virus infection: a case report.	30782130	Haemophagocytic lymphohistiocytosis is a life-threatening disease resulting from primary or secondary hyper-inflammatory disorders. The typical symptoms include persistent fever, splenomegaly, cytopenia and significant elevation of serum ferritin.We report a 30-year-old Chinese female patient who was diagnosed with chronic active Epstein-Barr virus infection more than 9 months prior and has since been presenting with cutaneous lymphoproliferative disorders mimicking hydroa vacciniforme and subsequent haemophagocytic lymphohistiocytosis. Exome sequencing suggested novel digenic heterozygous STXBP2 (c.592A > C, p.Thr198Pro) and LYST (c.830A > T, p.His277Leu) mutations.This is the first case report in which adult HLH was associated with novel digenic mutations of STXBP2 and LYST combined with Epstein-Barr virus infection. It could also be the first polygenic model report, given that the pathogenicity of other mutated genes still remains unclear. We additionally conducted an in-depth, two-generation pedigree analysis to further illustrate the mode of inheritance in this case.
783	Haploidentical Stem Cell Transplantation with Post-Transplant Cyclophosphamide for Primary Immune Deficiency Disorders in Children: Challenges and Outcome from a Tertiary Care Center in South India.	30778805	Haploidentical stem cell transplantation (haplo SCT) has emerged as an acceptable alternative to matched family donor transplantation for children diagnosed to have primary immune deficiency disorders (PIDs). We present data over 4 years on the challenges and efficacy of unmanipulated T cell replete haplo SCTs with post-transplant cyclophosphamide (PTCy) in children diagnosed to have PIDs. We performed a retrospective study in the pediatric blood and marrow transplantation unit where all children less than 18 years of age diagnosed to have PIDs and who underwent haplo SCT with PTCy from January 2014 to February 2018 were included in the study. Of the 16 transplants included in the study, 5 children were diagnosed to have Wiskott-Aldrich syndrome, 3 with congenital hemophagocytic lymphohistiocytosis, 2 each with Griscelli syndrome and Mendelian susceptibility to mycobacterial diseases, and one each with Chediak-Higashi syndrome, ORAI 1 mutation immune deficiency, severe combined immune deficiency, and Hyper IgM syndrome. The source of stem cells was PBSC in 62.5% and bone marrow in 32.5%. Engraftment by day 16-21 post hematopoietic stem cell transplantation was achieved in 75% transplants with 91% of these remaining in sustained complete chimerism. Acute skin and gut graft versus host disease of grade 2-3 were noted in 50% transplants and cytomegalovirus (CMV) reactivation in 43.7% transplants. One child with congenital HLH succumbed to refractory CMV, adenovirus, and BK virus infection. Cytokine release syndrome (CRS) was noted in 75% transplants with 2 children succumbing to the illness. Tocilizumab was successfully used early in one child. Overall mortality was found to be 37.5% with overall survival of 62.5% with a median follow-up of 23.3 months. In resource limited settings, PTCy has the potential to provide a cost-effective advantage in terms of accessibility of this curative procedure among children with PIDs.
784	Requirement for etoposide in the treatment of pregnancy related hemophagocytic lymphohistiocytosis: a multicenter retrospective study.	30777105	Hemophagocytic lymphohistiocytosis (HLH) is a rare severe clinical syndrome. HLH manifesting during pregnancy has been paid much attention in recent years. Despite the specificity of pregnancy-related HLH, there has not been any consensus regarding its treatment. According to a previous study, corticosteroid/IVIG is the mainstream therapy; however, the efficacy is controversial. Etoposide is an important agent in the HLH-94 regimen; nevertheless, its use is limited because of possible toxicity to the fetus.In this study, we summarized 13 cases from 4 medical institutions from April 2011 to April 2018. Treatment regimens and outcomes were observed.The median age was 26 (20-36) years old. The median gestational age was 28 (10-35) weeks. In these 13 patients, 10 were treated with methylprednisolone/IVIG and was effective in only two patients. In 6 patients who used etoposide during their treatment, all achieved remission. The median time from onset of disease to use of etoposide was 36 (17-131) days. Five of these 6 patients were treated with corticosteroids with/without IVIG before etoposide. One patient with pulmonary tuberculosis and one with lymphoma were treated according to etiology and achieved long survival.For treatment of pregnancy-related HLH, particularly for patients who do not respond to corticosteroids/IVIG therapy, etoposide should be used bravely. Nevertheless, suitable dosages and toxic and side-effects require further clinical observation.
785	The Immunology of Macrophage Activation Syndrome.	30774631	Synonymous with secondary hemophagocytic lymphohistiocytosis, macrophage activation syndrome (MAS) is a term used by rheumatologists to describe a potentially life-threatening complication of systemic inflammatory disorders, most commonly systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE). Clinical and laboratory features of MAS include sustained fever, hyperferritinemia, pancytopenia, fibrinolytic coagulopathy, and liver dysfunction. Soluble interleukin-2 receptor alpha chain (sCD25) and sCD163 may be elevated, and histopathology often reveals characteristic increased hemophagocytic activity in the bone marrow (and other tissues), with positive CD163 (histiocyte) staining. A common hypothesis as to the pathophysiology of many cases of MAS proposes a defect in lymphocyte cytolytic activity. Specific heterozygous gene mutations in familial HLH-associated cytolytic pathway genes (e.g., PRF1, UNC13D) have been linked to a substantial subset of MAS patients. In addition, the pro-inflammatory cytokine environment, particularly IL-6, has been shown to decrease NK cell cytolytic function. The inability of NK cells and cytolytic CD8 T cells to lyse infected and otherwise activated antigen presenting cells results in prolonged cell-to-cell (innate and adaptive immune cells) interactions and amplification of a pro-inflammatory cytokine cascade. The cytokine storm results in activation of macrophages, causing hemophagocytosis, as well as contributing to multi-organ dysfunction. In addition to macrophages, dendritic cells likely play a critical role in antigen presentation to cytolytic lymphocytes, as well as contributing to cytokine expression. Several cytokines, including tumor necrosis factor, interferon-gamma, and numerous interleukins (i.e., IL-1, IL-6, IL-18, IL-33), have been implicated in the cytokine cascade. In addition to broadly immunosuppressive therapies, novel cytokine targeted treatments are being explored to dampen the overly active immune response that is responsible for much of the pathology seen in MAS.
786	Clinical characteristics of extranodal NK/T-cell lymphoma-associated hemophagocytic lymphohistiocytosis.	30774427	Extranodal natural killer (NK) / T cell lymphoma is a subtype of non-Hodgkin's lymphoma (NHL) that usually has an aggressive clinical course. It is the predominant trigger of lymphoma-associated hemophagocytic syndrome (LAHS), which is highly lethal and with extremely poor prognosis. This study is aiming to characterize the associated clinical features and prognostic factors of the disease.Twenty-eight patients with extranodal NK/T cell lymphoma associated hemophagocytic lymphohistiocytosis (HLH) were retrospectively analyzed. The clinical records were collected, and the associations between clinical or laboratory parameters and overall survival (OS) were assessed.The most frequently clinical characteristics were fever (96.4%), and splenomegaly (81.5%). Concerning the laboratory findings, the most common features were hyperferritinemia (91.7%), grade III/IV thrombocytopenia (64.3%), hypertriglyceridemia (48%), severe anemia (46.4%), hypofibrinogenemia (45%), and grade III/IV neutropenia (32.1%). The interval between the diagnosis of NK/T LAHS and death / last contact was between 4 to 701 days with the median interval of 15 days. We found that higher serum lactic dehydrogenase (LDH) at HLH, hypofibrinogenemia, and splenomegaly were significantly associated with worse survival (P=0.002, 0.003, 0.003). Furthermore, Eastern Cooperative Oncology Group (ECOG) score, extra-upper aerodigestive tract NK/T cell lymphoma (EUNKTL) and cutaneous involvement were risk factors of HLH.Our data indicated that levels of LDH, fibrinogen, and presence of splenomegaly were prognostic factors of the disease. Higher ECOG scores, EUNKTL and cutaneous involvement were risk factors of NK/T LAHS. Additional independent, prospective clinical trials will be needed to explore optimal treatment.
787	Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation.	30770874	In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.
788	Macrophage Activation-Like Syndrome: A Distinct Entity Leading to Early Death in Sepsis.	30766533	Hemophagocytic lymphohistocytosis (HLH) is characterized by fulminant cytokine storm leading to multiple organ dysfunction and high mortality. HLH is classified into familial (fHLH) and into secondary (sHLH). fHLH is rare and it is due to mutations of genes encoding for perforin or excretory granules of natural killer (NK) cells of CD8-lymphocytes. sHLH is also known as macrophage activation syndrome (MAS). Macrophage activation syndrome (MAS) in adults is poorly studied. Main features are fever, hepatosplenomegaly, hepatobiliary dysfunction (HBD), coagulopathy, cytopenia of two to three cell lineages, increased triglycerides and hemophagocytosis in the bone marrow. sHLH/MAS complicates hematologic malignancies, autoimmune disorders and infections mainly of viral origin. Pathogenesis is poorly understood and it is associated with increased activation of macrophages and NK cells. An autocrine loop of interleukin (IL)-1β over-secretion leads to cytokine storm of IL-6, IL-18, ferritin, and interferon-gamma; soluble CD163 is highly increased from macrophages. The true incidence of sHLH/MAS among patients with sepsis has only been studied in the cohort of the Hellenic Sepsis Study Group. Patients meeting the Sepsis-3 criteria and who had positive HSscore or co-presence of HBD and disseminated intravascular coagulation (DIC) were classified as patients with macrophage activation-like syndrome (MALS). The frequency of MALS ranged between 3 and 4% and it was an independent entity associated with early mortality after 10 days. Ferritin was proposed as a diagnostic and surrogate biomarker. Concentrations >4,420 ng/ml were associated with diagnosis of MALS with 97.1% specificity and 98% negative predictive value. Increased ferritin was also associated with increased IL-6, IL-18, IFNγ, and sCD163 and by decreased IL-10/TNFα ratio. A drop of ferritin by 15% the first 48 h was a surrogate finding of favorable outcome. There are 10 on-going trials in adults with sHLH; two for the development of biomarkers and eight for management. Only one of them is focusing in sepsis. The acronym of the trial is PROVIDE (ClinicalTrials.gov NCT03332225) and it is a double-blind randomized clinical trial aiming to deliver to patients with septic shock treatment targeting their precise immune state. Patients diagnosed with MALS are receiving randomized treatment with placebo or the IL-1β blocker anakinra.
789	Calm in the midst of cytokine storm: a collaborative approach to the diagnosis and treatment of hemophagocytic lymphohistiocytosis and macrophage activation syndrome.	30764840	Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) were historically thought to be distinct entities, often managed in isolation. In fact, these conditions are closely related. A collaborative approach, which incorporates expertise from subspecialties that previously treated HLH/MAS independently, is needed. We leveraged quality improvement (QI) techniques in the form of an Evidence-Based Guideline (EBG) to build consensus across disciplines on the diagnosis and treatment of HLH/MAS.A multidisciplinary work group was convened that met monthly to develop the HLH/MAS EBG. Literature review and expert opinion were used to develop a management strategy for HLH/MAS. The EBG was implemented, and quality metrics were selected to monitor outcomes.An HLH/MAS clinical team was formed with representatives from subspecialties involved in the care of patients with HLH/MAS. Broad entry criteria for the HLH/MAS EBG were established and included fever and ferritin ≥500 ng/mL. The rheumatology team was identified as the "gate-keeper," charged with overseeing the diagnostic evaluation recommended in the EBG. First-line medications were recommended based on the acuity of illness and risk of concurrent infection. Quality metrics to be tracked prospectively based on time to initiation of treatment and clinical response were selected.HLH/MAS are increasingly considered to be a spectrum of related conditions, and joint management across subspecialties could improve patient outcomes. Our experience in creating a multidisciplinary approach to HLH/MAS management can serve as a model for care at other institutions.
790	Haemophagocytic Lymphohistiocytosis in a 16-Year-Old Boy.	30755576	Haemophagocytosis encompasses phagocytosis of erythrocytes, leukocytes, platelets and their precursors by macrophages in bone marrow and other tissues. Haemophagocytic lymphohistiocytosis (HLH) usually presents with high fever, pancytopenia, splenomegaly, lymphadenopathy, haemophagocytosis in bone marrow, liver, lymphnodes or CSF. We report coagulase negative Staphylococcus induced HLH in a 16 year old boy presenting with high grade fever, lymphadenopathy, hepatosplenomegaly, pancytopenia with neutropenic sepsis in the department of Medicine, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh on 28th November 2016. He responded to high dose dexamethasone and management of neutropenic sepsis. This might give a guidance in the management of haemophagocytic syndrome at the earliest time to prevent morbidity and mortality.
791	Disseminated CMV infection and HLH in a patient with well-controlled HIV and ulcerative colitis.	30755430	We present a case of haemophagocytic lymphohistiocytosis (HLH) in the context of disseminated cytomegalovirus (CMV) viraemia in a 50-year-old man with well-controlled HIV infection and ulcerative colitis (UC), for which he was receiving azathioprine. Peak CMV viral load was 371 000 copies/ml with evidence of end-organ CMV in the lungs and colon. A bone marrow biopsy showed evidence of haemophagocytosis of platelets, neutrophils and erythrocytes. The azathioprine was stopped, and he received intravenous ganciclovir and corticosteroids with suppression of the CMV viral load and resolution of the HLH.
792	Clinical and endoscopic complications of Epstein-Barr virus in inflammatory bowel disease: an illustrative case series.	30739187	Epstein-Barr virus (EBV) is a proposed trigger in the etiopathogenesis of inflammatory bowel disease (IBD) and is associated with lymphoproliferative diseases. Nevertheless, testing for EBV DNA in the intestinal mucosa and screening for EBV infection before initiation of a drug therapy are not routinely performed. The aim of this article is to increase awareness of the relevance of EBV infection in specific clinical situations.In this short communication, we describe the disease course of three IBD patients with EBV infection, varying from EBV reactivation during disease flare up to a trigger of EBV-related mucocutaneous ulcer (EBV-MCU) and haemophagocytic lymphohistiocytosis (HLH).Our first patient was diagnosed with EBV reactivation-associated severe colitis and showed a rapid clinical improvement after induction therapy with infliximab and azathioprine. Without antiviral treatment, the patient remained in complete remission and no complications of EBV were seen. After diagnosing EBV-MCU in the second patient, immunosuppressive medication was discontinued and four infusions of rituximab resulted in a rapid clinical recovery and eventually complete response. After discontinuation of the immunosuppression in our last patient with haemophagocytic lymphohistiocytosis, treatment with a combination of corticosteroid and antiviral therapy resulted in a complete recovery over a time span of several weeks.EBV infection has a wide variety of potentially life-threatening clinical manifestations in IBD patients. Testing for EBV in case of a flare up and screening for EBV before the start of immunosuppressive therapy will create awareness for EBV-related symptoms or complications during follow-up.
793	Exacerbation of Cushing's syndrome during pregnancy: stimulation of a cortisol-secreting adrenocortical adenoma by ACTH originating from the foeto-placental unit.	30738017	A 29-year-old G4A3 woman presented at 25 weeks of pregnancy with progressive signs of Cushing's syndrome (CS), gestational diabetes requiring insulin and hypertension. A 3.4 × 3.3 cm right adrenal adenoma was identified during abdominal ultrasound imaging for nephrolithiasis. Investigation revealed elevated levels of plasma cortisol, 24 h urinary free cortisol (UFC) and late-night salivary cortisol (LNSC). Serum ACTH levels were not fully suppressed (4 and 5 pmol/L (N: 2-11)). One month post-partum, CS regressed, 24-h UFC had normalised while ACTH levels were now less than 2 pmol/L; however, dexamethasone failed to suppress cortisol levels. Tests performed in vivo 6 weeks post-partum to identify aberrant hormone receptors showed no cortisol stimulation by various tests (including 300 IU hLH i.v.) except after administration of 250 µg i.v. Cosyntropin 1-24. Right adrenalectomy demonstrated an adrenocortical adenoma and atrophy of adjacent cortex. Quantitative RT-PCR analysis of the adenoma revealed the presence of ACTH (MC2) receptor mRNA, while LHCG receptor mRNA was almost undetectable. This case reveals that CS exacerbation in the context of pregnancy can result from the placental-derived ACTH stimulation of MC2 receptors on the adrenocortical adenoma. Possible contribution of other placental-derived factors such as oestrogens, CRH or CRH-like peptides cannot be ruled out. Learning points: Diagnosis of Cushing's syndrome during pregnancy is complicated by several physiological alterations in hypothalamic-pituitary-adrenal axis regulation occurring in normal pregnancy. Cushing's syndrome (CS) exacerbation during pregnancy can be associated with aberrant expression of LHCG receptor on primary adrenocortical tumour or hyperplasia in some cases, but not in this patient. Placental-derived ACTH, which is not subject to glucocorticoid negative feedback, stimulated cortisol secretion from this adrenal adenoma causing transient CS exacerbation during pregnancy. Following delivery and tumour removal, suppression of HPA axis can require several months to recover and requires glucocorticoid replacement therapy.
794	Pediatric Subcutaneous Panniculitis-like T-Cell Lymphoma With Hemophagocytosis Showing Complete Resolution With the BFM90 Protocol: Case Report and Review of Literature.	30730380	Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a distinct subtype of peripheral T-cell lymphoma associated with aggressive clinical behavior. Since its original description, it has continued to be a rare disease, and <200 cases have been reported in literature. We report an 11-year-old boy who presented with SPTCL and hemophagocytic lymphohistiocytosis (HLH) and responded to high-dose multiagent chemotherapy. He presented with steroid refractory erythematous, raised plaques over his face, trunk, and limbs over a period of 15 months treated elsewhere. Repeat evaluation in our center was consistent with SPTCL with features of HLH. He was initiated on therapy with the BFM90 protocol, which led to complete morphologic and biochemical remission. No single-best treatment regimen has been described for SPTCL with HLH in literature, and high-dose chemotherapy has shown good long-term remissions in the literature. The presence of SPTCL with HLH and systemic symptoms should prompt treatment with high-dose multiagent chemotherapy rather than Cyclophosphamide, Vincristine, Adriamycin, Prednisolone-like therapy. BFM90 is one such regimen that is well tolerated, and it can induce significant clinical and biochemical responses.
795	A Review of Neuropathological Features of Familial and Adult Hemophagocytic Lymphohistiocytosis.	30726926	Hemophagocytic lymphohistiocytosis (HLH) is a hematological disorder that can be due to genetic (primary HLH) causes or excessive activation of the immune system in association with infection, malignancy, rheumatologic disorders, or immune suppression (secondary HLH). Hemophagocytic lymphohistiocytosis remains an under-recognized condition among neuropathologists, especially the secondary forms, where it may be diagnosed only at brain biopsy or autopsy due to confounding comorbidities. The CNS is frequently affected, but neuropathological features are underappreciated. We place our own experience with HLH in context with review of neuropathological features from the literature. A 10-year database search for cases from our pediatric and adult hospitals with re-review of neuropathological features revealed 1 biopsy and 5 autopsies. Literature that reported neuropathological features was tabulated and 8 adult and 12 pediatric cases were identified. Children had predominantly secondary HLH: 5/12 co-associated with Epstein Barr (or dual) viral infections, 3/12 with malignancy. One biopsy showed florid lymphohistiocytic infiltrates and hemophagocytosis and served as first diagnosis; 2/5 CNS autopsies had originally been reported as negative for HLH, but on re-review had subtle lymphohistiocytic infiltrates with hemophagocytosis confined to leptomeninges. In conclusion, the neuropathological features are highly variable in HLH; features such as focal erythrophagocytosis may be histologically subtle in early phases, but should be sought.
796	A Unique Case of Severe Anemia Secondary to Copper Deficiency in an Adult Patient.	30723637	Anemia is a frequently encountered problem in the healthcare system. Common causes of anemia include blood loss, followed by impaired red blood cell production and red blood cell destruction. This case demonstrates the need for cognizance of the less frequent causes of anemia. A 27-year-old male with a history of traumatic brain injury and quadriplegia with chronic respiratory failure on home ventilator support presented to the emergency department with dyspnea and no bowel movements for three days. The patient received nutrition via percutaneous endoscopic gastostromy (PEG) tube. He was hypotensive with a mean arterial pressure (MAP) of 54 mm/Hg. There was no evidence of acute or ongoing blood loss. Initial lab data revealed hyperkalemia (K+ 6.1), severe anemia (Hb 1.5 g/dL), leukopenia (2.53 K/uL), neutropenia (ANC 700), and normal platelets. Peripheral smear revealed leukopenia with absolute neutropenia, marked anemia with anisopoikilocytosis with rare dacrocytes but no evidence of schistocytes. He responded to transfusion with improvement in hemoglobin from 1.5 to 9.1 within 24 hours. There was no evidence of hemolysis or vitamin deficiency. Ferritin and triglyceride levels were ordered to rule out hemophagocytic lymphohistiocytosis (HLH). Ferritin was elevated at 6506 ng/mL and triglycerides were 123 mg/dL. Soluble IL-2 receptor level was sent and found to be significantly elevated; however, this was felt to be more likely secondary to infection and inflammation, as the patient had no other clinical features of HLH, apart from cytopenias. Zinc supplementation was part of his wound care regimen. Copper levels were <10 ug/dL (normal: 70-140). Zinc supplements were stopped, and the patient was started on copper supplementation. At his three month follow-up clinic appointment, his anemia and leukopenia had resolved. Micronutrient deficiency is a potential cause of anemia, especially in a risk population and must be considered, as it is often easily correctible.
797	Transcriptome Analysis of Watermelon Leaves Reveals Candidate Genes Responsive to Cucumber green mottle mosaic virus Infection.	30708960	Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus, which cause diseases in cucurbits, especially watermelon. In watermelon, symptoms develop on the whole plant, including leaves, stems, peduncles, and fruit. To better understand the molecular mechanisms of watermelon early responses to CGMMV infection, a comparative transcriptome analysis of 24 h CGMMV-infected and mock-inoculated watermelon leaves was performed. A total of 1641 differently expressed genes (DEGs) were identified, with 886 DEGs upregulated and 755 DEGs downregulated after CGMMV infection. A functional analysis indicated that the DEGs were involved in photosynthesis, plant⁻pathogen interactions, secondary metabolism, and plant hormone signal transduction. In addition, a few transcription factor families, including WRKY, MYB, HLH, bZIP and NAC, were responsive to the CGMMV-induced stress. To confirm the high-throughput sequencing results, 15 DEGs were validated by qRT-PCR analysis. The results provide insights into the identification of candidate genes or pathways involved in the responses of watermelon leaves to CGMMV infection.
798	Identification of a novel CCDC22 mutation in a patient with severe Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis and aggressive natural killer cell leukemia.	30706328	Aggressive natural killer cell leukemia (ANKL) is a rare neoplasm characterized by the systemic infiltration of Epstein-Barr virus (EBV)-associated NK cells, and rapidly progressive clinical course. We report the case of a 45-year-old man with intellectual disability who developed ANKL, and describe the identification of a novel genetic mutation of coiled-coil domain-containing 22 (CCDC22). He presented with persistent fever, severe pancytopenia, and hepatosplenomegary. Following bone marrow aspiration, numerous hemophagocytes were identified. High EBV viral load was detected in NK cells fractionation by qPCR. The initial diagnosis was EBV-related hemophagocytic lymphohistiocytosis (EBV-HLH). A combination of immunosuppressive drugs and chemotherapy was administered, but was unsuccessful in controlling the disease. Therefore, he was treated with HLA-matched related allogeneic hematopoietic stem cell transplantation. However, his condition deteriorated within 30 days, resulting in fatal outcome. Autopsy revealed many EBV-infected NK cells infiltrating major organs, consistent with ANKL. Furthermore, whole-exome sequencing identified a novel missense mutation of the CCDC22 gene (c.112G>A, p.V38M), responsible for X-linked intellectual disability (XLID). CCDC22 has been shown to play a role in NF-κB activation. Our case suggests that CCDC22 mutation might be implicated in pathogenesis of EBV-HLH and NK-cell neoplasms as well as XLID via possibly affecting NF-κB signaling.
799	Buoyant wind-driven pollutant dispersion and recirculation behaviour in wedge-shaped roof urban street canyons.	30706269	The present study investigated the buoyant wind-driven pollutant plume dispersion and recirculation behaviour inside urban street canyons formed by buildings with wedge-shaped roofs. Numerical modelling was performed using a computational fluid dynamics (CFD) large eddy simulation (LES). Street canyon models with a strongly buoyant fire source located on the street and environmental winds perpendicular to the canyon were developed using the fire dynamics simulator (FDS). The complex interaction of buoyancy and wind, as well as their combined effects on the pollutant plume dispersion, was simulated inside the urban street canyon. The results showed that the flow pattern of pollutant plume dispersion inside the street canyon with increasing wind speed for different roof inclination angles could be divided into three regimes, including a recirculation regime, a quasi-recirculation regime and a non-recirculation regime. The pollutant levels in the street canyon, as indexed by carbon monoxide (CO) concentration, increased under the recirculation regime. For the quasi-recirculation regime, however, the leeward buildings primarily suffered from the higher pollutant levels. The critical wind speed needed to trigger recirculation was analysed for various roof inclination angles. A correlation was proposed to predict the critical wind speed of various wedge-shaped roof angles for recirculation regime and quasi-recirculation regimes.
800	Hemophagocytic lymphohistiocytosis followed by an episode of peritoneal dialysis associated peritonitis: a case report.	30696397	Hemophagocytic lymphohistiocytosis (HLH) is characterized by excessive activation of the immune system due to infection, autoimmune diseases, or malignancy. As an aggressive and life-threatening clinical syndrome, HLH secondary to peritoneal dialysis associated peritonitis (PDAP) has never been reported.A 34-year-old female peritoneal dialysis (PD) patient was hospitalized for fever, progressively multi-organ damage (including cytopenias, abnormalities of coagulation and liver enzyme) after an episode of organism-specific peritonitis. She was refractory to the broad-spectrum antimicrobial agent. Further tests found hemophagocytosis on the bone marrow examination, and extremely high level of sIL2-R and impaired activity of NK cell. The diagnosis of HLH was eventually established. After HLH-specific therapy, this patient recovered and discharged.The present case suggests that clinicians should to be aware of HLH in those patients apparently suspected with refractory or relapsing peritonitis, especially those accompanied with persist fever, hyperferritinemia, and cytopenias. HLH-specific therapy and supportive care should be applied without delay.
801	Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling.	30694174	To progress towards differentiation, progeny of stem cells need to extinguish expression of stem-cell maintenance genes. Failures in such mechanisms can drive tumorigenesis. In Drosophila neural stem cell (NSC) lineages, excessive Notch signalling results in supernumerary NSCs causing hyperplasia. However, onset of hyperplasia is considerably delayed implying there are mechanisms that resist the mitogenic signal. Monitoring the live expression of a Notch target gene, E(spl)mγ, revealed that normal attenuation is still initiated in the presence of excess Notch activity so that re-emergence of NSC properties occurs only in older progeny. Screening for factors responsible, we found that depletion of Mi-2/NuRD ATP remodeling complex dramatically enhanced Notch-induced hyperplasia. Under these conditions, E(spl)mγ was no longer extinguished in NSC progeny. We propose that Mi-2 is required for decommissioning stem-cell enhancers in their progeny, enabling the switch towards more differentiated fates and rendering them insensitive to mitogenic factors such as Notch.
802	Therapeutic plasma exchange for pediatric nonrenal disease indications and outcomes: A single-center experience.	30692797	Outcome data in pediatric plasma exchange, especially in nonrenal indications are scarce. We aimed to evaluate its role and outcome in our patients.A retrospective study of children admitted in the year 2016 to the Pediatric Intensive Care Unit requiring plasma exchange for nonrenal indications was undertaken. Plasma exchange was given as adjunctive therapy along with primary treatment for the disease concerned. Demographic and clinical data were studied, and descriptive statistical analysis was carried out.Ten children underwent plasma exchange during this 1-year period with a male: female ratio of 3:2 and a mean age of 10 years (range 3-16 years). The indications were acute disseminated encephalomyelitis (n = 2), acute neuromyelitis optica (n = 1), catastrophic antiphospholipid antibody syndrome secondary to systemic lupus erythematosus (SLE) (n = 1), severe SLE with cerebritis/hemophagocytic lymphohistiocytosis (HLH) (n = 2), severe dengue sepsis with HLH/multi-organ dysfunction syndrome (n = 2), and thrombotic microangiopathy secondary to snake bite envenomation (n = 2). All received either 1.5 or 2 times plasma volume exchange (mean sessions - 4, range = 1-6). The mean duration of stay in hospital was 17.2 days (range = 3-40 days), and follow-up was 78 days (range = 3-180 days), with the majority of children (8/10, 80%) survived from the catastrophic illness at the time of discharge. Two children (2/10, 20%) succumbed due to the disease per se in severe dengue sepsis in one and enterobacteriaceae sepsis (hospital-acquired pneumonia) in another.Plasma exchange was found to be beneficial as complementary therapy in a critical care setting, especially for nonrenal indications.
803	[Hemophagocytic syndrome in a patient followed for IgG4-related disease in intensive care unit].	30683033	IgG4-related disease (IgG4-RD) is a set of diseases that can affect multiple organs, produce an immune-mediated fibroinflammatory response, and lead to tissue destruction and organ failure. Hemophagocytic syndrome is a life-threatening hyperinflammatory fatal disease caused by defect and excessive macrophage activity in natural killer cells. The disease can often be confused with other immune-mediated diseases such as cancer, infection, interstitial lung disease, sjogren's syndrome, wegener's vasculitis, or temporal arteritis. Hemophagocytic syndrome is defined as hemophagocytic lymphohistiocytosis (HLH) which is a pathological and clinical condition caused by phagocytosis of erythrocyte, leukocyte, platelet and precursor cells which are the cellular elements of the macrophages which are activated due to various reasons. Although there are two types as primary (familial) and secondary (depending on infections), the clinical findings are the same. Presence of familial disease/known gene defect and/or at least 5 of 8 clinical and laboratory diagnostic criteria is required for diagnosis. The first target is the suppression of hyperinflammation urgently, the second is the elimination of the stimulus triggering the event. In secondary HLH, treatment should be planned according to the underlying cause. As a rare complication of a rare disease, a case with the diagnosis of IgG4-related disease complicated with hemophagocytic syndrome is presented with the literature.
804	Kawasaki disease shock syndrome complicated with macrophage activation syndrome in a 5-month old boy: A case report.	30681594	Kawasaki disease (KD) is an acute febrile systemic vasculitis of unknown etiology and often occurs in children under 5 years old. During the acute phase, approximately 5% of children with KD develop hypotension and shock, a severe condition termed KD shock syndrome (KDSS). Macrophage activation syndrome (MAS), another life-threatening complication, has been reported to be associated with KD, although rarely. KDSS complicated with MAS is extremely rare. In this article, we present our experience in the diagnosis and treatment of KDSS complicated with MAS.A 5-month boy with fever for 5 days was diagnosed with KD. After 2 doses of intravenous immunoglobulin and regular antiinflammatory treatment at a local hospital, the fever did not subdue. He was admitted to our department on the 10th day of illness. The boy developed KDSS on the 11th day of illness. In the mean time, the boy had hepatosplenomegaly, and laboratory tests showed hypertriglyceridemia, hypofibrinogenemia, decreased blood red cells and platelets, increased ferritin and soluble sIL2Rα, and reduced natural killer cell activity.The patient had KDSS complicated with MAS.Emergency antishock therapy along with high-dose steroid with a longer tapering course was carried out. Following these treatments, fever subsided and other symptoms and signs relieved, but progressive coronary dilatation occurred, warfarin was thereby administered.The patient was discharged 30 days after hospitalization. Echocardiography at the 2 month follow-up showed regression of coronary aneurysm.Laboratory testing is critical for the diagnosis of MAS and we recommend that 2009 HLH diagnostic criteria be used for the diagnosis of MAS in KD. Emergency treatment of shock and a longer course of high-dose steroid anti-inflammatory therapy are vital for the management of KDSS complicated with MAS.
805	Innovative analysis of predictors for overall survival from systemic non-Hodgkin T cell lymphoma using quantile regression analysis.	30681495	Non-Hodgkin T/NK cell lymphoma is a rare and widely variable type of lymphoma with the most dismal prognosis. This study aimed to investigate varied impact of the clinical indicators to the overall survival (OS).We conducted a retrospective study to identify the non-invasive clinical features of T cell lymphoma that can predict prognosis with an innovative analysis method using quantile regression. A total of 183 patients who visited a top-tier hospital in Beijing, China, were enrolled from January 2006 to December 2015. Demographic information and main clinical indicators were collected including age, erythrocyte sedimentation rate (ESR), survival status, and international prognostic index (IPI) score.The median age of the patients at diagnosis was 45 years. Approximately 80% of patients were at an advanced stage, and the median survival time after diagnosis was 5.1 months. Multivariable analysis of the prognostic factors for inferior OS associated with advanced clinical staging [HR=3.16, 95%CI (1.39-7.2)], lower platelet count [HR = 2.57, 95%CI (1.57-4.19), P < 0.001] and higher IPI score [HR = 1.29, 95%CI (1.01-1.66), P = 0.043]. Meanwhile, T cell lymphoblastic lymphoma [HR = 0.40, 95%CI (0.20-0.80), P = 0.010], higher white blood cell counts [HR = 0.57, 95%CI (0.34-0.96), P = 0.033], higher serum albumin level [HR = 0.6, 95%CI (0.37-0.97), P = 0.039], and higher ESR [HR = 0.53, 95%CI (0.33-0.87), P = 0.011] were protective factors for OS when stratified by hemophagocytic lymphohistiocytosis (HLH). Multivariable quantile regression between the OS rate and each predictor at quartiles 0.25, 0.5, 0.75, and 0.95 showed that the coefficients of serum β2-microglobulin level and serum ESR were statistically significant in the middle of the coefficient curve (quartile 0.25-0.75). The coefficient of IPI was negatively associated with OS. The coefficients of hematopoietic stem cell transplantation (HSCT) and no clinical symptoms were higher at the middle of the quartile level curve but were not statistically significant.The IPI score is a comparatively robust indicator of prognosis at 3 quartiles, and serum ESR is stable at the middle 2 quartiles section when adjusted for HLH. Quantile regression can be used to observe detailed impacts of the predictors on OS.
806	An 18-Year-Old Male With X-linked Lymphoproliferative Syndrome Type 1 Who Developed Primary Central Nervous System Lymphoma 6 Months After Primary Epstein-Barr Virus Infection.	30676439	X-linked lymphoproliferative syndrome type 1 (XLP1) is a rare congenital immunodeficiency disease. We report the case of an 18-year-old male who developed hemophagocytic lymphohistiocytosis (HLH) with neurologic complications after primary Epstein-Barr virus (EBV) infection and subsequently developed EBV-related central nervous system lymphoma (CNSL). Given the vulnerability to EBV, he was finally diagnosed with XLP1 and treated with whole-brain irradiation along with chemotherapy and subsequent allogeneic hematopoietic stem cell transplantation from a SH2D1A wild-type sibling donor. Although the prognosis for CNSL is generally dismal, reconstitution of the immune system from a normal donor contributed to the patient remaining in remission for 30 months.
807	A Case of Parvovirus-Related Haemophagocytic Lymphohistiocytosis in a Patient with HbH Disease.	30675165	Hemophagocytic lymphohistiocytosis (HLH) is rare and life-threatening medical emergency. Parvovirus infection is rarely associated with HLH. We report a case of parvovirus-related HLH in a patient with alpha thalassaemia (HbH disease). The patient responded well to a course of dexamethasone without the need of etoposide. Based on our literature search, this is the first case of parvovirus related HLH in a patient with HbH disease in the medical literature.
808	Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice.	30674681	Secondary hemophagocytic lymphohistiocytosis (sHLH) is a highly mortal complication associated with sepsis. In adults, it is often seen in the setting of infections, especially viral infections, but the mechanisms that underlie pathogenesis are unknown. sHLH is characterized by a hyperinflammatory state and the presence hemophagocytosis. We found that sequential challenging of mice with a nonlethal dose of viral toll-like receptor (TLR) agonist followed by a nonlethal dose of TLR4 agonist, but not other permutations, produced a highly lethal state that recapitulates many aspects of human HLH. We found that this hyperinflammatory response could be recapitulated in vitro in bone marrow-derived macrophages. RNA sequencing analyses revealed dramatic up-regulation of the red-pulp macrophage lineage-defining transcription factor SpiC and its associated transcriptional program, which was also present in bone marrow macrophages sorted from patients with sHLH. Transcriptional profiling also revealed a unique metabolic transcriptional profile in these macrophages, and immunometabolic phenotyping revealed impaired mitochondrial function and oxidative metabolism and a reliance on glycolytic metabolism. Subsequently, we show that therapeutic administration of the glycolysis inhibitor 2-deoxyglucose was sufficient to rescue animals from HLH. Together, these data identify a potential mechanism for the pathogenesis of sHLH and a potentially useful therapeutic strategy for its treatment.
809	Soluble ST2 and CD163 as Potential Biomarkers to Differentiate Primary Hemophagocytic Lymphohistiocytosis from Macrophage Activation Syndrome.	30671214	The differentiation of primary hemophagocytic lymphohistiocytosis (pHLH) and macrophage activation syndrome (MAS) poses a challenge to hematologists. The aim of this study was (1) to compare the levels of soluble ST2 (sST2), sCD163, IFN-γ, IL-10, IL-18, TNF-α and Serum soluble interleukin-2 receptor (sCD25) in patients with pHLH and MAS and (2) to investigate whether they can help differentiate the two diseases. A total of 52 participants were recruited in this study, including 12 pHLH patients, 20 MAS patients, and 20 healthy subjects. We measured the levels of sST2, sCD163 and sCD25 in serum by ELISA. The serum levels of IFN-γ, IL-10, IL-18, and TNF-α were detected using a Luminex 200 instrument. The serum levels of sST2 and sCD163 in MAS patients were markedly higher than that in pHLH patients (363.13 ± 307.24 ng/ml vs 80.75 ± 87.04 ng/ml, P = 0.004; 3532.72 ± 2479.68 ng/ml vs 1731.96 ± 1262.07 ng/ml, P = 0.046). There was no significant difference in the expression of IFN-γ (306.89 ± 281.60 pg/ml vs 562.43 ± 399.86 pg/ml), IL-10 (20.40 ± 30.49 pg/ml vs 8.3 ± 13.14 pg/ml), IL-18 (463.33 ± 597.04 pg/ml vs 1247.82 ± 1318.58 pg/ml), TNF-α (61.48 ± 84.69 pg/ml vs 106.10 ± 77.21 pg/ml), and sCD25 (21062.1 ± 18515.26 pg/ml vs 11074.78 ± 11149.96 pg/ml) between pHLH and MAS. Patients with pHLH and MAS show some differences in cytokine profiles. The elevated levels of IFN-γ, IL-10, TNF-α, IL-18, and sCD25 can contribute to the diagnosis of HLH, but may not discriminate pHLH from MAS. Levels of sST2 and sCD163 may serve as markers to distinguish pHLH from MAS.
810	Macrophage Activation Marker Soluble CD163 Associated with Fatal and Severe Ebola Virus Disease in Humans1.	30666927	Ebola virus disease (EVD) is associated with elevated cytokine levels, and hypercytokinemia is more pronounced in fatal cases. This type of hyperinflammatory state is reminiscent of 2 rheumatologic disorders known as macrophage activation syndrome and hemophagocytic lymphohistiocytosis, which are characterized by macrophage and T-cell activation. An evaluation of 2 cohorts of patients with EVD revealed that a marker of macrophage activation (sCD163) but not T-cell activation (sCD25) was associated with severe and fatal EVD. Furthermore, substantial immunoreactivity of host tissues to a CD163-specific antibody, predominantly in areas of extensive immunostaining for Ebola virus antigens, was observed in fatal cases. These data suggest that host macrophage activation contributes to EVD pathogenesis and that directed antiinflammatory therapies could be beneficial in the treatment of EVD.
811	Inhibitor of DNA binding proteins: implications in human cancer progression and metastasis.	30662638	Inhibitor of DNA binding (ID) proteins are a class of helix-loop-helix (HLH) transcription regulatory factors that act as dominant-negative antagonists of other basic HLH proteins through the formation of non-functional heterodimers. These proteins have been shown to play critical roles in a wide range of tumor-associated processes, including cell differentiation, cell cycle progression, migration and invasion, epithelial-mesenchymal transition, angiogenesis, stemness, chemoresistance, tumorigenesis, and metastasis. The aberrant expression of ID proteins has not only been detected in many types of human cancers, but is also associated with advanced tumor stages and poor clinical outcome. In this review, we provide an overview of the key biological functions of ID proteins including affiliated signaling pathways. We also describe the regulation of ID proteins in cancer progression and metastasis, and elaborate on expression profiles in cancer and the implications for prognosis. Lastly, we outline strategies for the therapeutic targeting of ID proteins as a promising and effective approach for anticancer therapy.
812	Unusual manifestation of disseminated herpes simplex virus type 2 infection associated with pharyngotonsilitis, esophagitis, and hemophagocytic lymphohisitocytosis without genital involvement.	30654754	Herpes simplex virus (HSV) has various presentations, depending on the patient's immune status, age, and the route of transmission. In adults, HSV type 1 is found predominantly in the oral area, and HSV type 2 (HSV-2) is commonly found in the genital area. HSV-2 infection without genital lesions is uncommon. Herein we report a unique case of pharyngotonsillitis as an initial manifestation of disseminated HSV-2 infection without genital involvement.A 46-year-old male was admitted to our hospital with a 1-week history of fever and sore throat. His past medical history included hypereosinophilic syndrome diagnosed at age 45 years. Physical examination revealed throat congestion, bilaterally enlarged tonsils with exudates, tender cervical lymphadenopathy in the left posterior triangle, and mild epigastric tenderness. The laboratory data demonstrated bicytopenia, elevated liver enzyme levels, and hyperferritinemia. A bone marrow smear showed hypocellular marrow with histiocytes and hemophagocytosis. The diagnosis of HLH was confirmed, and the patient was treated with methylprednisolone pulse therapy on days 1-3. On day 5, despite initial improvement of the fever and sore throat, multiple, new, small bullae developed on the patient's face, trunk, and extremities. Additional testing showed that he was positive for HSV-specific immunoglobulin M and immunoglobulin G. Disseminated HSV infection was suspected, and intravenous acyclovir (10 mg/kg every 8 h) was begun. A subsequent direct antigen test of a bulla sample was positive for HSV-2. Moreover, tonsillar and esophageal biopsies revealed viral inclusion bodies. Immunohistochemical staining and a quantitative real-time polymerase chain reaction (PCR) assay confirmed the presence of HSV-2. Disseminated HSV-2 infection with multiple bullae, tonsillitis, esophagitis, and suspected hepatic involvement was diagnosed. After a 2-week course of intravenous acyclovir, his hematological status and liver function normalized, and his cutaneous skin lesions resolved. He was discharged on day 22 in good general health and continued taking oral valacyclovir for viral suppression due to his immunosuppressed status.Disseminated HSV-2 infection should be considered as one of the differential diagnoses in patients with pharyngotonsillitis and impaired liver function of unknown etiology even if there are no genital lesions.
813	Case Report: Hemophagocytic Lymphohistiocytosis Caused by Disseminated Histoplasmosis in a Venezuelan Patient with HIV and Epstein-Barr Virus Reactivation Who Traveled to Japan.	30652668	We describe a Venezuelan visitor to Japan who was diagnosed with hemophagocytic lymphohistiocytosis (HLH). The patient was also diagnosed with human immunodeficiency virus (HIV) and Epstein-Barr virus infection by the Western blot and polymerase chain reaction (PCR) tests, respectively. The cause of HLH was considered to be these two infections at first; however, the patient did not recover with antiretroviral/anti-herpes virus therapy. Thereafter, diagnosis of disseminated histoplasmosis was confirmed with an antigen detection test, culture, and PCR test of blood, urine, and bone marrow, and the patient improved gradually after the initiation of liposomal amphotericin B. This case highlights the importance of ruling out endemic mycosis as a cause of HLH even if other probable causes exist in patients from endemic areas.
814	Haemophagocytic lymphohistiocytosis with collapsing lupus podocytopathy as an unusual manifestation of systemic lupus erythematosus with APOL1 double-risk alleles.	30642866	Haemophagocytic lymphohistiocytosis (HLH) is a potentially life-threatening syndrome caused by excessive immune activation. Secondary HLH has been described in autoimmune diseases. We detail the case of a 28-year-old African American woman who developed HLH in the setting of systemic lupus erythematosus with collapsing lupus podocytopathy superimposed on mesangial proliferative lupus nephritis class II. Genotyping for APOL1 risk alleles revealed the presence of double (G1/G2) risk alleles. Our patient achieved a complete renal recovery and resolution of HLH within 1 month of treatment with steroids and mycophenolate mofetil, highlighting the importance of prompt, aggressive therapy.
815	Chronic active EBV infection associated with NK cell lymphoma and hemophagocytic lymphohistiocytosis in a 27-year-old woman: A case report.	30633196	Chronic active Epstein-Barr virus infection (CAEBV) is a common infectious disease that often affects multiple organs or systems. However, it is liable to be neglected and misdiagnosed owing to its insidious onset, lack of specific findings in the early phase, and a general lack of awareness among clinicians. PATIENT CONCERNS:: a 27-year-old woman case has been described who was initially misdiagnosed as drug-induced liver injury due to onset presentation of mild splenomegaly, recurrent liver dysfunction, and disputable pathological evidence of liver biopsy.CAEBV complicated with natural killer (NK) cell lymphoma and hemophagocytic lymphohistiocytosis (HLH) was diagnosed by in situ hybridization of liver tissue section with EBV-encoded RNA -1 probe and flow cytometry of bone marrow.After admission, the patient received symptomatic treatment and antiviral therapy (combination of acyclovir and foscarnet sodium) as well as adjuvant treatment (thymosin alpha 1 and methylprednisolone); later, the patient received etoposide and dexamethasone for diagnosis of EBV associated HLH. Subsequently, the disease progressed to NK cell lymphoma and the patient received the revised EPOCH chemotherapy regimen [etoposide (100 mg/d, d1-5), dexamethasone (7.5 mg/d, d1-5; 5 mg/d, d6-14), cyclophosphamide (0.8 g/d, d1-2), and pegaspargase (3750 u/d, tid, d1-2)].Although the patient received a series of therapies and other comprehensive measures, finally she died of gastrointestinal hemorrhage and multiple organ failure.Liver is one of the main target organs of EBV infection. In the clinical setting of unexplained fever and liver injury, it is necessary to be aware of CAEBV, as well as its fatal complication such as EBV associated NK cell lymphoma and HLH.
816	Emapalumab: First Global Approval.	30623346	Emapalumab-Izsg (hereafter referred to as emapalumab) [Gamifant®] is a monoclonal antibody directed against interferon gamma that is available as an intravenous infusion. Emapalumab is being developed by Novimmune and Swedish Orphan Biovitrum for the treatment of haemophagocytic lymphohistiocytosis (HLH). In November 2018, emapalumab received its first global approval in the USA, for the treatment of paediatric (newborn and older) and adult patients with primary HLH, who have refractory, recurrent or progressive disease or intolerance to conventional HLH therapy. Emapalumab is under regulatory review in the EU for the treatment of primary HLH. This article summarizes the milestones in the development of emapalumab leading to this first global approval for HLH in the USA.
817	A Hidden Cause of MERS and HLH in a Girl: Unusual Presentation of Hantaviruses Infection.	30620949	NaN
818	Diagnostic utility of bone marrow examination in evaluation of fever: a descriptive study on 98 immunocompetent adults from a tertiary care institute in south India.	30614411	Five-year clinico-laboratory data from 99 (one HIV seropositive) adults (mean age = 41.3 ± 20.4 years) who underwent bone marrow examination for fever persisting for ≥ 1 week were analysed and correlated with microbiological characteristics. Infections, reactive marrow changes and haematolymphoid malignancies were most commonly associated with fever. A high concordance rate of 71% was noted between aspiration and trephine biopsies. Bone marrow granulomas (BMG) were seen exclusively on sections and were most commonly of tubercular and typhoidal in origin (two Salmonella Typhi, one Salmonella Paratyphi A). The common aetiologies associated with fever and cytopenia(s) were BMG, acute leukaemia and haemophagocytic lymphohistiocytosis (HLH; n = 3). The yield from bone marrow culture was inferior compared to other body fluids. In conclusion, bone marrow histology is superior to smears in the evaluation of prolonged fever. Marrow culture may not be useful in immunocompetent individuals other than if Salmonellosis is suspected.
819	Helix-loop-helix proteins and the advent of cellular diversity: 30 years of discovery.	30602438	Helix-loop-helix (HLH) proteins are dimeric transcription factors that control lineage- and developmental-specific gene programs. Genes encoding for HLH proteins arose in unicellular organisms >600 million years ago and then duplicated and diversified from ancestral genes across the metazoan and plant kingdoms to establish multicellularity. Hundreds of HLH proteins have been identified with diverse functions in a wide variety of cell types. HLH proteins orchestrate lineage specification, commitment, self-renewal, proliferation, differentiation, and homing. HLH proteins also regulate circadian clocks, protect against hypoxic stress, promote antigen receptor locus assembly, and program transdifferentiation. HLH proteins deposit or erase epigenetic marks, activate noncoding transcription, and sequester chromatin remodelers across the chromatin landscape to dictate enhancer-promoter communication and somatic recombination. Here the evolution of HLH genes, the structures of HLH domains, and the elaborate activities of HLH proteins in multicellular life are discussed.
820	Hemophagocytic Lymphohistiocytosis Associated With 2 Cases of Pediatric Lymphocyte-depleted Classic Hodgkin Lymphoma.	30601404	Hemophagocytic lymphohistiocytosis (HLH) is a rare and often fatal syndrome of abnormal T-cell activation and cytokine production, which can be familial or secondary in nature. Although HLH can occur concomitantly with lymphomas, the development of HLH alongside Hodgkin lymphoma in children is unusual. Here we report the diagnostic evaluation and clinical course of 2 pediatric cases of HLH secondary to lymphocyte-depleted classic Hodgkin lymphoma. These cases highlight the need to be vigilant for this rare presentation and the difficulties in managing these patients.
821	Stream biasing by different induction sequences: Evaluating stream capture as an account of the segregation-promoting effects of constant-frequency inducers.	30599694	Stream segregation for a test sequence comprising high-frequency (H) and low-frequency (L) pure tones, presented in a galloping rhythm, is much greater when preceded by a constant-frequency induction sequence matching one subset than by an inducer configured like the test sequence; this difference persists for several seconds. It has been proposed that constant-frequency inducers promote stream segregation by capturing the matching subset of test-sequence tones into an on-going, pre-established stream. This explanation was evaluated using 2-s induction sequences followed by longer test sequences (12-20 s). Listeners reported the number of streams heard throughout the test sequence. Experiment 1 used LHL- sequences and one or other subset of inducer tones was attenuated (0-24 dB in 6-dB steps, and ∞). Greater attenuation usually caused a progressive increase in segregation, towards that following the constant-frequency inducer. Experiment 2 used HLH- sequences and the L inducer tones were raised or lowered in frequency relative to their test-sequence counterparts (ΔfI  = 0, 0.5, 1.0, or 1.5 × ΔfT ). Either change greatly increased segregation. These results are concordant with the notion of attention switching to new sounds but contradict the stream-capture hypothesis, unless a "proto-object" corresponding to the continuing subset is assumed to form during the induction sequence.
822	Intensive care unit complications and outcomes of adult patients with hemophagocytic lymphohistiocytosis: A retrospective study of 16 cases.	30596029	To study the management, complications and outcomes of adult patients admitted with hemophagocytic lymphohistiocytosis (HLH) in the intensive care unit (ICU).We performed a retrospective observational study of adult patients with the diagnosis of "HLH" admitted to the two academic medical ICUs of Baylor College of Medicine between 01/01/2013 to 06/30/2017. HLH was diagnosed using the HLH-2004 criteria proposed by the Histiocyte Society.Sixteen adult cases of HLH were admitted to the medical ICUs over 4 years. Median age of presentation was 49 years and 10 (63%) were males. Median Sequential Organ Failure Assessment (SOFA) score at the time of ICU admission was 10. Median ICU length of stay (LOS) was 11.5 d and median hospital LOS was 29 d. Septic shock and acute respiratory failure accounted for majority of diagnoses necessitating ICU admission. Septic shock was the most common ICU complication seen in (88%) patients, followed by acute kidney injury (81%) and acute respiratory failure requiring mechanical ventilation (75%). Nine patients (56%) developed disseminated intravascular coagulation and eight (50%) had acute liver failure. 10 episodes of clinically significant bleeding were observed. Multi system organ failure was the most common cause of death seen in 12 (75%) patients. The 30 d mortality was 37% (6 cases) and 90 d mortality was 81% (13 cases). There was no difference in mortality based on age (above or less than 50 years), SOFA score on ICU admission (more than or less than 10), immunosuppression, time to diagnose HLH or direct ICU admission versus floor transfer.HLH is a devastating disease associated with poor outcomes in ICU. Intensivists need to have a high degree of clinical suspicion for HLH in patients with septic shock/multi system organ failure and progressive bi/pancytopenia who are not responding to standard management in ICU.
823	A mechanistic theory explaining hyperferritinaemia in haemophagocytic lymphohistiocytosis.	30593404	Intracellular ferritin is known to store iron, an essential element in an array of physiological processes. But what purpose does serum ferritin serve, what controls ultra-high hyperferritinaemia (ferritin levels above 4000 ng/ml), and finally why is it raised in inflammatory diseases, the most impressive being haemophagocytic lymphohistiocytosis? This paper proposes a hypothesis and model to explain the above questions. Serum ferritin has some unfamiliar physiological properties. It dampens severe inflammation by inhibiting lymphocyte activity, hinders bacterial growth by acting as an iron chelator and slows malignant growth by having an antiangiogenic effect. Therefore, serum ferritin is effective at modulating many conditions and could be a desired physiological process in malignancy or infection. The proposed theory is that phagocytic cells fuse with pathological native cells in much the same way as macrophages do when forming multinucleated giant cells. The pathological cells can be "infected" by intracellular pathogens or the malignant phenotype. These fusion cells result in a massive upregulation of exclusive features of macrophage activity such as phagocytosis, iron metabolism and ferritin release. Dysfunctional mitochondria are critical to this process, Pathological cells that contain dysfunctional mitochondria "infect" phagocytic mitochondrial following cellular fusion, resulting in ferritin release. The key evidence suporting this idea: macrophages are the source of ferritin secretion; they have intimate contact with known triggers of HLH, allowing for fusion hybrids, and they commonly fuse with cells. It is also argued that exclusively intracellular pathologies trigger HLH, which is a requirement for this model. HLH has features of a mitochondriopathy and finally it is debated that all the defective genes known to trigger familial-HLH can increase the likelihood of macrophage fusion. If this hypothesis is correct, the implications might discover novel diagnostic tools to detect HLH and point to avenues of targeted therapy. This is a bold theory with interesting perspectives on cell biology and clinical medicine. The paper discusses the evidence and challenges of this proposed mechanism in describing hyperferritinaemia and its relation to HLH.
824	Hemophagocytic Lymphohistiocytosis Due to Primary HHV-8 Infection in a Liver Transplant Recipient.	30584592	Human herpesvirus-8 (HHV-8) remains best known as an oncogenic virus, but nonneoplastic disease manifestations, such as bone marrow failure or hemophagocytic lymphohistiocytosis (HLH) have gained greater recognition in recent years. In organ transplantation, HHV-8 infection commonly occurs with reactivation of latent virus among recipients from endemic regions of the world or due to transmission from the organ donor. We describe a case of HHV-8-associated HLH in a liver transplant recipient at increased risk for primary infection. Our case highlights the risk of non-donor-derived, posttransplant primary HHV-8 infection, and demonstrates that HLH can be a life-threatening complication of this infection.
825	Clinical characteristics and effective treatments of scrub typhus-associated hemophagocytic lymphohistiocytosis in children.	30581619	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon and life-threatening disorder that may rarely complicate the clinical course of Orientia tsutsugamushi disease (scrub typhus). Here, we describe the clinical features, laboratory parameters, management, and outcome of 16 children with scrub typhus-associated HLH. All patients satisfied the HLH-2004 diagnostic criteria. All patients had fever of unknown origin and multisystem damage. Raised hepatic transaminases and abnormalities in routine blood test were observed in all children. Imaging tests showed abnormalities in 10 cases. Six patients were treated with intravenous azithromycin for 5 days, and 10 with intravenous chloramphenicol for 7-10 days because of non-response to 3-day azithromycin treatment. Five patients were treated with intravenous albumin and 3 with intravenous immunoglobulin. Two patients with severe symptoms (shortness of breath, cyanosis) were treated with dexamethasone (0.3 mg/kg/d). Fifteen patients recovered completely after 8-22 days of treatment. One patient died. The occurrence of severe complications draws attention to the need for early diagnosis and effective treatment. Anti-rickettsial antibiotic treatment (azithromycin or chloramphenicol) without the need for chemotherapy may be beneficial in such cases, instead of treatment according to the 2004 HLH protocol.
826	IFN-γ and CD25 drive distinct pathologic features during hemophagocytic lymphohistiocytosis.	30578871	Inflammatory activation of CD8+ T cells can, when left unchecked, drive severe immunopathology. Hyperstimulation of CD8+ T cells through a broad set of triggering signals can precipitate hemophagocytic lymphohistiocytosis (HLH), a life-threatening systemic inflammatory disorder.The mechanism linking CD8+ T-cell hyperactivation to pathology is controversial, with excessive production of IFN-γ and, more recently, excessive consumption of IL-2, which are proposed as competing hypotheses. We formally tested the proximal mechanistic events of each pathway in a mouse model of HLH.In addition to reporting a complete autosomal recessive IFN-γ receptor 1-deficient patient with multiple aspects of HLH pathology, we used the mouse model of perforin (Prf1)KO mice infected with lymphocytic choriomeningitis virus to genetically eliminate either IFN-γ production or CD25 expression and assess the immunologic, hematologic, and physiologic disease measurement.We found a striking dichotomy between the mechanistic basis of the hematologic and inflammatory components of CD8+ T cell-mediated pathology. The hematologic features of HLH were completely dependent on IFN-γ production, with complete correction after loss of IFN-γ production without any role for CD8+ T cell-mediated IL-2 consumption. By contrast, the mechanistic contribution of the immunologic features was reversed, with no role for IFN-γ production but substantial correction after reduction of IL-2 consumption by hyperactivated CD8+ T cells. These results were complemented by the characterization of an IFN-γ receptor 1-deficient patients with HLH-like disease, in whom multiple aspects of HLH pathology were observed in the absence of IFN-γ signaling.These results synthesize the competing mechanistic models of HLH pathology into a dichotomous pathogenesis driven through discrete pathways. A holistic model provides a new paradigm for understanding HLH and, more broadly, the consequences of CD8+ T-cell hyperactivation, thereby paving the way for clinical intervention based on the features of HLH in individual patients.
827	[Clinical features of children with Epstein-Barr virus-related acute liver failure: an analysis of four cases].	30572993	A retrospective analysis was performed for the clinical data of four children with Epstein-Barr virus (EBV)-related acute liver failure. There were two boys and two girls with a median age of 10 months (range 8.5-44 months). Of the four children, three were diagnosed with infectious mononucleosis (IM), among whom two met the diagnostic criteria of hemophagocytic lymphohistiocytosis (HLH), and one was diagnosed with past EBV infection. All the children had positive EBV DNA in blood and all had pyrexia, hepatomegaly, and jaundice on admission. Three children had the symptom of splenomegaly, ascites, or vomiting. Two children had enlargement of cervical lymph nodes, skin rash, or pleural effusion. One child had gastrointestinal bleeding or stage 2 hepatic encephalopathy. All the children had an abnormal lymphocyte count of <10%, and only one child had leukocytosis and thrombocytopenia. Among the four children, alanine aminotransferase level increased by 10-100 times; total bilirubin level increased by 3-5 times; lactate dehydrogenase level increased by many 10 times; prothrombin time prolonged significantly. All the children were given antiviral therapy with intravenously injected acyclovir or ganciclovir, as well as hepatocyte growth factor to promote hepatocyte growth and hormone to alleviate inflammatory response. Two children were given plasma exchange in addition, among whom one was given the combination of continuous venovenous hemodiafiltration. Two children with HLH were given chemotherapy according to the HLH-2004 regimen. Three children survived, and one child with HLH died of multiple organ failure. It is concluded that EBV infection can cause acute liver failure and that early use of multimodality therapy including blood purification may be beneficial for prognosis in these children.
828	Hemophagocytic lymphohistiocytosis in the setting of HELLP Syndrome.	30564350	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyper activation of the immune system. Rare cases associated with HELLP syndrome and other similar conditions in pregnancy have been reported. Despite the improved survival rates with etoposide and dexamethasone-based regimens, HLH remains a challenging disease. Experience in pregnant patients is exceedingly rare.
829	Life-Threatening Primary Varicella Zoster Virus Infection With Hemophagocytic Lymphohistiocytosis-Like Disease in GATA2 Haploinsufficiency Accompanied by Expansion of Double Negative T-Lymphocytes.	30564229	Two unrelated patients with GATA2-haploinsufficiency developed a hemophagocytic lymphohistiocytosis (HLH)-like disease during a varicella zoster virus (VZV) infection. High copy numbers of VZV were detected in the blood, and the patients were successfully treated with acyclovir and intravenous immunoglobulins. After treatment with corticosteroids for the HLH, both patients made a full recovery. Although the mechanisms leading to this disease constellation have yet to be characterized, we hypothesize that impairment of the immunoregulatory role of NK cells in GATA2-haploinsufficiency may have accentuated the patients' susceptibility to HLH. Expansion of a double negative T-lymphocytic population identified with CyTOF could be a further factor contributing to HLH in these patients. This is the first report of VZV-triggered HLH-like disease in a primary immunodeficiency and the third report of HLH in GATA2-haploinsufficiency. Since HLH was part of the presentation in one of our patients, GATA2-haploinsufficiency represents a potential differential diagnosis in patients presenting with the clinical features of HLH-especially in cases of persisting cytopenia after recovery from HLH.
830	Hemophagocytic Lymphohistiocytosis: Clinical Presentations and Diagnosis.	30557712	Hemophagocytic lymphohistiocytosis (HLH) is an overwhelming clinical syndrome associated with extreme immune activation. Familial HLH is caused by autosomal-recessive inheritance of gene mutations that cripple lymphocyte cytotoxicity. X-linked lymphoproliferative diseases and mutations in Nod-like receptor caspase activation and recruitment domain containing protein 4 (NLRC4) also feature HLH as a predominant manifestation. In addition, "secondary" HLH may occur in immunocompromized patients or in individuals with previously intact immune responses in the context of strong immunologic triggers such as EBV infection, malignancy, rheumatologic disease, and drug hypersensitivity. Regardless of the etiology, HLH is often fatal unless recognized and treated aggressively. Research over the last 20 years has led to many advances in diagnosis and treatment. Rapid testing strategies designed to quickly screen for immune activation and cytotoxic lymphocyte dysfunction are now clinically available and genetic panels/testing algorithms may accelerate a genetic diagnosis. Immunosuppressive treatment protocols have been refined, and experience is gaining with alternative and salvage approaches. However, these advances improve the outcome of patients only when the diagnosis of HLH is made. Ongoing education is needed to ensure medical providers can appropriately recognize and diagnose HLH. This Grand Rounds Review will summarize the clinical and diagnostic features of HLH and highlight known genetic causes.
831	Hemophagocytic Lymphohistiocytosis during the Postpartum Stage of Pregnancy: A Report of Eight Cases.	3053775730537710	Haemophagocytic lymphohistiocytosis (HLH) is a rare and severe clinical syndrome that can be classified as either primary or secondary. Secondary HLH can be triggered by a variety of diseases. Pregnancy-related HLH has already been observed clinically. However, most of these cases occur during pregnancy. Considering that the periods before and after delivery have different clinical features, we presented the first case series of HLH that presented during the postpartum stage of pregnancy. From these cases, we concluded that postpartum HLH is a common form of pregnancy-related HLH. Patients with postpartum HLH may suffer more complicated pathogenesis. Cytopenia was not as common as in other types of HLH, but liver dysfunction was present in almost all cases. The standard therapy of HLH-94/04 was very effective, and the outcomes of postpartum HLH were better.
832	Outcomes in children with hemophagocytic lymphohistiocytosis treated using HLH-2004 protocol in Japan.	30535855	Recent advances in intensive chemo- and immunotherapy have contributed to the outcome of hemophagocytic lymphohistiocytosis (HLH); however, the prognosis of HLH in children differs by HLH subtype. In Japan, secondary HLH, particularly Epstein-Barr virus-associated HLH (EBV-HLH), is the most common HLH subtype. The prognosis of HLH has improved in recent years. We here conducted a prospective study of 73 patients who were treated with HLH-2004 protocol in Japan. EBV-HLH, familial HLH (FHL), and HLH of unknown etiology were seen in 41, 9, and 23 patients, respectively. Patients with resistant or relapsed disease after HLH-2004 treatment and those with FHL received hematopoietic stem cell transplantation (HSCT). The induction rate after initial therapy was 58.9%, and the 3-year overall survival (OS) rate of all patients was 73.9% and differed significantly among those with EBV-HLH, FHL, and HLH of unknown etiology. Of the 17 patients who received HSCT, the 3-year OS rates of those with and without complete resolution before HSCT were 83.3% and 54.5%, respectively. Outcomes in children with HLH who were treated with the same protocol differed among HLH subtypes. Appropriate strategy for each subtype should be established in future studies.
833	A multicenter study of patients with multisystem Langerhans cell histiocytosis who develop secondary hemophagocytic lymphohistiocytosis.	30521100	Langerhans cell histiocytosis (LCH) is a rare myeloid neoplasm characterized by the presence of abnormal CD1a-positive (CD1a+ )/CD207+ histiocytes. Hemophagocytic lymphohistiocytosis (HLH) represents a spectrum of hyperinflammatory syndromes typified by the dysregulated activation of the innate and adaptive immune systems. Patients with LCH, particularly those with multisystem (MS) involvement, can develop severe hyperinflammation mimicking that observed in HLH. Nevertheless, to the authors' knowledge, little is known regarding the prevalence, timing, risk factors for development, and outcomes of children and young adults who develop HLH within the context of MS-LCH (hereafter referred to LCH-associated HLH).To gain further insights, the authors conducted a retrospective, multicenter study and collected data regarding all patients diagnosed with MS-LCH between 2000 and 2015.Of 384 patients with MS-LCH, 32 were reported by their primary providers to have met the diagnostic criteria for HLH, yielding an estimated 2-year cumulative incidence of 9.3% ± 1.6%. The majority of patients developed HLH at or after the diagnosis of MS-LCH, and nearly one-third (31%) had evidence of an intercurrent infection. Patient age <2 years at the time of diagnosis of LCH; female sex; LCH involvement of the liver, spleen, and hematopoietic system; and a lack of bone involvement each were found to be independently associated with an increased risk of LCH-associated HLH. Patients with MS-LCH who met the criteria for HLH had significantly poorer 5-year survival compared with patients with MS-LCH who did not meet the criteria for HLH (69% vs 97%; P < .0001).Given its inferior prognosis, further efforts are warranted to enhance the recognition and optimize the treatment of patients with LCH-associated HLH.
834	Fludarabine/Melphalan 100 mg/m2 Conditioning Therapy Followed by Allogeneic Hematopoietic Cell Transplantation for Adult Patients with Secondary Hemophagocytic Lymphohistiocytosis.	30508593	Our previous research indicated that a reduced-intensity conditioning regimen (fludarabine and melphalan at 100 mg/m2) was useful in allogeneic hematopoietic cell transplantation (HCT) for patients with lymphoma. This retrospective study evaluated the reduced-intensity conditioning regimen in allogeneic HCT for adult patients with hemophagocytic lymphohistiocytosis (HLH). Sixteen patients with HLH were evaluated, including 6 patients who were enrolled in a prospective clinical trial (NCT00772811) and 10 patients who received the same conditioning regimen (fludarabine at 30 mg/m2/day on days -6 to -2 and melphalan at 100 mg/m2 on day -2). The median age was 42 years (range, 18 to 64), and 12 patients had Epstein-Barr virus (EBV)-associated HLH. Donors were an HLA matched sibling for 10 patients, an unrelated matched volunteer for 4 patients, and a mismatched family member for 2 patients. After excluding 3 patients who died soon after HCT, 12 patients achieved an engraftment (neutrophil median, day 12; platelet median, day 16). Five patients experienced acute graft-versus-host disease (GVHD), including 1 case of grade II and 4 cases of grades III to IV. Chronic GVHD occurred in 3 patients (moderate, 1 case; severe, 2 cases). After a median follow-up of 33.8 months 1 patient progressed, 3 patients relapsed, and 9 patients died. Five deaths were unrelated to relapse or progression and were caused by infection (n = 3), bleeding (n = 1), and GVHD (n = 1). No deaths or relapses were observed at >124 days post-transplant. The overall survival rate was 48.6%, and significant differences were observed according to pretransplant ferritin level (P = .007) and cytopenia lineage (P = .021). Before allogeneic HCT 10 of 12 patients still tested positive for EBV DNA: 6 patients tested negative for EBV DNA after HCT, 2 patients had persistent EBV DNA, and 2 patients were unassessable because of early death. Conditioning therapy using a lower dose of melphalan combined with fludarabine appears to be promising in allogeneic HCT for adults with HLH. However, strategies are needed to reduce the risk of early death.
835	The rheumatology/hematology interface: CAPS and MAS diagnosis and management.	30504326	Catastrophic antiphospholipid antibody syndrome (CAPS) and macrophage activation syndrome (MAS) are both life-threatening hematologic disorders that infrequently afflict patients with rheumatologic disease. CAPS is characterized by fulminant multiorgan damage related to small vessel thrombosis in the setting of persistent antiphospholipid antibodies. It can occur in patients with rheumatologic diseases such as systemic lupus erythematosus but can also affect patients who do not have rheumatologic disease. By contrast, the term MAS is applied when patients with rheumatologic disease develop hemophagocytic lymphohistiocytosis (HLH); therefore, patients with MAS have an underlying rheumatologic disease by definition. Similar to CAPS, HLH/MAS can have a fulminant presentation, but the pathogenesis and manifestations are different. In both CAPS and MAS, management generally includes but is not limited to immunosuppression with steroids. Fatalities are relatively common and morbidity is often significant. Early recognition of these disorders and initiation of timely treatment are important. More effective therapies for both syndromes are urgently needed.
836	[The 466th case:myasthenia,fever,blurred vision and pancytopenia].	30486568	A 45-year-old woman was admitted to the Department of Rheumatology and Immunology, Peking Union Medical College Hospital, due to weakness of the upper limbs, fever, and blurred vision. She was clinically diagnosed as systemic lupus erythematosus overlapped primary biliary cirrhosis, with renal, retinal, hematological and musculoskeletal involvement, combined with severe pulmonary infection and respiratory failure. Treated with glucocorticoids, ursodeoxycholic acid, antibiotics and respiratory support, the patient got better. A couple of days later, her fever recurred and platelets count dropped to 30×10(9)/L, hemoglobin to 78 g/L, fibrinogen to<1.5 g/L, ferritin to 1 640 ng/ml, natural killer (NK) cell count to 8/μl, the activity of NK cells 2% (reference value 9.5%-23.5%), considering the occurrence of hemophagocytic lymphohistiocytosis (HLH). Cytomegalovirus pp65 antigenemia test: 13 positive cells/2×10(5) WBC. Considered the possibility of HLH caused by cytomegalovirus infection and treated by 250 mg ganciclovir intravenous drip twice a day for a full course. The temperature of the patient was gradually reduced to 36.5 ℃, the count of platelets were increased to 229 ×10(9)/L, the hemoglobin was increased to 94 g/L, and the fibrinogen was increased to 3.26 g/L. When there were unexplained critical signs of the primary disease during systemic lupus erythematosus treatment, severe complications such as infection, HLH, thrombotic thrombocytopenic purpura should be taken into account.患者女性，45岁。因双上肢无力、发热、视物模糊收入北京协和医院风湿免疫科。诊断系统性红斑狼疮（SLE）合并原发性胆汁性肝硬化，肾脏、视网膜、血液系统、肌肉受累，合并严重肺部感染、呼吸衰竭。予糖皮质激素、熊去氧胆酸及抗生素、呼吸支持治疗后短暂好转，再次出现发热，同时血小板下降至30×10(9)/L，血红蛋白下降至78 g/L，纤维蛋白原下降至<1.5 g/L，铁蛋白升高至1 640 ng/ml，自然杀伤细胞数8/μl，自然杀伤细胞活性2%（参考值9.5%~23.5%），考虑噬血细胞性淋巴组织细胞增生症（HLH）。患者巨细胞病毒pp65抗原检测，13个阳性细胞/2×10(5)WBC，考虑HLH由巨细胞病毒感染所致可能性大，加用更昔洛韦250 mg、2次/d静脉点滴治疗，患者体温逐渐降至36.5 ℃，血小板升至229×10(9)/L，血红蛋白升至94 g/L，纤维蛋白原升至3.26 g/L。SLE治疗过程中出现原发病不能解释的危急征象时，需警惕感染、HLH、血栓性血小板减少性紫癜等严重并发症。.
837	Complex Investigation of a Pediatric Haematological Case: Haemophagocytic Syndrome Associated with Visceral Leishmaniasis and Epstein⁻Barr (EBV) Co-Infection.	30486459	Visceral leishmaniasis (VL) is an anthropozoonosis caused by an intracellular parasite belonging to the genus Leishmania. In the Mediterranean region, L. donovani and L. infantum are responsible for VL and dogs are the main reservoir. Haemophagocytic lymphohistiocytosis (HLH) represents a complication of VL and consists of unrestrained activation and proliferation of lymphocytes and macrophages, leading to uncontrolled immune activation. Haemophagocytic lymphohistiocytosis may also develop during viral infection, and Epstein⁻Barr virus (EBV) infection is one of the main HLH causes. Macrophage haemophagocytosis in the bone marrow aspirate is pathognomonic.The case involves a 19-month-old male infant presenting with a high persistent fever with a fluctuating pattern, pancytopaenia, hepatosplenomegaly, and a high triglyceride level. Initial investigations showed an EBV infection. Considering the persistent signs and symptoms, bone marrow aspiration was performed and confirmed the suspicion of HLH. In addition, the presence of Leishmania infection was shown. The patient was treated with liposomal amphotericin B and had complete resolution of his symptoms.Diagnosis of VL represents a demanding challenge in endemic and non-endemic areas. Our case demonstrates that leishmaniasis should always be considered in the differential diagnosis in patients presenting with hepatosplenomegaly and cytopaenia with a persistent fever, even in cases of infectious mononucleosis. Moreover, the execution of bone marrow aspiration should not be delayed in order to diagnose and treat at an early stage the potential occurrence of VL, especially if complicated with HLH.
838	Characterization of bHLH/HLH genes that are involved in brassinosteroid (BR) signaling in fiber development of cotton (Gossypium hirsutum).	30482177	Basic helix-loop-helix/helix-loop-helix (bHLH/HLH) transcription factors play important roles in plant development. Many reports have suggested that bHLH/HLH proteins participate in brassinosteroid (BR) hormone signaling pathways to promote cell elongation. Cotton fibers are single-cells and derived from seed surface. To explore the roles of bHLH/HLH proteins in cotton fiber development progress by modulating BR signaling pathway, we performed a systematic analysis of the bHLH/HLH gene family in upland cotton (Gossypium hirsutum) genome.In this study, we identified 437 bHLH/HLH genes in upland cotton (G. hirsutum) genome. Phylogenetic analysis revealed that GhbHLH/HLH proteins were split into twenty six clades in the tree. These GhbHLH/HLH genes are distributed unevenly in different chromosomes of cotton genome. Segmental duplication is the predominant gene duplication event and the major contributor for amplification of GhbHLH/HLH gene family. The GhbHLH/HLHs within the same group have conserved exon/intron pattern and their encoding proteins show conserved motif composition. Based on transcriptome data, we identified 77 GhbHLH/HLH candidates that are expressed at relatively high levels in cotton fibers. As adding exogenous BR (brassinolide, BL) or brassinazole (Brz, a BR biosynthesis inhibitor), expressions of these GhbHLH/HLH genes were up-regulated or down-regulated in cotton fibers. Furthermore, overexpression of GhbHLH282 (one of the BR-response genes) in Arabidopsis not only promoted the plant growth, but also changed plant response to BR signaling.Collectively, these data suggested that these GhbHLH/HLH genes may participate in BR signaling transduction during cotton fiber development. Thus, our results may provide a valuable reference data as the basis for further studying the roles of these bHLH/HLH genes in cotton fiber development.
839	UrdA Controls Secondary Metabolite Production and the Balance between Asexual and Sexual Development in Aspergillus nidulans.	30477161	The genus Aspergillus includes important plant pathogens, opportunistic human pathogens and mycotoxigenic fungi. In these organisms, secondary metabolism and morphogenesis are subject to a complex genetic regulation. Here we functionally characterized urdA, a gene encoding a putative helix-loop-helix (HLH)-type regulator in the model fungus Aspergillus nidulans. urdA governs asexual and sexual development in strains with a wild-type veA background; absence of urdA resulted in severe morphological alterations, with a significant reduction of conidial production and an increase in cleistothecial formation, even in the presence of light, a repressor of sex. The positive effect of urdA on conidiation is mediated by the central developmental pathway (CDP). However, brlA overexpression was not sufficient to restore wild-type conidiation in the ΔurdA strain. Heterologous complementation of ΔurdA with the putative Aspergillus flavus urdA homolog also failed to rescue conidiation wild-type levels, indicating that both genes perform different functions, probably reflected by key sequence divergence. UrdA also represses sterigmatocystin (ST) toxin production in the presence of light by affecting the expression of aflR, the activator of the ST gene cluster. Furthermore, UrdA regulates the production of several unknown secondary metabolites, revealing a broader regulatory scope. Interestingly, UrdA affects the abundance and distribution of the VeA protein in hyphae, and our genetics studies indicated that veA appears epistatic to urdA regarding ST production. However, the distinct fluffy phenotype of the ΔurdAΔveA double mutant suggests that both regulators conduct independent developmental roles. Overall, these results suggest that UrdA plays a pivotal role in the coordination of development and secondary metabolism in A. nidulans.
840	Structural insights showing how arginine is able to be glycosylated by pathogenic effector proteins.	30463645	Glycosylation is one form of protein modification and plays a key role in protein stability, function, signaling regulation and even cancer. NleB and SseK are bacterial effector proteins and possess glycosyltransferase activity, even though they have different substrate preferences. NleB/SseKs transfer the GlcNAc sugar to an arginine residue of host proteins, leading to reduced NF-κB-dependent responses. By combining X-ray crystallography, NMR, molecular dynamics, enzyme kinetic assays and in vivo experiments, we demonstrated that a conserved HEN (His-Glu-Asn) motif in the active site plays a key role in enzyme catalysis and virulence. The lid-domain regulates the opening and closing of the active site and the HLH domain determines the substrate specificity. Our findings provide evidence for the enzymatic mechanism by which arginine can be glycosylated by SseK/NleB enzymes. [BMB Reports 2018; 51(12): 609-610].
841	Hematopoietic Stem Cell Transplantation for the Treatment of Epstein-Barr Virus-Associated T- or NK-Cell Lymphoproliferative Diseases and Associated Disorders.	30460216	Chronic active Epstein-Barr virus infection (CAEBV) is a prototype of EBV-associated T- and/or NK-cell (EBV+ T/NK-cell) lymphoproliferative disorders. Most subtypes of these are lethal. We established a unified treatment strategy composed of step 1 (immunochemotherapy: steroids, cyclosporine A, and etoposide), step 2 (multi-drug block chemotherapy), and step 3 (allogeneic hematopoietic stem cell transplantation; HSCT) for CAEBV and its related diseases. Allogeneic HSCT is the only cure for CAEBV with few exceptions. Primary-EBV infection-associated hemophagocytic lymphohistiocytosis (primary-EBV HLH) is also an EBV+ T/NK-cell lymphoproliferation. The nature of EBV+ T/NK cells in CAEBV and those in primary-EBV HLH differ. In primary-EBV HLH, most patients need step 1 only and some require step 2 for the successful induction of apoptosis in EBV-infected T cells; however, some exceptional patients require HSCT. We herein present our single institutional experience of CAEBV and primary-EBV HLH, together with that of post-transplant EBV+ T/NK-cell lymphoproliferative disease. We also discuss some practical points on HCST with a review of the literature.
842	Bilateral pulmonary nodules and intravascular pulmonary histiocytosis: A rare presentation of hemophagocytic lymphohistiocytosis secondary to Epstein-Barr Virus infection.	30456165	A 61-year-old male presented with worsening dyspnoea and constitutional symptoms for few weeks followed by bloody diarrhoea and loss and fever. Physical exam revealed tachycardia, respiratory distress, and splenomegaly without lymphadenopathy. Work up showed pancytopenia, hypofibrinogenemia, acute kidney injury requiring haemodialysis, high ferritin level, positive IgG and IgM for EBV and positive soluble CD25. Chest CT scan showed bilateral pulmonary nodules. Lung biopsy showed intravascular pulmonary histiocytosis while bone marrow biopsy was negative for hemophagocytes. A diagnosis of hemophagocytic lymphohistiocytosis (HLH) was made based on fulfilling the diagnostic criteria and systemic steroids were initiated, which improved the patient's condition gradually with resolution of dyspnoea, AKI and pancytopenia. Repeat chest CT scan showed resolution of bilateral pulmonary nodules. The patient was transferred to a tertiary centre to receive HLH-specific therapy. We present a rare presentation of HLH with steroid-responsive bilateral pulmonary nodules and a rare histopathologic finding of pulmonary intravascular histiocytosis, which has never been described in HLH or the lung tree.
843	Comparison of Different Diagnostic Guidelines for the Diagnosis of Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: Single Centre Experience.	30431724	Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic juvenile idiopathic arthritis (sJIA), caused by exaggerated but ineffective immune response. The aim of the study was to compare the capacity of the HLH-2004 guidelines with the capacity of the MAS guidelines from 2005, and with the new set of classification criteria from 2016 in diagnosing MAS complicating sJIA. The study included 35 children aged 1-18 diagnosed with sJIA according to ILAR criteria and treated at the Department of Pediatrics, Division of Immunology and Rheu-matology, Zagreb University Hospital Centre, in the period from 2009 to 2015. Out of 35 patients diagnosed with sJIA, there were 12 girls and 23 boys, with the mean age at disease onset (±SD) 5.51±3.65 years. Eight patients had flare of disease. With the guidelines from 2005, MAS was diag-nosed in six (17.1%) patients with sJIA. With the new set of classification criteria from 2016, MAS was diagnosed in four (11.4%) patients with sJIA. MAS was not diagnosed with the HLH-2004 guidelines. In our study, four out of six patients had MAS at the onset of sJIA, and in the rest two it occurred during relapse. Two patients with MAS developed full-blown clinical picture while another four had incomplete clinical features with minor laboratory alteration. Due to the use of different di-agnostic guidelines, we found difference in the prevalence of MAS. It was slightly higher in comparison to available studies, while other researched features, such as clinical characteristics, were similar.
844	Successful resolution of Hemophagocytic lymphohistiocytosis associated to brucellosis in the adult.	30430494	Hemophagocyticlymphohistocytosis (HLH) is a proliferation of histiocytes with importanthemophagocytosisoccurring in different organs such as the spleen and the bone marrow. HLH is now increasingly diagnosed in the context of infections, malignancies and connective tissue diseases. Although brucellosis is an endemic infection in Tunisia, its association with HLH is a very rare condition which should be considered in patients with splenomegaly and cytopenia. Here, we describe brucellosis associated HLH in a 31 year-old man. The patient was admitted to our hospital with fever, sweating, and fatigue. Physical and laboratory findings revealed splenomegaly, pancytopenia, elevated serum transaminases, triglycerides, lactate dehydrogenase, and ferritin, and bone marrow hemophagocytosis. The Brucella agglutination test was positive. The patient improved after treatment with Rifampin and doxycyclin.
845	Successful rescue of acute liver failure and hemophagocytic lymphohistiocytosis following varicella infection: A case report and review of literature.	30430121	Herein we report a case of acute liver failure (ALF) and hemophagocytic lymphohistiocytosis (HLH) induced by varicella infection, successfully rescued by a combination therapy of acyclovir, supportive care, and immunosuppression with dexamethasone and etoposide. A previously healthy 16-year-old boy presented with generalized rash, fever, severe abdominal pain, and abnormal liver function within 4 d. Chickenpox was suspected, and acyclovir and intravenous immunoglobulin were started on admission. However, the patient's condition deteriorated overnight with soaring transaminases, severe coagulopathy and encephalopathy. On the fourth day of admission, pancytopenia emerged, accompanied by hypofibrinogenemia and hyperferritinemia. The patient was diagnosed with ALF. He also met the diagnostic criteria of HLH according to the HLH-2004 guideline. Polymerase chain reaction (PCR) amplifications of varicella-zoster virus (VZV) were positive, confirming that VZV was a causative trigger for ALF and HLH. In view of the devastating immune activation in HLH, immunosuppression therapy with dexamethasone and etoposide was administered, in addition to high dose acyclovir. The patient's symptoms improved dramatically and he finally made a full recovery. To our knowledge, this is only the second report of a successful rescue of ALF associated with HLH, without resorting to liver transplantation. The first case was reported in a neonate infected by herpes simplex virus-1. However, survival data in older children and adults are lacking, most of whom died or underwent liver transplantation. Our report emphasizes the clinical vigilance for the possible presence of HLH, and the necessity of extensive investigation for underlying etiologies in patients presenting with indeterminate ALF. Early initiation of specific therapy targeting the underlying etiology, and watchful immunosuppression such as dexamethasone and etoposide, together with supportive therapy, are of crucial importance in this life-threatening disorder.
846	Hemophagocytic lymphohistiocytosis is a sign of poor outcome in pediatric Epstein-Barr virus-associated post-transplant lymphoproliferative disease after allogeneic hematopoietic stem cell transplantation.	30417487	EBV-related PTLD developing after HSCT is a potentially life-threatening disease. HLH is uncommon after allogeneic HSCT. Data on outcome of patients with PTLD and concomitant HLH after allogeneic HSCT are limited. In this retrospective study, we collected demographic, clinical, laboratory, and outcome data for 408 patients who underwent allogeneic HSCT from 2006 to 2015. Graft source included CB (n = 135; 33.1%), PBSCs (n = 34; 8.3%), and BM (n = 239; 58.6%). Eight out of 408 patients (2%) developed EBV-PTLD with a median age at HSCT of 5.9 years (range: 2.3-17.3). All eight patients received ATG as part of the conditioning regimen. Graft source was PBSC in three patients (37.5%), BM in four patients (50%), and CB in one patient (12.5%). Donors were matched unrelated in five patients (62.5%) and matched sibling in three patients (37.5%). Seven out of eight patients developed EBV-PTLD within the first 100-day post-HSCT. Lymph node biopsy revealed early lesions in three patients, polymorphic in three patients, and monomorphic PTLD in two patients. Three patients (37.5%) died within 1 month of EBV-PTLD diagnosis. All deceased patients developed HLH manifestations with two of them meeting HLH diagnostic criteria and one having an incomplete workup. PTLD after allogeneic HSCT with manifestations of HLH is associated with high mortality. Early identification and treatment of EBV-PTLD seems imperative to control the disease, especially if signs of HLH are evolving.
847	HLH: watch and wait, or act and cure?	3040989430104219	NaN
848	A tissue-specific enhancer of the C. elegans nhr-67/tailless gene drives coordinated expression in uterine stem cells and the differentiated anchor cell.	30404043	The nhr-67 nuclear receptor gene of Caenorhabditis elegans encodes the ortholog of the Drosophila tailless and vertebrate Tlx genes. In C. elegans, nhr-67 plays multiple roles in the development of the uterus during L2 and L3 larval stages. Four pre-VU cells are born in the L2 stage and form the precursor complement for the ventral surface of the mature uterus. One of the four pre-VU cells becomes the anchor cell (AC), which exits the cell cycle and differentiates, while the remaining three VU cells serve as stem cells that populate the ventral uterus. The nhr-67 gene functions in the development of both VU cell lineages and AC differentiation. Hypomorphic mutations in nhr-67 identify a 276bp region of the distal promoter that is sufficient to activate nhr-67 expression in pre-VU cells and the AC. The 276bp region includes 8 conserved potential cis-acting sites, including two E boxes and a nuclear receptor binding site. Mutational analysis demonstrates that the two E boxes are required for expression of nhr-67 in uterine precursor cells. The E/daughterless ortholog HLH-2 binds these sites as a homodimer, thus playing a central role in activating nhr-67 expression in the uterine precursors. At least two other binding activities, one of which may be the nhr-25/Ftz-F1 nuclear receptor transcription factor, also contribute to uterine precursor cell expression. The organization of the nhr-67 uterine precursor enhancer is compared to similar conserved enhancers in the egl-43, lag-2, and lin-3 genes, which contain the same HLH-2-binding E boxes and are similarly expressed in both pre-VU cells and the AC. This basic regulatory module allows the coordinated expression of at least four genes. Expression of genes in different cells that must coordinate to form a mature organ is driven by a shared set of promoter elements, which integrate multiple transcription factor inputs.
849	[Clinical features of adult patients with chronic active Epstein-Barr virus infection].	30392236	Objective: To investigate the clinical features of adult-onset chronic active Epstein-Barr virus infection (CAEBV). Methods: A total of 21 adult patients with CAEBV who were admitted to the department of General Internal Medicine at Peking Union Medical College Hospital from January 2006 to January 2016 were retrospectively analyzed. Demographic data, disease duration, clinical manifestations, laboratory findings, treatments and prognosis were reviewed. Results: Eighteen females and 3 males were enrolled with a mean age of 39 years. The most common clinical manifestations included fever in 20 patients, splenomegaly in 20 patients, lymphadenopathy in 18 patients, and hepatomegaly in 10 patients, followed by laryngopharyngeal disorders in 6 patients, pleural effusion and peritoneal effusion each in 5 patients, rash in 4 patients, interstitial lung disease in 3 patients, gastrointestinal hemorrhage in 2 patients, and peripheral neuropathy and pulmonary hypertension each in 1 patient. Six patients were complicated with hemophagocytic lymphohis-tioncytosis(HLH) that developed 5-17 (mean: 9) months following CAEBV onset, all of whom experienced hyperpyrexia, pancytopenia, lymphadenopathy, splenomegaly, and liver dysfunction, 3 with hepatomegaly. Nineteen of the 21 patients had received steroid therapy including 10 combined with immunosuppressive agents, 11 with antiviral therapy, and 8 with intravenous immunoglobulin. Thirteen patients died, including 10 of multiple organ failure, (including 6 of HLH) 2 of severe pulmonary infection, and 1 of lymphoma. Six patients remained on follow-up, yet 2 were missing. Conclusions: CAEBV is expected with severe condition and poor prognosis, which is likely to be complicated with HLH. Clinical physicians should pay attention to adult patients with fever, hepatosplenomegaly and lymphadenopathy, which suggests possible CAEBV.目的： 探讨成人慢性活动性EB病毒感染（chronic active Epstein-Barr virus infection, CAEBV）的临床特征，为临床诊治提供依据。 方法： 回顾性分析2006年1月至2016年1月于北京协和医院普通内科住院的成人CAEBV患者共21例。 结果： 患者年龄（39.0±16.7）岁，男女比例1∶6。17例（81.0%）以发热为首发症状。最常见临床表现包括发热（20例）、脾脏肿大（20例）、淋巴结肿大（18例）、肝脏肿大（10例），其次为咽喉部病变（6例）、胸腔积液和腹腔积液（各5例）、皮疹（4例）、肺间质病变（3例）、消化道出血（2例）、周围神经病变和肺动脉高压（各1例）。18例出现血白细胞减低，17例贫血，15例血小板减低，15例出现肝功能异常。有6例患者合并噬血细胞性淋巴组织细胞增生症（hemophagocytic lymphohistiocytosis, HLH），发生于CAEBV起病后5~17个月，平均9个月，临床表现为高热、全血细胞减少、淋巴结肿大、脾脏肿大、肝功能损害，3例合并肝脏肿大。21例患者中，19例患者接受过糖皮质激素治疗，其中10例联合免疫抑制剂，11例接受过抗病毒治疗，8例给予静脉丙种球蛋白治疗。13例患者死亡，死亡原因包括多器官功能衰竭10例（其中HLH 6例）、重症肺部感染2例、淋巴瘤1例；6例仍在随访中，2例失访。 结论： 成人CAEBV临床表现复杂，易合并HLH，病情重，预后差，临床医生应早期识别和重视该病，对于不明原因发热、伴有肝脾肿大和淋巴结肿大的成人患者，应警惕CAEBV可能。.
850	Thrombotic thrombocytopenic purpura and hemophagocytic lymphohistiocytosis in an elderly man: A case report.	30383662	Thrombotic thrombocytopenic purpura (TTP) and hemophagocytic lymphohistiocytosis (HLH) are rare hematologic conditions and have high mortality. Both TTP and HLH result from deregulation of the immune system. There are no published reports of coexisting TTP and HLH in elderly patients.A 67-year-old Asian male presented with altered consciousness and fever for 2 days. Physical examination revealed markedly pale, mild icterus with petechiae and purpura. Initially, TTP was recognized in this patient. Bone marrow studies are suggested for evaluating elderly patients to assess specific causes, especially infection and neoplasm.The TTP was diagnosed based on typical history-related symptoms and a specific laboratory result of very low ADAMTS13 level. The diagnosis of HLH was determined after detection of high levels of ferritin and lactase dehydrogenase, which were confirmed by the presence of hemophagocytosis in the bone marrow.Systemic corticosteroids and plasma exchange were initiated as specific treatment of the patient.The patient died in 3 weeks from ventilator-associated pneumonia.The HLH should be tested using bone marrow studies and specific laboratory tests in patients with TTP.
851	Langerhans cell histiocytosis complicated with hemophagocytic lymphohistiocytosis in a boy with a novel XIAP mutation: A case report.	30383659	X-linked lymphoproliferative syndromes (XLPs) are rare, yet often fatal primary immunodeficiency diseases, which rarely manifest as Langerhans cell histiocytosis (LCH) complicated with hemophagocytic lymphohistiocytosis (HLH). Clinical data of a case of XLP-2 manifesting as LCH complicated with HLH was retrospectively analyzed to determine the etiology and causal gene.The diagnosis of multisystem LCH was confirmed by skin biopsy and other examinations in a 13-month boy with recurrent ear discharge, fever and hemorrhagic papules for 3 months. A good therapeutic response to LCH-III protocol-directed induction chemotherapy was achieved but unremitting HLH developed 6 weeks after the initiation of induction chemotherapy. To identify possible underlying genetic causes, gene mutation analysis was undertaken. A novel XIAP gene mutation (c.99delT, p.F33fsX37) was documented.After the diagnosis of HLH had been confirmed, HLH-2004-directed chemotherapy was instituted.The clinical condition of the patient had become progressively deteriorating after 8-week chemotherapy of HLH-2004 protocol, requiring frequent infusions of RBC suspensions and apheresis platelets. His parents decided to receive no further therapy, and the patient died soon after discharge.Meticulous laboratory investigations including genetic studies should be undertaken in young children with LCH complicated with HLH and poor therapeutic response.
852	Acute myeloid leukemia with new complex t(8;21;22) induced hemophagocytic lymphohistiocytosis: A case report.	30383631	The balanced translocation t(8;21;22)(q22;q22;q11.2) is not reported previously, although t(8;21)(q22;q22) is seen in approximately 7% of adults and most frequent abnormality in children with newly diagnosed acute myeloid leukemia (AML). AML-associated hemophagocytic lymphohistiocytosis (HLH) is a rare event, reported only of limited numbers. The present study reports a very rare case of t(8;21;22)(q22;q22;q11.2) with AML, not reported previously, and developed HLH at the same time.A 15-year-old girl presented with a history of bleeding gums and high fever, leukocytosis, anemia, and thrombocytopenia. While waiting the result of bone marrow aspirate, the HLH-associated examinations were abnormal. Bone marrow aspirate showed a hypercellular marrow with 1% myeloblasts. The cytogenetic and molecular studies revealed the presence of abnormal karyotype-46, XX, t(8;21;22)(q22;q22;q11.2) and RUNX1-RUNX1T1 fusion gene. Genetic detections of HLH showed heterozygous genetic variants in lysosomal trafficking regulator (LYST). Hence, she was diagnosed with AML with t(8;21;22)(q22;q22;q11.2) and HLH.All HLH clinical symptoms disappeared after the 4 weeks treatment of HLH. Then the patient received standard AML induction chemotherapy and the leukemia relapsed after 2 cycles of high-dosed consolidation therapy. Eventually, the patient received emergent paternal haploidentical hematopoietic stem cell transplantation based on the complex variant translocation, leukemia replased state and HLH with compound heterozygotes mutation, and achieved sustained remission with RUNX1-RUNX1T1 negative for more than 1 year.Patients with some specific recurrent cytogenetic abnormalities should be diagnosed with AML regardless of the blast count, for example t(8;21). We should improve the understanding of complex variant translocations. HLH-related genetic mutations were not only found in primary HLH, but also in second HLH.
853	Adult onset haemophagocytic lymphohistiocytosis prognosis is affected by underlying disease: analysis of a single-institution series of 174 patients.	30378643	Haemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterised by activation of the mononuclear phagocytic system, and often leads to progressive multiple organ failure. The diagnosis of HLH is made late by most physicians.To confirm the diagnosis of acquired HLH made in a single-institution series of adult patients with HLH-04 criteria, we applied the HScore and evaluated prognostic factors associated with clinical outcome.A total of 174 patients with a median age of 51 years (range 17-90) were included. Male/female ratio was 111/63. In 92/174 (52.9%) patients, there were potential haematological diseases (4 acute leukaemia, 1 thrombotic thrombocytopenic purpura, 3 Hodgkin's lymphoma [HL], 17 B-cell non-Hodgkin's lymphoma [NHL], 67 T-cell NHL including 22 natural killer / T-cell NHL [NK/t-cell NHL). Six (3.4%) patients had autoimmune disease and 76 (43.7%) undiagnosed underlying disease. There were 44 (25.3%) patients with Epstein-Barr virus infection, 11 (6.3%) with cytomegalovirus, 1 (0.5%) syphilis, 9 (5.2%) hepatitis B virus and 3 (1.7%) human immunodeficiency virus. More than 95% of patients had hyperferritinaemia, high lactate dehydrogenase, fever and low albumin, whereas 89.1% of patients had bone marrow phagocytosis. By the HScore, 4/174 patients had a >50% and 16/174 patients had a >90% probability of not having HLH. All 174 patients fulfilled more than five of the HLH-04 diagnostic criteria, but 16 of them had a low probability of HLH by the HScore. In a multivariate analysis, lymphopenia and hypofibrinogenaemia were independent prognostic factors for death.In our study, viral infection was not an independent prognostic factor. NK/T-cell -NHL was associated with worse prognosis compared with B-cell NHL and T-cell NHL (p = 0.036) and similar to other aetiologies.
854	Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.	303740663040521530429576	Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.
855	Hemophagocytic lymphohistiocytosis with neurological manifestations in an infant with scrub typhus: a rare fatal occurrence.	30360694	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of unchecked activation of the immune system leading to phagocytosis of blood cells and proliferation of histiocytes in solid organs. HLH can be primary or secondary to infective, autoimmune and malignant conditions. Scrub typhus is an infective illness caused by Orientia tsutsugamushi, transmitted by mite. The illness ranges from mild fever with rash to severe multisystem illness. Scrub typhus has rarely been associated with secondary HLH. We report an infant with scrub typhus who progressed to develop HLH with central nervous system involvement with fatal outcome.
856	Epstein-Barr Virus-Positive T/NK-Cell Lymphoproliferative Diseases in Chinese Mainland.	30356785	Epstein-Barr virus-positive T/NK-cell lymphoproliferative disorders (EBV+ T/NK LPD) encompass a heterogeneous group of disorders, including chronic active Epstein-Barr virus infection (CAEBV), Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH), systemic EBV+ T-cell lymphoma of childhood and hydroa vacciniforme-like lymphoproliferative disorder (HVLPD) and so on, predominantly affecting children and young adults with high mortality. Patients with EBV+ T/NK LPD have overlapping clinical symptoms as well as histologic and immunophenotypic features. In this review, we summarized the clinical features of EBV+ T/NK LPD in Chinese patients from the published articles.
857	DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity.	30353013	The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat.
858	HLH - Unusual Trigger and Positive Outcome.	30341881	Hemophagocytic Lymphohistiocytosis (HLH), is an uncommon, aggressive and life threatening syndrome of excessive immune activation. We report an unusual case of HLH, in a 34 year old male, who was admitted with Subarachnoid hemorrhage and cerebellar contusion in a Neurosurgical Intensive care unit, whose trigger is not clear.
859	Safety of Follitropin Alfa/Lutropin Alfa for Stimulation of Follicular Development.	30341677	Recombinant human luteinizing hormone (r-hLH) is used in a fixed-ratio combination with recombinant human follicle-stimulating hormone (r-hFSH) for the stimulation of follicular development.The objective of this article was to conduct a review of safety data to evaluate the risks of r-hFSH/r-hLH treatment.Data were retrieved from the Global Safety Database (Merck KGaA, Darmstadt, Germany) including reports from healthcare professionals, patients, health authorities, clinical trials, non-interventional studies, and the literature. Reports of important risks (identified and potential) as per the risk management plan applicable at the time of data retrieval were obtained up to December 2017. The estimated patient exposure to r-hFSH/r-hLH in the post-marketing setting was 427,012 treatment cycles. Nine hundred patients received r-hFSH/r-hLH during company-sponsored clinical trials (pre- and post-marketing).We identified 72 case reports describing important risks related to r-hFSH/r-hLH use, including 46 cases of ovarian hyperstimulation syndrome (10.8 per 100,000 treatment cycles) and 24 of hypersensitivity reaction (5.6 per 100,000 treatment cycles). No thromboembolic events were reported. One congenital anomaly, not suspected to be related to r-hFSH/r-hLH use, was reported during a clinical trial; the event was resolved by corrective surgery. Two fatal cases were identified; one case of recurrent malignant melanoma (suspected to be related to r-hFSH/r-hLH use) and one case resulting from complications of ovarian hyperstimulation syndrome.Cumulative reporting rates of important identified and potential risks of r-hFSH/r-hLH during a 10-year surveillance period demonstrate the benefit-risk balance is positive. This post-marketing surveillance and continued surveillance of safety events should provide reassurance about the use of r-hFSH/r-hLH in clinical practice.
860	The role of inhibitor of binding or differentiation 2 in the development and differentiation of immune cells.	30340915	Inhibitor of binding or differentiation 2 (Id2), a member of helix-loop-helix (HLH) transcriptional factors, is recently reported as an important regulator of the development or differentiation of immune cells. It has been demonstrated that Id2 plays a critical role in the early lymphopoiesis. However, it has been discovered recently that Id2 displays new functions in different immune cells. In the adaptive immune cells, Id2 is required for determining T-cell subsets and B cells. In addition, Id2 is also involved in the development of innate immune cells, including dendritic cells (DCs), natural killer (NK) cells, and other innate lymphoid cells (ILCs). Here, we review the current reports about the role of Id2 in the development or differentiation of main immune cells.
861	Hemophagocytic lymphohistiocytosis, a rare condition in renal transplant - a case report.	30328466	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon and life-threating condition characterized by major immune activation and massive cytokine production by mononuclear inflammatory cells, due to defects in cytotoxic lymphocyte function. It is even more unusual in renal transplant recipients, in which it is often associated with uncontrolled infection. The mortality is high in HLH and differential diagnosis with sepsis is a challenge. The approach and management depend on the underlying trigger and comorbidities. We report a case of a 50-year-old renal transplant female admitted with fever and malaise 3 months post-transplant and presenting anemia, fever, hypertriglyceridemia, high levels of serum ferritin, and positive CMV antigenemia. Urine was positive for decoy cells and BKV-DNA. Graft biopsy showed CMV nephritis. Both blood and urine cultures where positive for E. coli. Hemophagocytosis was confirmed by bone marrow aspiration. Immunosuppression was reduced, and the patient received high-dose intravenous immunoglobulin and dexamethasone, with complete response after 3 weeks. We highlight the importance of early diagnosis and proper management of a rare and serious condition in a renal transplant patient, which can allow a favorable clinical course and improve survival rate.
862	Structural basis for arginine glycosylation of host substrates by bacterial effector proteins.	30327479	The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.
863	What's in a name? The heterogeneous clinical spectrum and prognostic factors in a cohort of adults with hemophagocytic lymphohistiocytosis.	30327177	Hemophagocytic lymphohistiocytosis (HLH) in adults is rare but frequently fatal. Diagnosis is often delayed and treatment approaches vary significantly in contrast to the protocol-driven approach typically used in pediatric HLH. To improve care of these complex patients, this study retrospectively examined the prevalence, clinical characteristics, therapies and outcomes of adult HLH patients at two large tertiary care centers.Adult patients with HLH confirmed by retrospective review of electronic medical records using HLH2004 criteria during admissions to the University of Texas Southwestern and Parkland Memorial Hospitals between June 2007 and June 2017 were studied.Of 31 patients included, 67.7% were male with mean age of 46 years. Average time from admission to diagnosis was 10.5 days. 48% of patients had malignancy, with T-cell lymphoma being most common. Infections were seen in 70%. Autoimmune disorders were found in 9.6%. In total, 13 patients survived (44.8%). Median survival was 8 months with increased mortality in malignancy-associated HLH (median 0.56 months versus 36.5 months, p < 0.001). T-cell lymphoma carried a worse prognosis than other malignancies. Central nervous system disease, hypoalbuminemia, elevated bilirubin, elevated soluble interleukin 2 receptor, and elevated lactate dehydrogenase, were also associated with poor survival. Treatment varied significantly. No individual treatment improved survival.This study corroborates prior limited data in adult HLH patients regarding poor survival, particularly in malignancy-associated HLH. Earlier recognition of this disease and a multidisciplinary approach to streamline diagnosis and optimize treatment are needed to improve outcomes in adult HLH patients.
864	Acute liver failure secondary to severe systemic disease from fatal hemophagocytic lymphohistiocytosis: Case report and systematic literature review.	30310541	To systematically review liver disease associated with hemophagocytic lymphohistiocytosis (HLH), propose reasonable contraindications for liver transplantation for liver failure in HLH, and report an illustrative case.Systematic review according to PRISMA guidelines of hepatic manifestations of HLH using computerized literature search via PubMed of articles published since 1980 with keywords ("hemophagocytic lymphohistiocytosis" or "HLH") AND ("liver" or "hepatic"). Two authors independently performed literature search and incorporated articles into this review by consensus. Illustrative case report presented based on review of medical chart, and expert re-review of endoscopic photographs, radiologic images, and pathologic slides.A 47-year-old Caucasian male, was hospitalized with high-grade pyrexia, rash, total bilirubin = 45 g/dL, moderately elevated hepatic transaminases, ferritin of 3300 ng/dL, leukopenia, and profound neutropenia (absolute neutrophil count < 100 cells/mm³). Viral serologies for hepatitis A, B, and C were negative. Abdominal computed tomography scan and magnetic resonance imaging revealed no hepatic or biliary abnormalities. Pathologic analysis of liver biopsy revealed relatively well-preserved hepatic parenchyma without lymphocytic infiltrates or macrophage invasion, except for sparse, focal hepatocyte necrosis. Bone marrow biopsy and aspirate revealed foamy macrophages engulfing mature and precursor erythrocytes, consistent with HLH. Interleukin-2 receptor (CD25) was highly elevated, confirming diagnosis of HLH according to Histiocytic Society criteria. Patient initially improved after high-dose prednisone therapy. Patient was judged not to be a liver transplant candidate despite model for end stage liver disease (MELD) score = 33 because liver failure was secondary to severe systemic disease from HLH, including septic shock, focal centrilobular hepatocyte necrosis from hypotension, bone marrow failure, and explosive immune activation from HLH. The patient eventually succumbed to overwhelming sepsis, progressive liver failure, and disseminated intravascular coagulopathy. Systematic review reveals liver injury is very common in HLH, and liver failure can sometimes occur. Data on liver transplantation for patients with HLH are very limited, and so far the results have shown a generally much worse prognosis than for other liver transplant indications. Liver transplantation should not be guided solely by MELD score, but should include liver biopsy results and determination whether liver failure is from intrinsic liver injury vs multisystem (extrahepatic) organ failure from HLH.This case report illustrates that liver transplantation may not be warranted when liver failure associated with HLH is primarily from multisystem failure from HLH. Liver biopsy may be very helpful in determining the severity and pathophysiology of the liver disease.
865	A high glucose diet induces autophagy in a HLH-30/TFEB-dependent manner and impairs the normal lifespan of C. elegans.	30299269	A high-glucose diet (HGD) is associated with the development of metabolic diseases that decrease life expectancy, including obesity and type-2 diabetes (T2D); however, the mechanism through which a HGD does so is still unclear. Autophagy, an evolutionarily conserved mechanism, has been shown to promote both cell and organismal survival. The goal of this study was to determine whether exposure of Caenorhabditis elegans to a HGD affects autophagy and thus contributes to the observed lifespan reduction under a HGD. Unexpectedly, nematodes exposed to a HGD showed increased autophagic flux via an HLH-30/TFEB-dependent mechanism because animals with loss of HLH-30/TFEB, even those with high glucose exposure, had an extended lifespan, suggesting that HLH-30/TFEB might have detrimental effects on longevity through autophagy under this stress condition. Interestingly, pharmacological treatment with okadaic acid, an inhibitor of the PP2A and PP1 protein phosphatases, blocked HLH-30 nuclear translocation, but not TAX-6/calcineurin suppression by RNAi, during glucose exposure. Together, our data support the suggested dual role of HLH-30/TFEB and autophagy, which, depending on the cellular context, may promote either organismal survival or death.
866	Histopathologic Correlates of Familial Hemophagocytic Lymphohistiocytosis Isolated to the Central Nervous System.	30295794	Familial hemophagocytic lymphohistiocytosis (HLH) is an immune hyperactivation syndrome caused by mutations in genes associated with cytotoxic T-cell and NK-cell function. While neurological manifestations frequently accompany systemic inflammation at initial presentation, isolated central nervous system (CNS) involvement is rare, and the histological correlates are not well described. We present 3 patients (ages 5, 6, and 7 years) with CNS-isolated familial HLH, who presented with a variety of neurological symptoms and underwent brain biopsies for multifocal enhancing supratentorial and infratentorial lesions. Biopsy slides from all 3 patients revealed similar findings: perivascular lymphocytes, predominantly CD3+ T-cells (CD4>CD8) with occasional intramural infiltration of small vessels; scattered histiocytes without hemophagocytosis; parenchymal and leptomeningeal inflammation varying from mild and focal to severe and sheet-like with associated destructive lesions. There was no evidence of demyelination, neoplasia, or infection. Genetic testing identified compound heterozygous mutations in PRF1 (Patients 1 and 2) and UNC13D (Patient 3), with no evidence of systemic disease except decreased NK-cell function. All 3 patients were treated with hematopoietic stem cell transplantation with marked improvement of symptoms. These findings combined with the poor outcomes associated with delayed diagnosis and lack of aggressive treatment highlight the need to consider HLH in the differential diagnosis of inflammatory brain lesions.
867	[Clinical and pathological features of 13 children with Epstein-Barr virus-positive lymphoproliferative disease].	30293280	Objective: To summarize the clinical characteristics, virological and histopathological features, clinical outcome of Epstein-Barr virus-positive lymphoproliferative disease (EBV(+)LPD) in children. Methods: The clinical and follow-up data of 13 children histopathologically diagnosed as EBV(+)LPD in the Department of Infectious Disease of Beijing Children's Hospital between January 2011 and December 2016 were summarized. Results: Of the 13 patients, 5 were males and 8 females. The median age of disease onset was 6.0 years (range 1.3 to 15.0 years). The median duration between disease onset and diagnosis was 3 months (range 1 to 24 months). All the 13 patients had fever, 9 cases had hepatosplenomegaly and lymphoadenopathy, 4 cases had only lymphoadenopathy, 7 cases had reduced peripheral blood cells, 7 cases had lung involvement, 3 cases had central nervous system involvement, 3 cases had cardiac involvement, 3 cases had intestinal involvement, 2 cases had skin involvement and 1 case had abdominal mass. All the 13 patients underwent whole blood EBV-DNA PCR examination and the copies ranged from 1×10(8)/L to 1×10(11)/L. Pathology of lymph node confirmed 6 cases, skin pathology confirmed 2 cases, lung pathology, ileum mucosa pathology, liver pathology, abdominal mass pathology and bone marrow pathology confirmed 1 case each. Among 13 patients, 9 cases presented with EBV-positive T cell lymphoproliferative disease(EBV(+) T-LPD), 2 cases with hydroa vacciniforme (HV) and 2 cases with EBV-positive diffuse large B-cell lymphoma (EBV(+) DLBCL) . All the patients were followed up for 2 days to 65 months after discharge. Among 9 cases of EBV(+)T-LPD, 1 case died in a short time, 1 case died after evolved to T-cell lymphoma, 2 cases recovered after hematopoietic stem cell transplantation, 1 case recovered after the chemotherapy of hemophagocytic lymphohistiocytosis(HLH) 2004 protocol and 4 cases were stable now. Of 2 cases of HV patients, 1 case died after evolved to HV like lymphoma and the other still have symptoms. Among 2 cases of EBV(+) DLBCL, 1 case died shortly after discharge and the other was still stable after chemotherapy. Conclusions: Chronic recurrent fever, lymphadenopathy and hepatosplenomegaly are the most common clinical manifestations in children with EBV(+)LPD. Involvement of lung, central nervous system, intestinal tract, skin and other organs are also involved frequently. For children with chronic fever of unknown cause and accompanied by lymphadenopathy and (or) hepatosplenomegaly, EBV (+) LPD should be considered highly when the whole blood EBV-DNA load continues to increase significantly, early biopsy of the proliferative lesion should be performed to make a definite diagnosis. The prognosis of EBV (+) LPD is poor, and some evolve to lymphoma, hematopoietic stem cell transplantation is an effective way to treat this disease.目的： 探讨儿童EB病毒阳性淋巴组织增殖性疾病（EBV(+)LPD）的临床表现、病毒学特征、组织病理学特点和临床转归。 方法： 对北京儿童医院感染内科2011年1月至2016年12月收治的13例组织病理学确诊的儿童EBV(+)LPD的临床资料及随访情况进行回顾性病例总结。 结果： 13例患儿中，男5例、女8例。中位年龄6.0岁（范围1.3~15.0岁）。从发病到诊断的中位时间为3个月（1~24个月）。13例均有发热，9例肝脾淋巴结肿大，4例仅有淋巴结肿大；7例外周血细胞降低，7例肺受累，3例中枢神经系统受累，3例心脏受累，3例肠道受累，2例皮肤受累，腹部包块1例。13例均行全血EB病毒-DNA检查，拷贝数1×10(8)/L~1×10(11)/L。病理学检查：6例为淋巴结病理，2例为皮肤病理，肺、回肠黏膜、肝脏、腹腔肿物、骨髓组织病理各1例；13例中9例为系统性EB病毒阳性T细胞淋巴组织增殖性疾病（EBV(+)T-LPD），2例为种痘样水疱病（HV），2例为EB病毒阳性弥漫性大B细胞淋巴瘤（EBV(+) DLBCL）。对13例患儿分别随访至出院后2 d~65个月，9例系统性EBV(+)T-LPD，其中1例短期内死亡，1例进展为T细胞淋巴瘤死亡，2例行造血干细胞移植痊愈，1例行噬血细胞综合征2004方案化疗后痊愈，4例病情尚稳定；2例HV患儿1例发展为种HV样淋巴瘤死亡，另外1例仍有症状；2例EBV(+) DLBCL，1例出院后短期死亡，1例化疗后病情稳定。 结论： 儿童EBV(+)LPD常见表现为慢性反复发热、淋巴结、肝脾肿大，另外可累及肺、中枢神经系统、肠道、皮肤等脏器；对于慢性反复不明原因发热伴有淋巴结和（或）肝脾肿大的，伴全血EB病毒-DNA载量持续显著增高时，应警惕EBV(+)LPD，尽早行增殖病灶组织活检明确诊断；本病预后差，部分进展为淋巴瘤，造血干细胞移植治疗有效。.
868	Establishment of a protein biochip to detect serum IgG antibodies against IL-2 and soluble CD25 in hemophagocytic lymphohistiocytosis.	30292629	Interleukin-2 (IL-2) and soluble CD25 (sCD25) are among the most important cytokines and diagnostic biomarkers in hemophagocytic lymphohistiocytosis (HLH). Detecting serum level of IL-2 and sCD25 is valuable for making clinical diagnosis and treatment decision in HLH.Since tests showing serum IgG antibody against IL-2 or sCD25 have never been carried out, a new protein biochip, which was modified with cysteine and activated sophorolipid (Cys-SL), was developed.Limits of detection on the biochip were 78 pg/ml for IL-2 and 39 pg/ml for sCD25, respectively. The data showed that on-chip seroimmunological responses to IL-2 and sCD25 proteins were 20.8% and 83.1% and the seroprevalence of IL-2 and sCD25 IgG antibodies were 45.5% and 57.2%, respectively. Data collection for the seroprevalence of serum antigen-antibody complex of sCD25 was 68.8%. The new biochip model shared similar sensitivity and specificity to chemiluminescent immunoassay (CLIA) in its measuring capacity of serum sCD25.We addressed and confirmed the involvement of serum IgG antibodies against IL-2 and sCD25 as well as Ag-Ab complex of sCD25 in HLH patients. Therefore, this biochip platform would offer a new technological substitution for clinical serological diagnosis of HLH.
869	Fatal case of hemophagocytic lymphohistiocytosis associated with group B streptococcus sepsis: A case report.	30290591	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease characterized by an excessive systemic inflammatory response. HLH is classified as primary or secondary, where the latter may occur in association with many infections. However, no case of HLH has been previously associated with group b streptococcus (GBS) sepsis.We present a fatal case of HLH in a 5-year-old girl with GBS sepsis.The present patient met 5 of the HLH criteria: fever, splenomegaly, bicytopenia, hypertriglyceridemia and/or hypofibrinogenemia, and hyperferritinemia. GBS was identified in 2 sets of peripheral blood bacterial cultures.Empirical antibiotics, inotropes, and immunoglobulins were administered.The clinical course of the patient was fulminant and the patient died of septic shock 10 hours after admission to the hospital.We suggest GBS infection can cause HLH and early awareness of HLH associated with GBS infection and proper effective treatment are necessary to reduce mortality.
870	Haemophagocytic lymphohistiocytosis complicating pembrolizumab treatment for metastatic breast cancer in a patient with the PRF1A91V gene polymorphism.	30287596	Immune checkpoint inhibitor therapy is a modern breakthrough in medical oncology, but it can precipitate inflammatory and autoimmune adverse effects. Among the most serious of these toxicities is haemophagocytic lymphohistiocytosis (HLH), a life-threatening disorder of unbridled immune activation that results in injury to multiple organ systems.Description of a case of pembrolizumab-associated HLH in a patient with a proposed underlying genetic risk factor for its occurrence.We describe a patient with aggressive metastatic breast cancer who developed HLH while undergoing experimental treatment with pembrolizumab, resulting in critical illness and multiorgan system failure. Pembrolizumab discontinuation and high-dose corticosteroids were effective in managing HLH. Subsequent next-generation sequencing of 15 genes associated with HLH revealed a germline polymorphism in perforin-1 (PRF1), PRFA91V, that may have predisposed the patient to develop HLH. The patient has had no evidence of malignancy for 2 years following recovery despite receiving no further cancer-directed treatment.HLH is a rare but serious complication of immune checkpoint blockade. Patients with underlying hypomorphic alleles in PRF1 may be predisposed to develop this toxicity. Further studies are necessary to confirm a possible link between perforin gene mutations and immune checkpoint blockade-associated HLH.
871	Hemophagocytic Lymphohistiocytosis in the Elderly.	302788753041324630528945	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease of massive, dysregulated cytokine release and secondary multiorgan failure and is associated with high mortality. Primary HLH occurs predominately in infants and young children with a genetic predisposition. Acquired HLH is less well characterized and usually occurs in younger adults in the setting of severe inflammation triggered by infection or malignancy. Little is known about the disease in elderly. We report 3 patients >50 years old who presented with multiorgan failure and shock without an identifiable source and were ultimately diagnosed with acquired HLH. We performed a literature review of HLH in adults >50 years of age and identified an additional 68 cases. Mean age was 62 years, with male predominance. Most cases were triggered by infection (49%) followed by malignancy (27%). Nineteen patients were treated with the HLH-94 protocol, 11 received corticosteroids and the remainder received non-HLH specific interventions. Overall mortality was 62%.
872	Hemophagocytic Lymphohistiocytosis Associated with Scrub Typhus: Systematic Review and Comparison between Pediatric and Adult Cases.	30274417	Scrub typhus is a mite-borne bacterial infection caused by Orientia tsutsugamushi. Hemophagocytic lymphohistiocytosis (HLH) is a potential severe complication. Most reported cases of HLH associated with scrub typhus were single cases or case series with a small sample sizes. Thus, no clear consensus exists on clinical manifestations and differences between pediatric and adult cases of this condition.a systematic search of English and Japanese articles from PubMed, PubMed Central, and Directory of Open Access Journals databases was performed from 3 December 2016 to 28 December 2017. The primary outcome was mortality in patients with HLH associated with scrub typhus; secondary outcomes were differences in clinical symptoms, laboratory findings, and treatment between pediatric and adult patients with HLH associated with scrub typhus.thirty cases of HLH associated with scrub typhus were identified (age range: 2 months to 75 years; median age: 21.5 years, male:female ratio, 1:1). Eschar was frequently observed in the pediatric group (p = 0.017), whereas acute kidney injury was more prevalent in the adult group (p = 0.010). Two patients died of intracranial hemorrhage complicated with multiple organ failure; overall mortality rate was 6.7%.HLH associated with scrub typhus could be cured with remarkable improvement using single antibiotic therapy in approximately half the cases, with the mortality rate being relatively lower than that of HLH associated with other secondary causes.
873	Limitations of HLH-2004 criteria in distinguishing malignancy-associated hemophagocytic lymphohistiocytosis.	30272386	Hemophagocytic lymphohistiocytosis (HLH) is characterized by dysregulated immune activation.  Primary HLH involves hereditary deficits in cytotoxic lymphocytes while secondary HLH is triggered by extrinsic factors. The HLH-2004 criteria are widely used for clinical diagnosis, yet their specificity for HLH or their ability to differentiate primary from secondary disease is unclear, potentially leading to inappropriate treatment. We describe several cases where fulfillment of HLH-2004 criteria obscured the diagnoses of underlying malignancies and delayed curative management. These issues are remedied without waiting for genetic testing results through an alternative diagnostic approach using flow cytometry-based immunologic assays and a thorough investigation for malignancy.
874	HLH like disease vs. true HLH in tropical disease.	302699012962578730266533	NaN
875	The rational specimen for the quantitative detection of Epstein-Barr virus DNA load.	30267627	Background Epstein-Barr virus (EBV) DNA load monitoring in blood is essential for the diagnosis of EBV-associated diseases. However, the best-suited blood compartment for detection is still under discussion. The aim of this study was to evaluate the diagnostic value of EBV-DNA load in peripheral blood mononuclear cells (PBMC), plasma and whole blood (WB) samples. Methods A total of 156 patients, including 45 patients with infectious mononucleosis (IM), 57 patients with EBV-associated hemophagocytic lymphohistiocytosis (HLH) and 54 patients with post-transplant lymphoproliferative disorders (PTLD), were enrolled in this study. The EBV-DNA load in PBMC, plasma and WB samples were measured with real-time quantitative polymerase chain reaction (PCR). Results EBV-DNA load of patients with HLH showed no statistical difference in PBMC, plasma and WB samples, while patients with IM and PTLD showed a higher viral load in PBMC samples. The strongest correlation of EBV-DNA level was found between PBMC and WB samples among patients with IM, HLH and PTLD. The follow-up of EBV-DNA showed that the viral load became negative along with the recovery from the disease, while that in WB and PBMC would remain positive for a long time. Conclusions For the diagnosis and monitoring of EBV-DNA, the type of specimen should be chosen reasonably according to the disease. As for IM and HLH, plasma is recommended to quantify the EBV-DNA load, while PBMC and plasma are preferred in PTLD.
876	Finding "intermediate" ground in transplant and HLH.	3026258329997222	NaN
877	Tuberculosis-associated hemophagocytic lymphohistiocytosis with subsequent unmasking cryptococcal immune reconstitution inflammatory syndrome (IRIS) in an HIV-negative man.	30259307	A 22-year-old HIV-negative man from Ghana was diagnosed with severe hemophagocytic lymphohistiocytosis (HLH) induced by multiorgan tuberculosis with peritoneal, hepatic, pericardial, myocardial, pleural, pulmonary, and bone manifestation. His body mass index was 12.9 m2/kg. Bioptic material of a peritoneal biopsy grew M. tuberculosis, sensitive to all first-line antituberculous drugs. HLH resolved with antituberculous therapy, without additional anti-inflammatory therapy being given. The initial CT scan of his brain was normal. After 5 months of antituberculous treatment, he developed a paralysis of the left arm. A cerebral MRT showed ring-enhanced lesions. Blood cultures and lumbar puncture revealed Cryptococcus neoformans var. grubi. The HIV test was repeatedly negative. Antituberculous treatment was continued for a total of 9 months, and additional treatment with antifungal therapy was established. He recovered fully after 14 months of antifungal treatment.
878	Sudden Cardiac Arrest Due to Hemophagocytic Lymphohistiocytosis in a Child.	30253954	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon hyperinflammatory condition in children that may acutely mimic septic shock. Sudden out-of-hospital cardiac arrest in children is also uncommon and may be of unclear etiology upon initial presentation.A 10-year-old previously healthy child presented with sudden cardiac arrest after an insidious course of throat pain, fever, and progressive altered mental status. He was subsequently diagnosed with Epstein-Barr virus-associated HLH and suffered cerebral edema and death. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: HLH has not previously been described as a cause of sudden out-of-hospital cardiac arrest in children. Rapid diagnosis of underlying cause of an unexpected cardiac arrest may help guide appropriate therapy to salvage organ function.
879	Secondary hemophagocytic lymphohistiocytosis in a patient with rheumatoid arthritis and vasculitis: a case report and review of the literature.	32185318	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder characterized by excessive systemic inflammation, caused by uncontrolled activation of lymphocytes and macrophages, which secrete increased amounts of cytokines. HLH may be caused by gene mutations (primary HLH) or associated with malignancy, immunodeficiency, infection or autoimmune disease (secondary HLH). A 58-year-old woman with seropositive rheumatoid arthritis (RA) presented with fever, ulcers on the left foot and in the intergluteal cleft and increased inflammation markers. Clinical and laboratory evaluation, combined with findings from intra-arterial digital subtraction angiography of the lower limbs, pointed towards the diagnosis of vasculitis. Intravenous administration of low-dose cyclophosphamide resulted in recession of fever and decrease of inflammation markers. However, the patient later developed pancytopenia, hepatomegaly, hyperferritinemia, hypofibrinogenemia and hypertriglyceridemia, while bone marrow aspiration demonstrated hemophagocytosis. The diagnosis of HLH was established. An extensive workup excluded malignancies, systemic infections and immunodeficiencies. HLH in our patient was attributed to activation of RA and presentation of vasculitis. Treatment with corticosteroids and intravenous immunoglobulin led to resolution of fever, correction of pancytopenia and complete healing of the ulcers. Timely diagnosis and treatment of HLH is highly important for a favorable outcome for the patients. Treatment of secondary HLH associated with autoimmune diseases involves corticosteroids and/or other immunomodulatory agents, such as intravenous immunoglobulin.
880	Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives.	30237760	The neuropeptide gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Vertebrates possess multiple GnRH forms that are classified into three main groups, namely GnRH1, GnRH2, and GnRH3. In order to gain more insights into the GnRH gene family in vertebrates, we sought to identify which paralogs of this family are present in cartilaginous fish. For this purpose, we searched the genomes and/or transcriptomes of three representative species of this group, the small-spotted catshark, Scyliorhinus canicula, the whale shark, Rhincodon typus and the elephant shark Callorhinchus milii. In each species, we report the identification of three GnRH genes. In catshark and whale shark, phylogenetic and synteny analysis showed that these three genes correspond to GnRH1, GnRH2, and GnRH3. In both species, GnRH1 was found to encode a novel form of GnRH whose primary structure was determined as follows: QHWSFDLRPG. In elephant shark, the three genes correspond to GnRH1a and GnRH1b, two copies of the GnRH1 gene, plus GnRH2. 3D structure prediction of the chondrichthyan GnRH-associated peptides (GAPs) revealed that catshark GAP1, GAP2, and elephant shark GAP2 peptides exhibit a helix-loop-helix (HLH) structure. This structure observed for many osteichthyan GAP1 and GAP2, may convey GAP biological activity. This HLH structure could not be observed for elephant shark GAP1a and GAP1b. As for all other GAP3 described so far, no typical 3D HLH structure was observed for catshark nor whale shark GAP3. RT-PCR analysis revealed that GnRH1, GnRH2, and GnRH3 genes are differentially expressed in the catshark brain. GnRH1 mRNA appeared predominant in the diencephalon while GnRH2 and GnRH3 mRNAs seemed to be most abundant in the mesencephalon and telencephalon, respectively. Taken together, our results show that the GnRH gene repertoire of the vertebrate ancestor was entirely conserved in the chondrichthyan lineage but that the GnRH3 gene was probably lost in holocephali. They also suggest that the three GnRH neuronal systems previously described in the brain of bony vertebrates are also present in cartilaginous fish.
881	Prognostic Factors of Death in 151 Adults With Hemophagocytic Syndrome: Etiopathogenically Driven Analysis.	30225460	To characterize the etiologies and clinical features at diagnosis of patients with hemophagocytic lymphohistiocytosis (HLH) and correlate these baseline features with survival using an etiopathogenically guided multivariable model.The Spanish Group of Autoimmune Diseases HLH Study Group, formed in 2013, is aimed at collecting adult patients with HLH diagnosed in internal medicine departments between January 3, 2013, and October 28, 2017.The cohort consisted of 151 patients (91 men; mean age, 51.4 years). After a mean follow-up of 17 months (range, 1-142 months), 80 patients died. Time-to-event analyses for death identified a worse survival curve for patients with neoplasia (P<.001), mixed microbiological infections (P=.02), and more than 1 infection (P=.01) and glucocorticoid monotherapy (P=.02). According to univariate analyses, platelets of less than 100,000/mm3 (hazard ratio [HR], 3.39; 95% CI, 1.37-8.40), leukopenia (HR, 1.81; 95% CI, 1.01-3.23), severe hyponatremia (HR, 1.61; 95% CI, 1.02-2.54), disseminated intravascular coagulation (HR, 1.87; 95% CI, 1.05-3.34), bacterial infection (HR, 1.99; 95% CI, 1.09-3.63), mixed microbiological infections (HR, 3.42; 95% CI, 1.38-8.46), and 2 or more infectious triggers (HR, 2.95; 95% CI, 1.43-6.08) were significantly associated with death. In contrast, peripheral adenopathies (HR, 0.63; 95% CI, 0.40-0.98) and the immunosuppressive drug/intravenous immunoglobulin/biological therapies (HR, 0.44; 95% CI, 0.20-0.96) were protective against all-cause mortality. Multivariable Cox proportional hazards regression analysis identified 2 or more infectious triggers (HR, 3.14; 95% CI, 1.28-7.68) as the only variable independently associated with death.The mortality rate of adult patients diagnosed with HLH exceeds 50%. Infection with more than 1 microbiological agent was the only independent variable associated with mortality irrespective of the underlying disease, epidemiological profile, clinical presentation, and therapeutic management.
882	Early Testing of Serum Ferritin Facilitates Hemophagocytic Lymphohistiocytosis Diagnosis in Children.	30222642	The clinical and laboratory features of hemophagocytic lymphohistiocytosis (HLH) are nonspecific that makes the definitive diagnosis of HLH very challenging. The disease is almost universally fatal in the absence of early recognition and appropriate therapy. Elevated serum ferritin level is one of the diagnostic markers of HLH disease. We report the value of testing serum ferritin level early in the disease process in 3 pediatric patients who presented with persistent fever and sepsis-like features. Detection of elevated serum ferritin levels facilitated further testing to confirm the diagnosis of HLH and initiate early therapy with good outcomes.
883	A Case of Recurrent Pregnancy-Induced Adult Onset Familial Hemophagocytic Lymphohistiocytosis.	30220951	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal disease primarily of children, characterized by a severe hyperinflammatory state. We describe a case of adult onset familial HLH with a novel exon 19, c.1607G>T (p.Arg536Leu) heterozygous mutation of the UNC13D gene in a 40-year-old woman who developed HLH during her first and second pregnancies, both episodes occurring during the first trimester. Our patient was treated successfully both times with HLH-94 protocol following spontaneous abortions and is currently in the process of getting a bone marrow transplant. We also discuss pregnancy as a potential trigger for late onset familial HLH.
884	Hemophagocytic lymphohistiocytosis associated with an IgG Cold agglutinin.	30220463	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease eventually caused or reactivated by a viral infection, which can also lead to the production of cold agglutinins (CA). The nature of these autoantibodies is usually an IgM, less frequently an IgA or IgG, they agglutinate red blood cells at low temperatures. They can interfere with hematological parameters causing interpretation difficulties. We report a case of a 4-year-old boy who developed an IgG CA during recurrent HLH reactivated by EBV infection. The purpose of this observation is to underline HLH criteria and to analyze CA interference as well as its biological and clinical characteristics.
885	[Hemophagocytic lymphohistiocytosis: tortuous experience in the diagnosis and treatment of one lymphoma].	30220287	The etiology of hemophagocytic lymphohistiocytosis (HLH) is complicated and difficult to diagnose, unexplained HLH often with hematological malignancies. Invasive biopsy can help to find etiology, the results may be affected by the technique and the location of the puncture site. Multiangle puncture can improve the success rate, but the corresponding risk increases. A patient with HLH was admitted to Affiliated Hospital of Zunyi Medical College. The etiology was unknown. Active symptomatic support treatment was conducted, at the same time, finding the evidence of viral infection, autoimmune disease related detection, blood culture, bone marrow puncture smear and spleen biopsy were performed respectively to find the pathogen basis. Spleen hemorrhage was not being controlled after spleen biopsy in patients, and emergency splenectomy was adopted to stop bleeding for saving lives. Finally, the patients died of low protein, pulmonary edema and respiratory failure. The bone marrow puncture and spleen biopsy failed to provide the basis for tumor invasion, while the spleen pathological slices plus immunohistochemical indicate diffuse large B cell lymphoma (DLBCL) after splenectomy, which was identified as malignant tumor-associated hemophagocytic syndrome. Underscoring the high risk of bleeding after tumor-associated splenomegaly puncture and the importance of having emergency plans. Through analyzing the clinical characteristics, diagnosis and treatment of this patient, we hope to improve the clinicians' understanding of HLH and lymphoma.
886	Natural killer/T-cell lymphoma and secondary haemophagocytic lymphohistiocytosis in pregnancy.	30219776	Haemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal disorder. It is challenging to diagnose due to its rarity and variation in clinical presentation, laboratory abnormalities and underlying aetiologies. A reproductive-aged woman, gravida 2 para 1001 at 27 weeks gestation presented with fever, hypotension and subacute upper respiratory infection. She delivered a male infant by caesarean section secondary to fetal distress. Subsequently, she was diagnosed with T-cell lymphoma and secondary HLH. Despite management with supportive care and multiple chemotherapeutic agents, she ultimately died of multiorgan failure. Patients with HLH secondary to malignancy have a particularly poor prognosis. This case highlights the importance of considering secondary HLH in the differential diagnosis of a patient with fever, pancytopenia and systemic symptoms of unclear aetiology in pregnancy.
887	Cytomegalovirus-associated haemophagocytic lymphohistiocytosis: a rare cause of febrile neutropenia during cancer chemotherapy.	30217800	Febrile neutropenia (FN) is a common complication in patients with cancer during treatment with antineoplastic drugs. The initial cause is usually bacterial, and treatment of FN follows well-defined algorithms. We report a case of a 62-year-old patient with chronic lymphocytic leukaemia (CLL), who developed FN, which was unresponsive to both empirical antibacterial and empirical antifungal therapy. Surprisingly, a diagnosis of the life-threatening condition haemophagocytic lymphohistiocytosis (HLH) associated with cytomegalovirus (CMV) infection was made and treated successfully. CMV-associated HLH has not previously been described in patients with CLL treated with rituximab and bendamustine. It is concluded that HLH should be considered in patients with cancer with FN not responding to conventional antibiotic therapy.
888	Macrophage activation syndrome: early diagnosis is key.	30214327	Macrophage activation syndrome (MAS) is a life-threatening condition, and it is a subset of hemophagocytic lymphohistiocytosis (HLH). The clinical features include a persistent high-grade fever, hepatosplenomegaly, lymphadenopathy, hemorrhagic manifestations, and a sepsis-like condition. From the clinical features, it is usually difficult to differentiate between a true sepsis, disease flare-ups, or MAS. Although the laboratory abnormalities are similar to those of a disseminated intravascular coagulation, which shows pancytopenia, coagulopathy, hypofibrinogenemia, and an elevated d-dimer test, it can also be a late stage of MAS. Currently, MAS is still underrecognized and usually results in delayed in diagnosis, which leads to high morbidity and mortality. This literature review was conducted in the context of the clinical manifestations and the laboratory abnormalities in MAS, which might provide some clues for an early diagnosis. The best ways for an early recognition and a satisfactory diagnosis were based on the relative changes in the overall parameters from the baseline, together with a thorough and continuous physical examination for these kinds of patients. At present, diagnostic criteria have been proposed for HLH, MAS-associated systemic juvenile idiopathic arthritis, and an MAS-associated systemic lupus erythematosus. Therefore, selecting the proper diagnostic criteria for use is essential because not all of the criteria are suitable for every autoimmune disease.
889	Hemophagocytic Lymphohistiocytosis in Adults: Low Incidence of Primary Neoplasm as a Trigger in a Case Series from Turkey.	30210740	Hemophagocytic Lymphohistiocytosis (HLH) is an indicator of an exaggerated immune response and eventually adverse outcomes. This study aimed to investigate the clinical and laboratory features and outcomes of patients with HLH. The medical records of 26 HLH adult patients (≥ 16 years of age) were retrospectively analyzed. Gender, age, the duration of fever, time to diagnosis, etiology and laboratory data were extracted from the records. The mean age was 38 ± 18 years, and 15 (58%) patients were female. A total of nine cases had infectious diseases; four cases had rheumatologic diseases, three cases had hematological malignancies while nine cases could not have a definitive diagnosis. The median time to detection of HLH was 20 days (IQR: 8-30 d). Of the 25 patients, 11 (44%) died. The erythrocyte sedimentation rates of the surviving and non-surviving patients were 39 ± 22 mm/h and 15 ± 13 mm/h, respectively. When a long-lasting fever is complicated by bicytopenia or pancytopenia (especially), clinicians should promptly consider the possibility of HLH syndrome to improve patients' prognosis.
890	Hemophagocytic lymphohistiocytosis and congenital factor VII deficiency: a case report.	30208845	Hemophagocytic lymfohistiocytosis (HLH) is a rare, life-threatening hyperinflammation, characterized by immune system over-activation resulting in hemophagocytosis. HLH could appear as a primary disease caused by mutations of immune-regulatory genes, or develop as a result of viral or bacterial infections, or malignancy. Congenital factor VII (FVII) deficiency is a rare autosomal recessive disorder characterized by prolonged prothrombin time (PT) and low FVII, which may increase bleeding risk.A 50-year-old woman was admitted for a fever persisted for 20 days, presenting with cytopenia, high hyperferritinemia, low activity of NK cells. Bone marrow aspiration showed hemophagocytosis. CT scanning found pulmonary infection. EBV and CMV were not detected. Genetic scanning did not find pathogenic mutation of a HLH NGS panel including 26 genes. This patient was treated as recommended by the HLH 2004 Guidelines. Coagulation tests identified FVII deficiency. Genetic analysis of F7 gene in the patient and her family members identified recurrent compound heterozygous F7 c.64 + 5G > A and c.1224 T > G (p.His408Gln) mutations in this patient and her brother who showed postoperative hemorrhage after surgical resection of renal cell carcinoma. Heterozygotes in this family were asymptomatic.To our knowledge, this is the first report of HLH in combination with congenital FVII deficiency in Chinese population.
891	Oral features of Griscelli syndrome type II: A rare case report.	30207398	Griscelli syndrome (GS) is an autosomal-recessive disorder of the vesicle transport and membrane trafficking system first identified by Griscelli et al in 1978. The three types of GS have specific genetic defects and systemic manifestations apart from classic partial pigmentary dilution, resulting in hypopigmentation of skin and silvery hair. GS-II occurs due to a defect in the Rab27a gene and is characterized by primary immune deficiency along with accelerated phases of a hemophagocytic lymphohistiocytosis (HLH) crisis. This rare disorder has been widely studied for dermatological, hematological, and neurological manifestations; however, the oral features and presentations have not been elucidated in detail. This report presents a case of a 4-year-old male with known mutation c.550C > T or p.R184X mutation (ENST00000396307) in Rab27a with oral features.
892	Extreme hyperferritinaemia, soluble interleukin-2 receptor, and haemophagocytic lymphohistiocytosis.	3020383729672840	NaN
893	Recommendations for the Use of Etoposide-Based Therapy and Bone Marrow Transplantation for the Treatment of HLH: Consensus Statements by the HLH Steering Committee of the Histiocyte Society.	30201097	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome requiring aggressive immunosuppressive therapy. Following 2 large international studies mainly targeting pediatric patients with familial disease and patients without underlying chronic or malignant disease, the HLH-94 protocol is recommended as the standard of care when using etoposide-based therapy by the Histiocyte Society. However, in clinical practice, etoposide-based therapy has been widely used beyond the study inclusion criteria, including older patients and patients with underlying diseases (secondary HLH). Many questions remain around these extended indications and published reports do not address several practical issues. To tackle these concerns, the HLH Steering Committee of the Histiocyte Society decided to issue guidance for use of the HLH-94 protocol. The group convened in a structured consensus finding process to define recommendations that are based largely on expert opinion backed up by available data from the literature. The recommendations address all main elements of HLH-94 including corticosteroids, cyclosporin, etoposide, intrathecal therapy, and hematopoietic stem cell transplantation (HSCT) and consider various forms of HLH and all age groups. Aspects covered include indications, applications, dosing, side effects, duration of therapy, salvage therapy, and HSCT. These recommendations aim to provide a framework to guide treatment decisions in this severe disease.
894	Hemophagocytic lymphohistiocytosis (HLH) presenting as fever of unknown origin and acute liver failure.	30191126	A 51-year-old previously healthy woman presenting with two-weeks of fever, flu-like symptoms, jaundice, and abdominal pain was found to have pancytopenia, transaminitis, and significantly elevated ferritin in the setting of an Epstein-Barr Virus (EBV) infection. Bone marrow biopsy revealed phagocytic macrophages consistent with findings of hemophagocytic lymphohistiocytosis (HLH). Given bone marrow findings and that the patient had five of the eight clinical criteria supporting the diagnosis of HLH, chemotherapy was initiated as per the HLH-94 protocol with initial improvement in patient's symptoms and overall functional status. This case demonstrates a classic presentation of HLH and displays the importance of correct diagnosis and prompt treatment.
895	Hemophagocytic lymphohistiocytosis presenting with annular erythema multiforme-like eruptions in a patient with angioimmunoblastic T cell lymphoma: A case report.	30186440	Angioimmunoblastic T cell lymphoma (AITL)-associated hemophagocytic lymphohistiocytosis (HLH) rarely occurs with annular erythema multiforme-like rashes. The present case report describes a patient who was misdiagnosed with erythema multiforme at an early stage of the disease due to annular erythema multiforme-like eruptions. However, antihistamine treatment was ineffective. The patient progressed rapidly with high fever, hepatosplenomegaly and pharyngitis. The number of copies of Epstein-Barr virus DNA continuously increased. Accompanied by the swelling of lymph nodes, the blood cell count decreased. Further bone-marrow examination and biopsy of the lymph nodes were conducted. The patient was eventually diagnosed with AITL-associated HLH, and treated with etoposide together with cyclophosphamide, doxorubicin, vincristine and prednisolone. The patient was successfully treated with several courses of chemotherapy. In view of the fact that AITL-associated HLH with annular erythema multiforme-like rashes is relatively rare worldwide and is associated with a high mortality rate, the data on previous cases were reviewed with the hope of providing clinical bases for early diagnosis and treatment of AITL-associated HLH.
896	Daphnane diterpenes inhibit the metastatic potential of B16F10 murine melanoma cells in vitro and in vivo.	3015778530227834	Melanoma is one of the most invasive and aggressive types of cancer with a very poor prognosis. Surgery remains the most efficient treatment prior melanoma invasion and metastasis formation. However, therapy becomes a challenge once the cancer cells colonized other tissues. At present, there are two main classes of therapies acting with a certain efficiency on metastatic melanoma: immune check point inhibitors (anti-PD1/PDL1) and targeted therapy such as Vemurafenib. Unfortunately, these therapies are not fully responsive, induce resistance and/or generate unwanted side effects. In this respect, it is important to continue to discover new cancer therapeutics. Here, we show that daphnane diterpenes type of compounds can prevent melanoma metastasis by inhibiting metastasis-associated matrix metalloproteinases expression without cytotoxicity.Evaluation of the anti-metastasis effect of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) and its bioactive component gnidilatidin was carried out in vitro using B16 murine melanoma cells and in vivo using male C57BL/6 J mice. Global gene expression in B16 cells was done using DNA microarray, validated using real-time PCR, to further understand the effect of daphnane diterpenes, specifically daphnane diterpenoid gnidilatidin.Oral administration of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) suppressed MMP2 and MMP9 expression, decreasing lung tumor in mice injected with B16 murine melanoma cells. Validation of these observations in vitro showed reduced B16 cells migration, adhesion, and invasion. Results of microarray analysis of B16 cells treated with daphnane diterpenoid gnidilatidin from TH revealed an upregulation of tumor suppressor Egr1 while inhibiting metastasis-associated genes Id2 and Sytl2 expression. A downregulation of the melanoma oncogene microphthalmia-associated transcription factor (Mitf) was observed, and most likely caused by the inhibition of Id2, a gene that regulated HLH transcription factors such as MITF and also reported to promote tumor cell migration and invasion.Daphnane diterpenes have inhibitory effect on the metastatic potential of B16 melanoma cells, and the results of this study provided evidence for their potential for use in the prevention and inhibition of melanoma metastasis.
897	Fueling the FIRES: Hemophagocytic lymphohistiocytosis in febrile infection-related epilepsy syndrome.	30132834	Although secondary hemophagocytic lymphohistiocytosis (HLH) has been reported in children with critical illness of various etiologies, it has not been reported in patients with febrile infection-related epilepsy syndrome (FIRES). We describe a series of patients with concurrent HLH and FIRES in an effort to establish common pathophysiologic abnormalities.Five patients with FIRES who were assessed for HLH were identified from a neurocritical care database. All were previously healthy and had extensive diagnostic testing. All had clinical deterioration with multiorgan dysfunction prompting HLH screening 20-29 days after hospitalization. Markers for inflammatory dysregulation were assessed in cerebrospinal fluid (CSF) and serum at various time points. Outcomes were assessed 6 months after presentation.Three patients met clinical criteria for secondary HLH. Elevation of specific cytokines/chemokines was variable. CSF neopterin, high mobility group box 1 (HMGB1), and C-X-C motif chemokine ligand 8 (CXCL8) were significantly elevated in all. Interleukin-1β (IL-1β) and IL-18 were not elevated in any of the samples. Treatment and outcomes were variable.We describe 3 patients with HLH and FIRES. The co-occurrence of these 2 rare disorders suggests the possibility of a common immune dysregulation phenotype prolonging epileptogenesis. HLH screening in critically ill patients with FIRES may yield a broader understanding of shared inflammatory processes.
898	Hemophagocytic Lymphohistiocytosis Complicating Systemic Sarcoidosis.	30131931	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening hyperinflammatory syndrome characterized by histiocyte proliferation and hemophagocytosis. Primary HLH is caused by genetic defects, whereas secondary HLH occurs in the setting of underlying diseases, such as infections, malignancies, or rheumatic diseases. Rheumatic diseases, such as systemic juvenile arthritis or systemic lupus erythematosus, have been associated with HLH. However, the association between sarcoidosis and HLH has been rarely reported in the literature. Herein, we report a case of a 36-year-old female who was recently diagnosed with sarcoidosis, and she developed fatal HLH that was not responsive to high-dose steroids.
899	Autoimmune hemolytic anemia: An unusual presentation of hemophagocytic lymphohistiocytosis in a pediatric post-liver transplant patient.	30129086	Hemophagocytic lymphohistiocytosis (HLH), a rare condition characterized by immune dysfunction with uncontrolled activation of macrophages and hypersecretion of cytokines, has only been reported in a small number of pediatric patients following solid organ transplant (SOT). The diagnosis of HLH after SOT is especially difficult, as several of the diagnostic criteria, including fever, splenomegaly, and cytopenias, are nonspecific and can be seen with other post-transplant complications. Autoimmune hemolytic anemia (AIHA) has also been reported after pediatric SOT and is thought to be related to immunosuppression, specifically tacrolimus. Although HLH and AIHA have been separately described following SOT, there have been no reports of them occurring together in post-liver transplant (LT) patients. We report the first case of autoimmune hemolysis as the presenting symptom of HLH in a pediatric post-LT patient.
900	[Clinical Analysis of EB Virus Infection Complicated with Hemophagocytic Syndrome and Hodgkin's Lymphoma].	30111410	To investigate the clinical characteristics and outcome of parhents with EBV infection conbined with hemophagocytic syndrome and Hodgkin's lymphoma.The morphotogy of bone marrow cells was observed by bone marrow smear and light microscopy, the pathologic changes of bone marrow ware analyzed by bone marrow biopsy and immunohistochemistry methord, the pathologic changes of lymphonudes ware detected by immunohistochemical methord, the paticnts were treated with ABVD （epirubicin, bleomycin, vincristine and dacarbazine） chemotherapeutic regimen.Fever complicatid with pancytopenia, obvious increase of ferritin and sCD25, hypofibrinogenemia, hemophogocytic phenomen of bone marrow, increase of EBV-DNA copy number ware observed, which all accorded with the criteria EBV righted hemophagocytic syndrome. The curative efficacy of amtiinfective treatmatnt was poor, After treatment with HLH-2004 regimen, the fever symptome and the laboratory indicaters such as whole blood cells, ferritin and fibrinogen all were recovered to normal levels. Left mandibular lymphadenctasis was confirmed as Hodgkin's lymphoma （mixed cell type） by pathological examination. The patient achieved complete molecular remission after 1 course chemotherapy with ABVD regimen. The level of EBV-DNA copy number were also decreased. As the reshlt, the patient's hemophagocytic syndrome had bean effectively controlled, and the Hodgkin's lymphoma is still in complete remission.Epstein-Barr virus-ratated hemophagocytic syndrome and Hodgkin's lymphoma are rare, and their long-term prognosis needs to be further explored.
901	Dengue hemophagocytic syndrome: A systematic review and meta-analysis on epidemiology, clinical signs, outcomes, and risk factors.	30109914	We systematically searched and meta-analyzed the epidemiological characteristics, frequency of clinical signs, and outcomes of dengue-associated hemophagocytic lymphohistiocytosis. Ten electronic databases were searched systematically plus manual search of reference lists to identify relevant articles published until May 2017. The highest number of reported cases were from South-East Asia region (62 cases), followed by Western Pacific region (20 cases), and America (31 cases). The term "dengue hemorrhagic fever" predominated in studies that used the World Health Organization 1997 definition (59.7%), whereas "severe dengue" predominated in studies using the World Health Organization 2009 definition (76.8%). Among 122 cases, fever, splenomegaly, hepatomegaly, anemia, thrombocytopenia, and serum ferritin ≥500 μg/L were likely to report by articles representing by large sample size. The pooled proportion of these findings were as follows: fever 97.2%, hepatomegaly 70.2%, splenomegaly 78.4%, thrombocytopenia 90.1%, anemia 76.0%, and serum ferritin ≥500 μg/L 97.1%. This study highlighted a high case fatality rate (14.6%) and co-infection among dengue hemophagocytic lymphohistiocytosis patients. We suggest that long fever duration, persistent thrombocytopenia, elevated serum ferritin, and lactate dehydrogenase levels could be good diagnostic indicators for dengue-associated hemophagocytic syndrome. Bone marrow aspiration could be used as one criterion for diagnosis but is not obligatory. Further research is needed to examine the possible risk difference for development of hemophagocytic syndrome and to explore potential relationships between specific dengue classifications and dengue-associated hemophagocytic syndrome.
902	Epstein-Barr-positive classical Hodgkin lymphoma-associated haemophagocytic lymphohistocytiosis: a rare case.	30108117	Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition, which is usually triggered by autoimmune disorders, viral infections and malignancy, like lymphomas. We present a 60-year-old Hispanic woman with a medical history of hypertension and systemic lupus erythematosus presenting with fever, generalised weakness and shortness of breath for 3 weeks. She was hypotensive on presentation, and a CT scan of abdomen/chest showed multiple irregular hepatic and splenic hypodense lesions. A liver biopsy revealed Classical Hodgkin lymphoma (cHL) with positive Epstein-Barr virus (EBV) staining that was later confirmed with high serum EBV DNA levels. Incidentally, a liver biopsy disclosed haemophagocytosis in some cells. HLH-associated cHL is an uncommon condition that can cause severe systemic symptoms acting as the perfect mimic of septic shock, deviating the clinical eye toward treating with antibiotics and not addressing in a timely manner the real aetiology of the patient's condition.
903	Treatment dilemmas in asymptomatic children with primary hemophagocytic lymphohistiocytosis.	3010421930409894	Asymptomatic carriers (ACs) of pathogenic biallelic mutations in causative genes for primary hemophagocytic lymphohistiocytosis (HLH) are at high risk of developing life-threatening HLH, which requires allogeneic hematopoietic stem cell transplantation (HSCT) to be cured. There are no guidelines on the management of these asymptomatic patients. We analyzed the outcomes of pairs of index cases (ICs) and subsequently diagnosed asymptomatic family members carrying the same genetic defect. We collected data from 22 HSCT centers worldwide. Sixty-four children were evaluable. ICs presented with HLH at a median age of 16 months. Seven of 32 ICs died during first-line therapy, and 2 are alive after chemotherapy only. In all, 23/32 underwent HSCT, and 16 of them are alive. At a median follow-up of 36 months from diagnosis, 18/32 ICs are alive. Median age of ACs at diagnosis was 5 months. Ten of 32 ACs activated HLH while being observed, and all underwent HSCT: 6/10 are alive and in complete remission (CR). 22/32 ACs remained asymptomatic, and 6/22 have received no treatment and are in CR at a median follow-up of 39 months. Sixteen of 22 underwent preemptive HSCT: 15/16 are alive and in CR. Eight-year probability of overall survival (pOS) in ACs who did not have activated HLH was significantly higher than that in ICs (95% vs 45%; P = .02), and pOS in ACs receiving HSCT before disease activation was significantly higher than in ACs receiving HSCT after HLH activation (93% vs 64%; P = .03). Preemptive HSCT in ACs proved to be safe and should be considered.
904	Ligand and solvent control of selectivity in the C-H activation of a pyridylimine-substituted 1-naphthalene; a combined synthetic and computational study.	30101960	The pyridylimine-substituted 1-naphthalenes, 2-(1-C10H7)-6-{CR[double bond, length as m-dash]N(2,6-i-Pr2C6H3)}C5H3N (R = Me HLMe, H HLH), react with Na2[PdCl4] in acetic acid at elevated temperature to afford either ortho-C-Hnaphthyl activated (LMe)PdCl (2ortho) or the unactivated adduct (HLH)PdCl2 (1b). Alternatively, 1b and its ketimine analogue (HLMe)PdCl2 (1a), can be prepared by treating (MeCN)2PdCl2 with either HLMe or HLH in chloroform at room temperature. Regio-selective ortho-C-H activation to form 2ortho can also be initiated by the thermolysis of 1a in acetic acid, while no reaction occurs under similar conditions with 1b. Interestingly, the C-H activation of HLMe to give 2ortho is found to be reversible with 100% deuteration of the peri-site occurring on reacting Na2[PdCl4] with HLMe in acetic acid-d4. By contrast, heating 1a in toluene gives a 55 : 45 mixture of 2ortho and its peri-activated isomer 2peri. Pure 2peri can, however, be obtained either from (LMe)PdOAc (3peri) by OAc/Cl exchange or by the sequential reactions of 1a with firstly silver acetate then with aqueous sodium chloride. Intriguingly, a peri to ortho interconversion occurs on heating 2peri in acetic acid to give 2ortho. DFT calculations have been used to investigate the C-H activation steps and it is found that in acetic acid ortho-C-H activation is kinetically and thermodynamically favoured but peri-CH activation is kinetically accessible (ΔΔG‡ = 2.4 kcal mol-1). By contrast in toluene, the reaction appears to be irreversible with the difference in barrier height for ortho- and peri-C-H activation being very small within the error of the method (ΔΔG‡ = 0.7 kcal mol-1), findings that are in agreement with the empirically observed product distribution for 2ortho and 2peri. Single crystal X-ray structures are reported for 1a, 1b, 2ortho and 2peri.
905	E proteins sharpen neurogenesis by modulating proneural bHLH transcription factors' activity in an E-box-dependent manner.	30095408	Class II HLH proteins heterodimerize with class I HLH/E proteins to regulate transcription. Here, we show that E proteins sharpen neurogenesis by adjusting the neurogenic strength of the distinct proneural proteins. We find that inhibiting BMP signaling or its target ID2 in the chick embryo spinal cord, impairs the neuronal production from progenitors expressing ATOH1/ASCL1, but less severely that from progenitors expressing NEUROG1/2/PTF1a. We show this context-dependent response to result from the differential modulation of proneural proteins' activity by E proteins. E proteins synergize with proneural proteins when acting on CAGSTG motifs, thereby facilitating the activity of ASCL1/ATOH1 which preferentially bind to such motifs. Conversely, E proteins restrict the neurogenic strength of NEUROG1/2 by directly inhibiting their preferential binding to CADATG motifs. Since we find this mechanism to be conserved in corticogenesis, we propose this differential co-operation of E proteins with proneural proteins as a novel though general feature of their mechanism of action.
906	[Hemophagocytic Lymphohistiocytosis].	30086687	Hemophagocytic Lymphohistiocytosis Abstract. Hemophagocytic lymphohistiocytosis (HLH) is a group of rare diseases characterized by over-activation of the immune system. They form two groups: primary and secondary HLH. Primary HLH are linked to mutations impairing lymphocyte cytotoxicity. Secondary HLH are triggered by infections, autoimmune diseases or neoplasia, the remaining cases being labeled idiopathic. HLH manifest as febrile states, cytopenias and hepatosplenomegaly. In the absence of treatment, they quickly lead to multiple organ failure. The diagnosis is currently based on the presence of several clinical and biological markers. Treatment consists of suppression of the triggering factor, organ support and immunosuppression. Primary forms, affecting a pediatric population, have been the subject of intense research, and are nowadays treated with established therapeutic protocols. Several recent retrospective studies have improved our knowledge of secondary HLH, which affects mostly adults and whose incidence seems to be increasing. Thus, new diagnostic criteria are currently being studied for secondary HLH, and several treatment protocols have just been published or are being evaluated.Zusammenfassung. Die hämophagozytische Lymphohistiozytose (HLH) umfasst eine Gruppe seltener Krankheiten, welche mit einer schwerwiegenden Entzündungsreaktion durch ungezügelte Aktivierung des Immunsystems einhergehen. Für das Erscheinungsbild ist auch die Bezeichnung Makrophagenaktivierungssyndrom gebräuchlich. Man unterscheidet primäre und sekundäre Formen der HLH. Erstere treten bereits im Säuglingsalter auf und sind durch Mutationen im Bereich der zytotoxischen Lymphozytenfunktion bedingt. Die sekundären Formen werden durch Infekte, Autoimmunerkrankungen und bösartige Bluterkrankungen und Tumoren hervorgerufen oder sind idiopathisch. Die verschiedenen Formen der HLH manifestieren sich einheitlich mit hohem Fieber, einer Hepatosplenomegalie und Zytopenien. Ohne Behandlung kommt es zum Multiorganversagen mit meist tödlichem Verlauf. Die Diagnose beruht zurzeit auf der Zusammenstellung mehrerer klinischer und biologischer Merkmale. Die Behandlung basiert auf der Entfernung der Ursache, Unterstützung des Kreislaufes und der Organfunktion und der Verabreichung von Immunosuppressiva. Die primären HLH wurden intensiv erforscht und gut etablierte Therapieprotokolle liegen für pädiatrische Fälle vor. Es liegen nun auch mehrere retrospektive Studien über die sekundären HLH vor, welche vor allem Erwachsene betreffen und eine steigende Inzidenz verzeichnen. So werden nun neue Diagnosekriterien und moderne Behandlungsansätze vorgeschlagen, welche in diesem Artikel zusammengefasst sind.
907	[CME: Acquired Hemophagocytic Lymphohistiocytosis].	30086677	CME: Acquired Hemophagocytic Lymphohistiocytosis Abstract. Acquired hemophagocytic lymphohistiocytosis comprises a heterogenous group of hyperinflammatory immunoreactions often resulting in uncontrolled immune responses, mainly throughout proliferation of cytotoxic T cells and hemophagocytosis by macrophages. Hemophagocytic lymphohistiocytosis is often underdiagnosed, contributing to its high morbidity and mortality. A systematic diagnostic approach and the use of established diagnostic criteria should lead to an early diagnosis, which is crucial for any therapeutic attempt to achieve a curative state of the disease.Zusammenfassung. Sekundäre hämophagozytische Lymphohistiozytose-Syndrome beschreiben eine vom Erscheinungsbild heterogene Gruppe überschiessender entzündlicher Reaktionen des Immunsystems, die durch Hyperinflammation mit Vermehrung zytotoxischer T-Lymphozyten und Makrophagen bei gesteigerter Hämophagozytose-Aktivität reagieren. Das sekundäre hämophagozytische Lymphohistiozytose-Syndrom wird häufig unterdiagnostiziert, was zu einer hohen Morbidität und Mortalität beiträgt. Die Abfrage der etablierten diagnostischen Kriterien in einem systematischen Abklärungsalgorithmus soll helfen, durch frühzeitige Diagnosestellung und Initiation einer passenden Therapie höhere Heilungsraten zu erreichen.
908	Contribution of Inhibitor of Differentiation and Estrogenic Endocrine Disruptors to Neurocognitive Disorders.	30081481	The devastating growth in the worldwide frequency of neurocognitive disorders and its allied difficulties, such as decline in memory, spatial competency, and ability to focus, poses a significant psychological public health problem. Inhibitor of differentiation (ID) proteins are members of a family of helix-loop-helix (HLH) transcription factors. ID proteins have been demonstrated to be involved in neurodevelopmental and depressive diseases and, thus, may influence neurocognitive deficiencies due to environmental exposure. Previously, it has been demonstrated that environmental factors, such as estrogenic endocrine disruptors (EEDs), have played an essential role in the influence of various neurocognitive disorders such as Alzheimer's, dementia, and Parkinson's disease. Based on this increasing number of reports, we consider the impact of these environmental pollutants on ID proteins. Better understanding of how these ID proteins by which EED exposure can affect neurocognitive disorders in populations will prospectively deliver valuable information in the impediment and regulation of these diseases linked with environmental factor exposure.
909	A prospective quality improvement initiative in adult hemophagocytic lymphohistiocytosis to improve testing and a framework to facilitate trigger identification and mitigate hemorrhage from retrospective analysis.	30075527	Hemophagocytic lymphohistiocytosis (HLH) is a highly fatal, hyperinflammatory syndrome in adults triggered by an underlying illness in most cases. As such, suspicion of HLH dictates further investigation to identify the HLH trigger and determine treatment. HLH is clinically challenging due to diverse presentations and underlying triggers, provider unfamiliarity, and bleeding complications. Clinically, we observed diagnostic error from incorrect testing and cognitive biases (interleukin-2 confused with soluble interleukin-2 receptor and natural killer cell quantification confused with functional assays).This study reports our single institutional experience with adult HLH with the aim to reduce erroneous testing with a quality improvement (QI) project, and to facilitate trigger discovery and mitigate hemorrhage. Provider education on HLH testing was the prospective intervention, followed by mistaken test removal. HLH triggers and diagnostic utility were determined by retrospective chart review. Risk factors for hemorrhage were determined by multivariable analysis.Erroneous HLH testing was reduced from 74% to 24% of patients (P < .001) by the QI intervention. These changes were projected to save $11,700 yearly. The majority (64%) of patients evaluated for HLH were on non-hematology/oncology services, highlighting the need for vigilance in hematology consultation. Sixty-three patients met classic HLH-2004 criteria for HLH. Malignancy (38%), infection (27%), Epstein-Barr virus (EBV) (14%), or autoimmune disease (8%) triggered most HLH cases. HLH triggers were most commonly identified by serologic testing (27%) and bone marrow biopsy (19%). Biopsy of other affected organs based on PET-CT imaging after unsuccessful initial diagnostic measures was helpful, and focal fluorodeoxyglucose uptake was predictive of an underlying malignancy (likelihood ratio 8.3, P = .004). Major hemorrhage occurred in 41% of patients. On multivariable analysis the odds ratios (OR) for major hemorrhage were increased for patients with intensive care unit level care (OR 10.47, P = .005), and disseminated intravascular coagulation in the first week of admission (OR 10.53, P = .04).These data are incorporated into a framework to encourage early HLH recognition with the HScore, facilitate trigger identification, identify those at risk for hemorrhage, and minimize low-yield or erroneous testing.
910	Hemophagocytic lymphohistiocytosis after certolizumab treatment in a patient with rheumatoid arthritis.	30071926	Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening syndrome that may be triggered by hereditary factors, autoimmune and immunologic disorders, infectious diseases, malignancies and medications. Suspicion of the disease and early treatment is of paramount importance. Since the presentation of HLH with only skin involvement is rare, early diagnosis may be difficult. A pathologically confirmed HLH case that presented with maculopapular skin lesions after certolizumab treatment is being reported in this presentation.
911	Is an infectious trigger always required for primary hemophagocytic lymphohistiocytosis? Lessons from in utero and neonatal disease.	30070073	In this report, we evaluate the hypothesis that hemophagocytic lymphohistiocytosis in patients with defects of lymphocyte cytotoxicity is usually triggered by infections. We show that in the majority of patients, extensive virus PCR panels performed in addition to routine microbiological investigations remain negative and summarize 25 patients with onset of hemophagocytic lymphohistiocytosis in utero or within the first 10 days of life, in none of which an associated bacterial or viral infection was reported. These observations, even though preliminary, invite to consider a key role of lymphocyte cytotoxicity in controlling T-cell homeostasis also in the absence of apparent infectious stimuli.
912	Lymphoma associated hemophagocytic syndrome: A single-center retrospective study.	30061947	To improve the understanding of lymphoma associated hemophagocytic syndrome (LAHS) and find an effective treatment for this fatal disease, 57 patients with LAHS were retrospectively reviewed. The most common histopathological type was extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKL) (45.61%). Patients with B-cell LAHS were significantly older (P<0.001), and exhibited a higher triglyceride level (P=0.012), lower serum ferritin level (P=0.014) and lower plasma Epstein-Barr virus DNA (P<0.001) compared with patients with T/NK-cell LAHS. The median survival time of all patients was 43 days, and patients with B-cell (n=14) and T/NK-cell (n=43) LAHS had a median survival time of 55 and 40 days, respectively (P=0.797). Compared with patients who were treated based on HLH-2004 protocols combined with multidrug chemotherapy, those without chemotherapy had a reduced prognosis (P=0.002). The patients that underwent hematopoietic stem cell transplantation (HSCT) following chemotherapy had a significantly improved overall survival (OS) compared with patients that did not undergo HSCT (P=0.001). Patients with B-cell LAHS treated with rituximab (P=0.015) and patients with ENKL treated with L-asparaginase/pegaspargase (L-asp/peg) (P=0.009) had an improved prognosis compared with patients not treated with these drugs. In the T/NK-cell LAHS group, patients treated with chemotherapy containing gemcitabine did not exhibit an improved OS compared with those not treated with gemcitabine (P=0.326). Furthermore, multivariate analysis demonstrated that long diagnosis time and poor performance status were independent prognosis factors for all patients with LAHS. The present study indicated that survival time does not differ between patients with B-cell LAHS and patients with T/NK-cell LAHS. Early diagnosis and appropriate immunochemotherapy plus HSCT are essential to achieve improved outcomes. The outcome of patients with B-cell LAHS may be significantly improved following treatment with rituximab. L-asp/peg-containing regimens are promising treatments for patients with NK/T-cell LAHS.
913	Central Nervous System Involvement in 179 Chinese Children with Hemophagocytic Lymphohistiocytosis.	30058574	Central nervous system (CNS) involvement is found in many patients with hemophagocytic lymphohistiocytosis (HLH). In this study, we mainly analyzed neurological symptoms, imaging findings, cerebrospinal fluid (CSF), and their relationship with outcomes of HLH children.Related data of 179 Chinese pediatric patients with HLH admitted to our center from January 2010 to December 2015 were analyzed retrospectively. Diagnosis and treatment were based on the HLH-2004 protocol. Two-tailed Chi-squared test was used to compare between different groups, and Kaplan-Meier survival curves were used to analyze the overall survival (OS) of patients with HLH.In the present study, 21.2% (38/179) of total patients had neurological symptoms including seizure, irritability, somnolence, and unconsciousness. There were 80 (50.0%, excluding 19 patients without imaging data) patients with cranial imaging abnormalities. There were 14.7% (17/116, excluding 63 patients who did not accept lumbar puncture) of patients with abnormal CSF results. CNS involvement is defined as abnormalities in one or more of CNS symptoms, radiological findings, and CSF. Thus, 60.3% of them had CNS involvement. As for the prognosis, the median follow-up time was 3.2 years (17 lost to follow-up). The probable 3-year OS of children was higher without CNS involvement (86.0% ± 4.6%) than those with CNS involvement (68.9% ± 4.9%, hazard ratio [HR] = 2.286, P = 0.019). Among them, the probable 3-year OS of children without CNS symptoms was 76.0% ± 3.8%, higher than with CNS symptoms (59.5% ± 8.1%, HR = 2.147, P = 0.047). The 3-year OS of children with abnormal CSF was 64.7% ± 11.6%, compared with normal CSF (85.1% ± 3.7%, HR = 0.255, P = 0.038).HLH patients with CNS involvement might have worse outcomes compared with those without CNS involvement, and CNS symptoms and CSF changes are more important to access the prognosis than imaging abnormality.对179例诊断嗜血细胞性淋巴组织细胞增生症患儿中枢神经系统受累情况的临床分析背景：嗜血细胞性淋巴组织细胞增生症（Hemophagocytic Lymphohistiocytosis, HLH）是一种严重威胁患儿生命的临床综合征，许多HLH患儿合并中枢神经系统（CNS）受累。本文主要分析了HLH患儿的CNS症状、影像学表现、脑脊液和预后之间的关系。 方法：回顾性分析了2010年1月至2015年12月之间就诊于我中心的179例HLH患儿的相关数据。诊断和治疗均根据HLH-2004方案。卡方检验用于分析各组之间的构成比，Kaplan-Meier生存曲线用于分析预后。 结果：在这179例HLH患儿中，21.2%（38/179）存在神经系统症状，包括抽搐、易激惹、嗜睡、意识障碍等，80名患儿（50%，排除19例未做影像检查者）存在影像学异常，14.7%（17/116，排除63例未行腰穿者）具有脑脊液异常。存在症状、影像学、脑脊液三者其一异常即被认为具有CNS受累。因此，60.3%的HLH患儿具有CNS受累。预后方面，平均随访时间为3.2年（17名失访）。合并CNS受累的患儿3年生存率（68.9%±4.9%）明显低于无CNS受累组（86.0%±4.6%,风险比[HR]=2.286, p=0.019）。其中，存在CNS临床症状患儿的生存率（59.5%±8.1%）亦低于无CNS症状组（76.0%±3.8%,HR=2.147, p=0.047）。脑脊液异常患儿生存率（64.7%±11.6%）低于脑脊液正常组（85.1%±3.7%,HR=0.255, p=0.038）。 结论：存在CNS受累的HLH患儿预后明显差于无CNS受累者，并且合并CNS临床症状和CSF异常相对于影像学改变更具意义。.
914	Parvoviral disease in childhood cancer: Clinical outcomes and impact on therapy at a tertiary cancer center in India.	30058287	Parvovirus-B19 disease in immunocompromised children can cause myelosuppression and therapeutic delays. We studied the clinical profiles of children having symptoms suggestive of parvoviral disease at our institution, a large tertiary cancer center.Children below age 15 years undergoing treatment for malignancies with clinical features suggestive of parvoviral infection, and/or unexplained drop in hemoglobin, and/or prolonged cytopenia were screened for parvovirus infection using DNA-PCR for parvovirus-B19 (PB19) in the peripheral blood. Patients testing positive from September 2014 till February 2017 were studied.Of the 59 patients (36 patients with hematolymphoid malignancies, 23 with solid tumors) screened for suspected parvoviral infections, 27 tested positive. Median age was 9.6 years (2.25-15 years), 18 (66%) had hematolymphoid malignancies, while 7 (33%) had solid tumors. Six patients (26%) were on intensive phases, 16 (60%) patients developed the symptoms during maintenance chemotherapy, and 4 (15%) after completion of therapy. Isolated anemia was the commonest feature seen in 10 patients (37%) while bicytopenia and pancytopenia were noticed in 8 (30%) and 9 (33%) patients respectively. Fifty percent of patients those who received rituximab (3/6) developed persistent parvoviremia (>4 weeks) as compared with 24% (5/21) of those who did not. Two patients (7%) developed hemophagocytic lymphohistiocytosis (HLH). Treatment delay by more than 14 days was encountered in a majority (62%), with 5 patients requiring treatment modification or even suspension.Parvoviral infection in children who are on or have recently completed chemotherapy can lead to multiple cytopenias and significant treatment delays. Rituximab exposure may lead to persistent parvoviral disease (p < 0.05). HLH, though occasional, can be a serious complication.
915	Adult-onset hemophagocytic lymphohistiocytosis type 2 presenting as a demyelinating disease.	30053752	Familial hemophagocytic lymphohistiocytosis (HLH) is a rare autosomal recessive life-threatening multisystem inflammatory disorder. It is characterized by excessive production of cytokines and uncontrolled activation of lymphocytes and macrophages leading to widespread organ infiltration and tissue destruction. Central nervous system involvement is common occurring in approximately 75% of patients. The neurological symptoms often develop during the course of the disease. However, they can be the initial presenting manifestation. In this article, we describe a patient with adult-onset familial HLH who presented solely with neurological involvement with lack of the initial classical presentation of HLH. He was initially misdiagnosed as acute disseminated encephalomyelitis and later on as multiple sclerosis. This paper indicates that familial HLH may present with pure neurological involvement. A high index of suspicion should be practiced with patients who present with vague recurrent neurological symptoms associated with abnormal non-specific neuroradiological findings. Genetic testing should be included in the investigations of such cases. Steroids and plasma exchange are non-specific therapies that may mask certain conditions including HLH or may be part of the treatment regimen. This may cause a delay in the diagnosis of the underlying causative disease.
916	Type 2 familial hemophagocytic lymphohistiocytosis in half brothers: A case report.	30045285	We describe a novel case of half-brothers suffering from type 2 familial hemophagocytic lymphohistiocytosis (FHL).A 15-year-old Chinese child was admitted to the hematology department. PRF1 gene coding revealed that he was c.282C>A/p.N94K heterozygous and had a c.1349C>T/p.T450M heterozygous mutation. One year later, his younger halfbrother suffered from the same disease. PRF1 gene coding revealed that the younger brother was c.282C>A/p.T450M heterozygous with a c.1349C>T/p.T450M heterozygous mutation. His mother and grandfather were confirmed to have c.1349C>T/p.T450M heterozygous mutations in exon 3.Half-brothers were diagnosed for type 2 familial hemophagocytic lymphohistiocytosis INTERVENTIONS:: To our knowledge, this is a possible FHL and the children's mother may be a pathogenic gene carrier.After being treated with the HLH-04 schedule, the symptoms of half-brothers were all improved.Therefore, once FHL is diagnosed, HSCT needs to be done early, even if no perfect match is found.
917	Successful Haploidentical Stem Cell Transplant With Posttransplant Cyclophosphamide for Hemophagocytic Lymphohistiocytosis.	30044345	Allogeneic hematopoietic stem cell transplant (HSCT) has been known to be a curative therapy for patients with hemophagocytic lymphohistiocytosis (HLH) but donor availability is an issue. Haploidentical HSCT with posttransplant cyclophosphamide (PTCy) has been investigated as a feasible option for various malignant and nonmalignant conditions with reduced incidence of acute graft versus host disease (GVHD) and graft rejection. However, its use has not been described in children with HLH and here we describe 2 boys who underwent successful haploidentical HSCT with PTCy. None had acute GVHD and 1 had limited chronic GVHD. Both are alive and disease-free at follow-up of 912 and 239 days, respectively. Haploidentical HSCT with PTCy is a feasible option for children with HLH lacking a matched sibling donor.
918	Transfusion requirements and 30-day mortality predictors for adult hemophagocytic lymphohistiocytosis.	30043331	Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by an uncontrolled hyper-inflammatory response. We assessed the transfusion requirements and predictors of 30-day mortality for adult HLH patients. We identified all adult patients with a diagnosis of HLH at a large academic hospital from October 2003 through February 2017. We extracted patients' clinical and laboratory data, including transfusion requirements, from their medical records. One-hundred sixteen patients were identified. Their median age was 48 years (range 18-82); 72(62%) were male. Median duration of hospital stay was 19 days (range 1-89 days). At 30 days from admission, 81(70%) patients were alive. Death was attributed to sepsis in 21 patients, lymphoma in six, bleeding in four, GVHD in one, liver failure in one, metastatic solid tumor in one, and unknown in one. Transfusion requirements at 30 days from admission were as follows: RBC, 86% of patients, median 6 units (range 1-58); platelets, 74% of patients, median 6 units (1-67); plasma, 40% of patients, median 4 units (1-56). Renal failure (OR = 4.39; P = 0.008) and hypofibrinogenemia (OR = 4.07; P = 0.009) correlated with 30-day mortality. The transfusion requirements for adult HLH patients were high. Our study indicated that renal insufficiency and hypofibrinogenemia are predictors of early death in adult HLH.
919	Prognostic Factors and Outcomes in Adults With Secondary Hemophagocytic Lymphohistiocytosis: A Single-center Experience.	30037588	Hemophagocytic lymphohistiocytosis (HLH) is a disorder caused by severe immune activation. There are no specific criteria to establish the diagnosis in adults; however, the HLH-04 criteria are among the most commonly used. The HScore is a non-validated tool that can also be useful for HLH diagnosis.We describe the prognostic factors and outcomes of 64 adults diagnosed with HLH in a reference medical center in Mexico City. We included patients ≥ 18 years with HLH, diagnosed and treated at our institution from 1998 to 2016.The median age was 35 years (range, 18-77 years). The underlying cause of HLH was lymphoma in 33 (51.56%) patients (MA-HLH). Cutaneous involvement was more frequent in MA-HLH (33.33%), when compared with patients with non-malignancy associated HLH (NM-HLH) (9.68%) (P = .022). Neurologic symptoms were more frequent in NM-HLH (25.81%) versus MA-HLH (6.06%) (P = .032). After a median follow-up of 14 months (range, 0-216 months), 30-day mortality was 26.56%. Three-year overall survival (OS) was higher for patients with MA-HLH compared with patients with NM-HLH (41% vs. 22.5%; P = .046). Multivariate analysis showed that the presence of nosocomial infection and neurologic symptoms were statistically significant predictors of inferior OS (P = .034 and P = .033, respectively).In this series of adults with HLH, patients with nosocomial infections and neurologic symptoms had a statistically significant worse OS. In the largest series in Latin America, the most common cause of HLH was T-cell lymphoma. In our population, NM-HLH presented a higher mortality.
920	Lateral inhibition: Two modes of non-autonomous negative autoregulation by neuralized.	30028887	Developmental patterning involves the progressive subdivision of tissue into different cell types by invoking different genetic programs. In particular, cell-cell signaling is a universally deployed means of specifying distinct cell fates in adjacent cells. For this mechanism to be effective, it is essential that an asymmetry be established in the signaling and responding capacities of the participating cells. Here we focus on the regulatory mechanisms underlying the role of the neuralized gene and its protein product in establishing and maintaining asymmetry of signaling through the Notch pathway. The context is the classical process of "lateral inhibition" within Drosophila proneural clusters, which is responsible for distinguishing the sensory organ precursor (SOP) and non-SOP fates among adjacent cells. We find that neur is directly regulated in proneural clusters by both proneural transcriptional activators and Enhancer of split basic helix-loop-helix repressors (bHLH-Rs), via two separate cis-regulatory modules within the neur locus. We show that this bHLH-R regulation is required to prevent the early, pre-SOP expression of neur from being maintained in a subset of non-SOPs following SOP specification. Lastly, we demonstrate that Neur activity in the SOP is required to inhibit, in a cell non-autonomous manner, both neur expression and Neur function in non-SOPs, thus helping to secure the robust establishment of distinct cell identities within the developing proneural cluster.
921	Kikuchi-Fujimoto disease in children: two case reports and a review of the literature.	30021595	Kikuchi-Fujimoto disease is a rare, idiopathic and generally self-limiting cause of lymphadenitis of unknow etiology with a low recurrence rate. The typical clinical signs are cervical lymphadenopathy, fever, and symptoms of respiratory infection, and less frequently chills, night sweats, arthralgia, rash, and weight loss.Here we describe two case reports of Kikuchi Fujimoto disease presenting in Milan within the space of a few months. The first involved the recurrence of KFD in a young boy from Sri Lanka; the second was a rare case of severe KFD complicated by HLH.Pediatricians must consider KFD in the differential diagnosis of fever of unknown origin in children, even in western countries. Although rare, recurrence and severe complications are possible. Where symptoms suggest KFD, a systematic diagnostic approach is key. Since no guidelines on the management of KFD are available, further studies should be conducted to investigate the therapeutic options and long term outcome in children.
922	The b-HLH transcription factor Hes3 participates in neural plate border formation by interfering with Wnt/β-catenin signaling.	30016640	Hes3 belongs to the Hes basic helix-loop-helix family of transcriptional repressors that play central roles in maintaining progenitor cells and regulating binary cell fate decisions in the embryo. During Xenopus laevis development, hes3 is expressed in the embryonic ectoderm in a horseshoe shape domain at the edge of the developing neural pate. Hes3 mis-expression at early neurula stage blocks neural crest (snai2, sox8, sox9 and sox10) and cranial placode (six1 and dmrta1) gene expression, and promotes neural plate (sox2 and sox3) fate. At tailbud stage, these embryos exhibited a massive up-regulation of both sox8 and sox10 expression, associated with an increase in genes important for melanocytes differentiation (mitf and dct). Using a hormone inducible construct we show that Hes3 does not induce a pigment cell differentiation program de novo, rather it maintains progenitor cells in an undifferentiated state, and as Hes3 expression subsides overtime these cells adopt a pigment cell fate. We demonstrate that mechanistically Hes3 mediates its activity through inhibition of Wnt/β-catenin signaling, a molecular pathway critical for neural crest specification and pigment cell lineage differentiation. We propose that Hes3 at the edge of the neural plate spatially restricts the response to mesoderm-derived Wnt ligands, thereby contributing to the establishment of sharp boundaries of gene expression at the neural plate border.
923	Child Neurology: Siblings with infantile epilepsy and developmental delay: A circuitous path to genomic diagnosis.	30012655	Chromosome 14q11-q22 deletion syndrome (OMIM 613457) is a rare genomic disorder with a variable phenotype.We report 2 full siblings, a brother and sister, with a unique familial 2.4 Mb microdeletion at 14q13.1-14q13.3 by microarray (first identified in the brother, Mayo Clinical Laboratories, 2010).Both children presented with infantile spasms that evolved to intractable epilepsy and profound developmental delay. They share distinctive dysmorphic features: long expressionless facies, full cheeks, flattened midface, full lips, and generalized hypotonia. Only the sister has hemophagocytic lymphohistiocytosis (HLH). Testing in the brother revealed 3 variants of unknown significance (VUS) (Greenwood Genetics, epilepsy/seizure panel, 145 genes, 2015). The sister had normal results with a different gene panel (GeneDx, infantile epilepsy panel, 75 genes, 2016) but it did not include the 3 genes in which VUS were identified in her brother. Whole exome sequencing in the mother, father, and both siblings was negative without VUS (GeneDx, XomeDx, 2016). There were no variants within the deleted interval in the intact allele for both children. Parental fluorescent in situ hybridization studies for 14q13.1-14q13.3, done in 2017, were normal. Haplotype analysis of the intact chromosome 14 in the sister supported paternal origin for the deletion and likely germline mosaicism in the father. Haploinsufficiency of genes in the deleted region has not been associated with an abnormal phenotype.These children have a specific, recognizable neurodevelopmental phenotype and 14q13 microdeletion. This report highlights the challenges of coordinating and interpreting genetic testing in syndromic epilepsy.
924	Severe hemophagocytic lymphohistiocytosis in a melanoma patient treated with ipilimumab + nivolumab.	30012206	Treatment of metastatic melanoma patients with immune checkpoint inhibitors is an important standard of care. Side effects are due to immune activation, can affect virtually all organ systems, and are occasionally severe. Although hematologic toxicity has been reported, we present a case of hemophagocytic lymphohistiocytosis (HLH) due to immune checkpoint inhibitor therapy.A patient with metastatic melanoma was treated with one course of ipilimumab + nivolumab and presented 3 weeks later with severe anemia and hyperferritinemia. A bone marrow biopsy revealed necrotic tumor cells, infiltrating T cells, and hemophagocytosis. The patient was treated with high-dose steroids; 12 months later, the patient remains off all therapy and in complete remission of both HLH and metastatic melanoma.The hemophagocytic syndromes are attributable to dysregulated immune activation and share pathophysiologic mechanisms with immune activation from checkpoint inhibitors. Increasing use of regimens that include immune checkpoint inhibition require vigilant monitoring for immune-activating side effects as they can occasionally be life threatening, as in this case of HLH.
925	Hemophagocytic Lymphohistiocytosis: Potentially Underdiagnosed in Intensive Care Units.	30010630	Hemophagocytic lymphohistiocytosis in adults (aHLH) is a rare life-threatening hyperinflammatory syndrome caused by excessive activation of macrophages and CD8+ T-cells. Due to the clinical overlap with severe sepsis, aHLH often remains undiagnosed resulting in poor outcome. Here, we present a retrospective study of incidence, clinical findings, and the outcome of aHLH in intensive care units (ICUs).This retrospective analysis was performed at the university hospital Charité - Universitätsmedizin Berlin. We gathered data from 556 out of 46,532 patients admitted to our anesthesiological ICUs between 2006 and 2013, who had at least one plasma ferritin measurement during ICU treatment, and were at least 18 years old. Of these, 244 patients with ferritin at least 500 μg/L and available datasets of at least 4 HLH-2004 criteria were included. HLH-2004 diagnostic criteria and the recently published HScore were used. An aHLH expert team retrospectively reviewed the potential aHLH cases.Seventy-one of the included 244 patients died; 9 out of the 244 patients were retrospectively classified as aHLH of whom 4 patients had died (44.4%). Two of the 9 aHLH patients had been correctly diagnosed and had received specific aHLH treatment. Thus, 7 out of 9 patients (77.8%) remained undetected. ICU patients with at least 1 captured ferritin value and hyperferritinemia showed an aHLH rate of 3.7%, which rises up to 5.6% when only deceased patients are considered. Mortality in this selected cohort is 44.4%.Overall, 7 out of 9 patients (77.8%) suffering from aHLH remained undiagnosed. Awareness of this life-threatening syndrome, especially in ICUs, should be raised. The inclusion of ferritin into the admission lab panel for ICU is warranted.Clinical trial registered with www.ClinicalTrials.gov (NCT02854943) on August 1, 2016. As this is a retrospective study, trial registration was after final data collection date.
926	Reduced-intensity conditioning for hematopoietic cell transplant for HLH and primary immune deficiencies.	2999722230262583	Allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning for disorders associated with excessive inflammation such as hemophagocytic lymphohistiocytosis (HLH) is associated with early mortality. A multicenter prospective phase 2 trial of reduced-intensity conditioning with melphalan, fludarabine, and intermediate-timing alemtuzumab was conducted for HLA matched or single HLA locus mismatched related or unrelated donor HCT in a largely pediatric cohort. Graft-versus-host disease (GVHD) prophylaxis was cyclosporine with methylprednisolone. The primary end point was 1-year overall survival (OS). Thirty-four patients with HLH and 12 with other primary immune deficiencies were transplanted. With a median follow-up of 20 months, the 1-year OS for transplanted patients was 80.4% (90% confidence interval [CI], 68.6%-88.2%). Five additional deaths by 16 months yielded an 18-month OS probability of 66.7% (90% CI, 52.9%-77.3%). Two patients experienced primary graft failure, and 18 patients either experienced a secondary graft failure or required a second intervention (mostly donor lymphocyte infusion [DLI]). At 1 year, the proportion of patients alive with sustained engraftment without DLI or second HCT was 39.1% (95% CI, 25.2%-54.6%), and that of being alive and engrafted (with or without DLI) was 60.9% (95% CI, 45.4 %-74.9%). The day 100 incidence of grade II to IV acute GVHD was 17.4% (95% CI, 8.1%-29.7%), and 1-year incidence of chronic GVHD was 26.7% (95% CI, 14.6%-40.4%). Although the trial demonstrated low early mortality, the majority of surviving patients required DLI or second HCT. These results demonstrate a need for future approaches that maintain low early mortality with improved sustained engraftment. The trial was registered at Clinical Trials.gov (NCT 01998633).
927	Primary bone marrow lymphoma: A hematological emergency in adults with fever of unknown origin.	29984910	Primary bone marrow lymphoma (PBML) represents non-Hodgkin lymphoma (NHL) that primarily arises in the bone marrow (BM) without lymphadenopathy. This condition has various definitions and can be masked by prolonged fever, leading to delayed diagnosis. We aimed to identify clinical features and risk indicators of PBML. We enrolled 269 adults with fever of unknown origin (FUO) who underwent a BM study for potential PBML. Thirty patients were diagnosed with PBML (26 and 4 patients in the training and validation cohort, respectively), and 20 patients (67%) showed initial manifestation of hemophagocytic lymphohistiocytosis (HLH). Among PBML patients in the training cohort, their median overall survival is short (8 days), with pneumonia being the most common direct cause of early mortality, followed by life-threatening HLH. Despite extremely poor prognoses, some B-cell PBML patients who survived 30 days after BM studies achieved long-term survival with rituximab-based treatment. To assist general practitioners in early PBML diagnosis when approaching adults with naïve FUO, we identified several risk indicators, including elevated serum alkaline-phosphate levels, lowered serum immunoglobulin-G levels, cytopenia in ≥2 lineages, and peripheral blood leukoerythroblastosis. Our recently published scoring system, which can predict hematological BM disease in FUO adults, showed excellent ability in recognizing PBML early, with high sensitivity and specificity. We conclude that PBML is a specific "clinical" phenotype of NHL; moreover, we have identified diagnostic clues for early identification of FUO adults with underlying PBML, which should be considered a hematological emergency once suspected in any adult with FUO.
928	A case of developing progressive multifocal leukoencephalopathy while using rituximab and mycophenolate mofetil in refractory systemic lupus erythematosus.	29983569	Progressive multifocal leukoencephalopathy (PML) is a central nervous system infection caused by John Cunningham (JC) virus reactivation in an immunocompromised patient. PML has various neurologic symptoms and has very poor prognosis. A 36-year-old man developed transverse myelitis and had a psychiatric disorder at the age of 26. He was diagnosed with systemic lupus erythematosus (SLE) and neuropsychiatric SLE (NPSLE), on the basis of leukopenia and presence of anti-DNA and anti-nuclear antibodies. Treatment with glucocorticoid (GC) was started, and remission was introduced. Six months before PML onset, his condition was complicated with hemophagocytic lymphohistiocytosis (HLH) due to exacerbation of SLE. Remission re-induction therapy by GC, cyclosporine-A, intravenous cyclophosphamide, and rituximab (RTX) was initiated and HLH improved. However, interleukin-6 levels of the cerebrospinal fluid (CSF) continued to rise. We thought that the disease activity of NPSLE worsened; thus, we introduced mycophenolate mofetil (MMF) 4 months before the PML onset. He developed progressive dysarthria and right hemiplegia. He was diagnosed with PML via magnetic resonance imaging and JC virus polymerase chain reaction in CSF. Considering that immunosuppressants, including RTX and MMF, are precipitating factors of PML, we discussed the RTX removal using plasma exchange (PEx), but we did not introduce PEx, because it was expected that the concentration of RTX was already lowered when he was diagnosed with PML. Treatment for PML with mefloquine and mirtazapine saved his life, but severe residual disabilities remained. This is the first report of a patient who developed PML during combination therapy with RTX and MMF.
929	HLH: genomics illuminates pathophysiological diversity.	2997677929632024	NaN
930	Macrophage activation syndrome in adult systemic lupus erythematosus: report of seven adult cases from a single Italian rheumatology center.	29976044	The aim was to describe the macrophage activation syndrome (MAS), a life-threatening syndrome characterized by excessive immune activation that can be triggered by conditions affecting immune homeostasis, in a cohort of adult Italian patients with systemic lupus erythematosus (SLE). This was a monocentric retrospective evaluation. The utility of the H-score, developed to estimate the individual risk of having reactive MAS in adult patients, was assessed. Among 511 patients with SLE, 7 cases (1.4%) of MAS (all females) were identified and their medical records reviewed. In all cases, MAS was simultaneous to the onset of SLE. All patients had fever, lymphadenopathy, hematological involvement, and high titer of anti-dsDNA antibodies. Workup for infections and malignancies was negative. In all cases, the H-score was higher than the cut-off suggested for the classification of reactive MAS. All cases required hospital admission, and 2 patients were admitted to the intensive care unit. Most patients were treated successfully with high doses of corticosteroids and with immunosuppressive drugs, whereas the full therapeutic regimen developed for primary hemophagocytic lymphohistiocytosis HLH was used only in one case. No death from MAS was observed. MAS is a rare and severe disorder that complicated the onset of SLE in our cohort. The H-score may be useful in the classification of these patients.
931	[Human parvovirus B19-induced hemophagocytic lymphohistiocytosis and myocarditis in an adult patient with hereditary spherocytosis].	29973443	Human parvovirus B19 (HPV-B19) causes hemophagocytic lymphohistiocytosis (HLH). Here we describe a 35-year-old female with hereditary spherocytosis (HS) who developed HLH due to HPV-B19 infection. Upon admission, she had high fever and diarrhea. Laboratory findings included severe pancytopenia and elevated serum triglyceride and ferritin levels. Moreover, high HPV-B19 levels in the peripheral blood and increased reactive lymphocytosis in the bone marrow led to a diagnosis of HLH due to HPV-B19 infection. With supportive therapy and a blood transfusion, HLH symptoms, including fever and myelosuppression, improved in 1 week. However, symptoms of heart failure (HF) suddenly developed, and an echocardiography revealed diffuse systolic dysfunction, suggesting viral myocarditis due to HPV-B19 infection. Conservative management with diuretics gradually improved HF symptoms over a period of 2 weeks. HPV-B19 infection in adult patients with HS rarely results in severe HLH, but conservative therapy may improve the symptoms. Nonetheless, a careful follow-up is required after HLH improves because viral myocarditis can develop, as was seen in our patient.
932	Hemophagocytic lymphohistiocytosis in adults.	29966474	Hemophagocytic lymphohistiocytosis (HLH), a rare but life-threatening condition characterized by uncontrolled inflammation, is increasingly recognized in adults. The management of adult onset HLH is challenging, in part due to gaps in current state of knowledge on etiology, clinical presentation, diagnosis, and management. HLH secondary to triggers such as infections, autoimmune disorders, and malignancy are more commonly seen in adults although cases of familial form have also been reported. Underlying conditions such as sepsis, or malignancy could pose as major confounders while applying universal diagnostic criteria, and therefore could lead to delay in diagnosis. Despite advent of newer therapeutic agents, outcomes of adults continue to remain poor. Future efforts need to be orchestrated to develop evidence-based tailored therapies to improve outcomes of this under recognized heterogeneous entity.
933	Hemophagocytic Lymphohistiocytosis: A Case Series.	29963339	Hemophagocytic lymphohistiocytosis (HLH) has been recognized as an inflammatory endpoint for a variety of conditions including autoimmune diseases, malignancies and infections. It can be further classified as primary and secondary HLH. Primary HLH is also known as familial HLH. It usually presents in childhood and can be associated with gene mutations. Secondary HLH is also known as acquired HLH and usually presents in adulthood. In comparison to children, it is difficult to diagnose adults with HLH since it occurs with a variety of different diseases and most of the literature on HLH is derived from a pediatric population. In this case series, we report two cases of HLH that illustrate the difficulty of making this diagnosis and a brief review of the literature on its pathophysiology, clinical presentation, diagnosis, and a subsequent therapeutic approach.
934	[Cytomegalovirus myositis complicated with hemophagocytic lymphohistiocytosis, acute renal failure, and colitis].	29962438	A 60-years-old previously healthy man presented with acute renal failure and hemophagocytic lymphohistiocytosis (HLH). Both conditions improved after immunotherapies, but severe limb weakness with elevation of serum CK developed. Needle EMG showed myogenic changes with spontaneous activities and muscle weakness thereafter improved without adding further immunotherapies, suggesting that our patient had viral myositis. After the stabilization of limb weakness, cecal perforation occurred due to cytomegalovirus (CMV) enteritis and temporal significant change of anti-CMV IgG antibody titer was confirmed using paired serum samples. Upregulation of MHC-class I molecule and numerous regenerative muscle fibers were observed in muscle biopsy, but no evidence of direct CMV infection in muscle fibers were seen. Although CMV infection may cause either myositis, acute renal failure, HLH or colitis in individual patient, this is the first case which had been complicated by all these conditions subsequent to CMV infection.
935	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in adults and adolescents-a life-threatening disease: analysis of 133 cases from a single center.	29957156	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis is the most common type of infection-associated HLH. Previous studies were focused on pediatric EBV-HLH patients, therefore there lack of adult data.We performed a retrospective analysis of 133 EBV-HLH patients (≥14 years old) in Beijing Friendship Hospital from March 2009 to April 2016 to evaluate the clinical manifestation and the effects and prognosis of existing regimens of EBV-HLH in adult and adolescents.Of these patients, 91 male and 42 female cases had a median age of 26 (14-77) years. EBV-DNA load on admission was at a median of 6.6E + 05 IU/ml. The one-year mortality of these patients was 78%. 112 patients received the HLH-94/04 regimen as the initial treatment, 52 patients (46.43%) had response. Of the 6 patients who received the L-DEP regimen as the initial treatment, 5 patients (83.33%) had response. The rest 15 patients received initial treatment without etoposide, 5 cases achieved PR. 69 refractory or relapsed patients received DEP or L-DEP regimen, 55 (79.71%) cases had response. In addition, who received the L-DEP regimen, with the overall response rate significantly higher than the DEP regimen (88.37% VS 65.38%, P = 0.031). 36 out of 133 EBV-HLH patients eventually received allo-HSCT, with the overall survival rate of 52.78%. In summary, EBV-HLH is a highly lethal disease.DEP/L-DEP was a good salvage treatment. L-DEP might be a more effective first-line initial regimen than HLH-94/04 regimen for EBV-HLH. Finally, allo-HSCT is an effective radical treatment for EBV-HLH.
936	Identification of open chromosomal regions and key genes in prostate cancer via integrated analysis of DNase‑seq and RNA‑seq data.	29956775	Prostate cancer is a type of adenocarcinoma arising from the peripheral zone of the prostate gland, and metastasized prostate cancer is incurable with the current available therapies. The present study aimed to identify open chromosomal regions and differentially expressed genes (DEGs) associated with prostate cancer development. The DNase sequencing data (GSE33216) and RNA sequencing data (GSE22260) were downloaded from the Gene Expression Omnibus database. DNase I hypersensitive sites were detected and analyzed. Subsequently, DEGs were identified and their potential functions were enriched. Finally, upstream regulatory elements of DEGs were predicted. In LNCaP cells, following androgen receptor activation, 244 upregulated and 486 downregulated open chromosomal regions were identified. However, only 1% of the open chromosomal regions were dynamically altered. The 41 genes with upregulated open chromosomal signals within their promoter regions were primarily enriched in biological processes. Additionally, 211 upregulated and 150 downregulated DEGs were identified in prostate cancer, including eight transcription factors (TFs). Finally, nine regulatory elements associated with prostate cancer were predicted. In particular, inhibitor of DNA binding 1 HLH protein (ID1) was the only significantly upregulated TF which exhibited motif enrichment in the promoter regions of upregulated genes. CCCTC‑binding factor (CTCF) and ELK1 ETS transcription factor (ELK1), enriched in the open promoter regions of downregulated genes, were potential upstream regulatory elements. Furthermore, reverse transcription‑quantitative polymerase chain reaction analysis confirmed that ID1 expression was significantly upregulated in LNCaP cells and 5α‑dihydrotestosterone (DHT)‑treated LNCaP cells compared with that in BPH1 cells, while CTCF and ELK1 expression was significantly downregulated in LNCaP cells and DHT‑treated LNCaP cells. In conclusion, ID1, CTCF and ELK1 may be associated with prostate cancer, and may be potential therapeutic targets for the treatment of this disease.
937	Downregulation of CD5 and dysregulated CD8+ T-cell activation.	29920868	CD5 is a cell surface molecule that is expressed on most circulating T cells and a small population of B cells, and is involved in modulation of antigen-specific receptor-mediated activation. Downregulation of CD5 on CD8+ T cells is a poorly understood but increasingly recognized phenomenon that may be associated with dysregulated T-cell activation. An increased subpopulation of activated CD8+ T cells with downregulation of CD5 has recently been described in patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (HLH) and familial HLH caused by perforin deficiency and Munc 13-4 deficiency. These cells were detectable only in the acute phase of HLH, in which patients exhibited hypercytokinemia, and declined progressively after successful treatment in parallel with improvement of systemic inflammation. It is unknown whether CD8+ T cells from HLH with other causes have similar profiles. Assessment of CD5 expression on T cells has the potential to assist in the understanding of the diagnosis and pathogenesis of human inflammatory diseases such as HLH.
938	ROS is the major player in regulating altered autophagy and lifespan in sin-3 mutants of C. elegans.	29912629	SIN3, a transcriptional corepressor has been implicated in varied functions both as transcription activator and repressor. Recent studies associated Sin3 with the macroautophagic/autophagic process as a negative regulator of Atg8 and Atg32. Though the role of SIN3 in autophagy is being explored, little is known about the overall effect of SIN3 deletion on the survival of an organism. In this study using a Caenorhabditis elegans sin-3(tm1279);him-5(e1490) strain, we demonstrate that under in vivo conditions SIN-3 differentially modulates autophagy and lifespan. We provide evidence that the enhanced autophagy and decreased lifespan observed in sin-3 deletion mutants is dependent on ROS and intracellular oxidative stress. Inability of the mutant worms to maintain redox balance along with dysregulation of enzymatic antioxidants, depletion of GSH and NADP reserves and elevation of ROS markers compromises the longevity of the worms. It is possible that the enhanced autophagic process observed in sin-3(tm1279);him-5(e1490) worms is required to compensate for oxidative stress generated in these worms.cat: catalase; DCFDA: 2',7'-dichlorodihydrofluoroscein diacetate; GSH: reduced glutathione; GSSG: oxidized glutathione; H2O2: hydrogen peroxide; HDAC: Histone deacetylase; HID: HDAC interacting domain; him-5: high incidence of males; HLH-30: Helix Loop Helix-30; HNE: 4-hydroxyl-2-noneal; LIPL: LIPase Like; MDA: malondialdehyde; NGM: nematode growth medium; PAH: paired amphipathic α-helix; PE: phosphatidylethanolamine; RFU: relative fluorescence unit; ROS: reactive oxygen species; sin-3/SIN3: yeast Switch Independent; SOD: superoxide dismutase; NADP: nicotinamide adenine dinucleotide phosphate; SQST-1: SeQueSTosome related-1; ATG: AuTophaGy related.
939	Hemophagocytic lymphohistiocytosis.	29904309	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of widespread inflammation due to massive amounts of cytokines released from activated macrophages. The most common trigger for HLH is infection from a virus, most commonly Epstein-Barr virus. Here we report an adult case of this rare and life-threatening syndrome.
940	Infection-associated Hemophagocytic Lymphohistiocytosis: An Unusual Clinical Masquerader.	29900092	Hemophagocytic lymphohistiocytosis (HLH) secondary to an infection is a great impersonator. It is caused by hyperimmune activation, which leads to a wide array of hematological abnormalities. If the disease is untreated, it is usually fatal. We report the case of a four-year-old girl who presented to our tertiary care hospital with high-grade fever, frequent loose stools, and bleeding from the lips and gums. Investigations showed pancytopenia, hyperferritinemia, hypofibrinogenemia, and hypertriglyceridemia whereas the bone marrow biopsy revealed hemophagocytosis with trilineage suppression. Blood cultures grew Salmonella typhi. After ruling out other possibilities, the diagnosis of HLH was made as per the HLH-2004 diagnostic criteria. The patient responded well to culture-sensitive antibiotics and supportive treatment. We discuss the diagnosis and clinical course of this unique case and strive to create awareness about secondary HLH induced by common diseases, such as typhoid fever.
941	Malignancy associated hemophagocytic lymphohistiocytosis in children.	29893316	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder of immune dysregulation resulting in abnormal T-cell activation and inflammatory cytokine production which produces a constellation of clinical features unique to HLH. Pediatric secondary HLH is usually triggered by infection, malignancy, or rheumatological disorders. The diagnosis of malignancy-associated HLH (MA-HLH) poses a difficult challenge as clinical features may be attributed to the underlying disease or chemotherapy. Our study aimed to see the occurrence of this rare entity at our centre.Data were collected from all pediatric oncology patient treated at our center with the diagnosis of MA-HLH from January 2012 to December 2014. Data were collected for age, sex, underlying disease, treatment protocol, stage of chemotherapy, any underlying infection, treatment given for HLH, and outcome.There were five patients with a diagnosis of MA-HLH in the study period. Age ranged from 18 months to 9 years. Of the five MA-HLH, two patients had acute lymphoblastic leukemia, two acute myeloid leukemia, and one had Hodgkin lymphoma. The three patients who had documented microbiological infection also did not improve after appropriate treatment. Two patients died during treatment. One patient improved completely on steroid alone. One patient received HLH 2004 induction.The delay in the diagnosis of MA-HLH in pediatric patients may be due to decrease awareness about the condition the timely diagnosis of MA-HLH is crucial for a better outcome. Herein, we discuss our experience with this rare entity in pediatric oncology patients with review of literature.
942	BMP and FGF signaling interact to pattern mesoderm by controlling basic helix-loop-helix transcription factor activity.	29877796	The mesodermal germ layer is patterned into mediolateral subtypes by signaling factors including BMP and FGF. How these pathways are integrated to induce specific mediolateral cell fates is not well understood. We used mesoderm derived from post-gastrulation neuromesodermal progenitors (NMPs), which undergo a binary mediolateral patterning decision, as a simplified model to understand how FGF acts together with BMP to impart mediolateral fate. Using zebrafish and mouse NMPs, we identify an evolutionarily conserved mechanism of BMP and FGF-mediated mediolateral mesodermal patterning that occurs through modulation of basic helix-loop-helix (bHLH) transcription factor activity. BMP imparts lateral fate through induction of Id helix loop helix (HLH) proteins, which antagonize bHLH transcription factors, induced by FGF signaling, that specify medial fate. We extend our analysis of zebrafish development to show that bHLH activity is responsible for the mediolateral patterning of the entire mesodermal germ layer.
943	Macrophage activation syndrome associated with griscelli syndrome type 2: case report and review of literature.	29875956	Macrophage activation syndrome (MAS) is a severe and potentially fatal life-threatening condition associated with excessive activation and expansion of T cells with macrophages and a high expression of cytokines, resulting in an uncontrolled inflammatory response, with high levels of macrophage colony-stimulating factor and causing multiorgan damage. This syndrome is classified into primary (genetic/familial) or secondary forms to several etiologies, such as infections, neoplasias mainly hemopathies or autoimmune diseases. It is characterised clinically by unremitting high fever, pancytopaenia, hepatosplenomegaly, hepatic dysfunction, encephalopathy, coagulation abnormalities and sharply increased levels of ferritin. The pathognomonic feature of the syndrome is seen on bone marrow examination, which frequently, though not always, reveals numerous morphologically benign macrophages exhibiting haemophagocytic activity. Because MAS can follow a rapidly fatal course, prompt recognition of its clinical and laboratory features and immediate therapeutic intervention are essential. However, it is difficult to distinguish underlying disease flare, infectious complications or medication side effects from MAS. Although, the pathogenesis of MAS is unclear, the hallmark of the syndrome is an uncontrolled activation and proliferation of T lymphocytes and macrophages, leading to massive hypersecretion of pro-inflammatory cytokines. Mutations in cytolytic pathway genes are increasingly being recognised in children who develop MAS in his secondary form. We present here a case of Macrophage activation syndrome associated with Griscelli syndrome type 2 in a 3-years-old boy who had been referred due to severe sepsis with non-remitting high fever, generalized lymphoadenopathy and hepato-splenomegaly. Laboratory data revealed pancytopenia with high concentrations of triglycerides, ferritin and lactic dehydrogenase while the bone marrow revealed numerous morphologically benign macrophages with haemophagocytic activity that comforting the diagnosis of a SAM according to Ravelli and HLH-2004 criteria. Griscelli syndrome (GS) was evoked on; consanguineous family, recurrent infection, very light silvery-gray color of the hair and eyebrows, Light microscopy examination of the hair showed large, irregular clumps of pigments characteristic of GS. The molecular biology showed mutation in RAB27A gene confirming the diagnosis of a Griscelli syndrome type 2. The first-line therapy was based on the parenteral administration of high doses of corticosteroids, associated with immunosuppressive drugs, cyclosporine A and etoposide waiting for bone marrow transplantation (BMT).
944	Hemophagocytic Lymphohistiocytosis in Cutaneous T-Cell Lymphoma.	29874360	Hemophagocytic lymphohistiocytosis (HLH) has been reported as a serious complication of cutaneous T-cell lymphoma (CTCL). Despite available diagnostic guidelines, it remains a diagnostic and therapeutic challenge in this patient population.To examine the characteristics of CTCL associated with HLH and analyze the presenting signs and symptoms, therapeutic options, and outcome.In this case series, patients diagnosed with CTCL and HLH who were treated at a single institution from January 1, 2014, through December 31, 2017, were studied.The HLH-2004 trial criteria, HScore, and various clinical and histopathologic variables were applied to and analyzed in the cohort.Subtype of CTCL, treatment administered for HLH, and patient outcome were assessed.Seven patients (4 men and 3 women; median age, 50 years; range, 34-77 years) were identified from the database and included in the study. Cytotoxic subtypes of CTCL that involve the deep dermis and subcutaneous tissue were most commonly associated with HLH. Four patients met 5 or more HLH-2004 trial criteria, and 5 had an HScore probability greater than 85% at presentation. Common presenting HLH symptoms were fever and malaise. Cyclosporine, polychemotherapy, and systemic corticosteroids were the most common treatments. Patients receiving allogeneic stem cell transplants had the best outcomes, with all 3 of these patients alive and in complete remission.Hemophagocytic lymphohistiocytosis is a life-threatening complication of CTCL associated with rare cytotoxic CTCL subtypes that primarily involve the subcutaneous tissue. Because these cases may resemble a granulomatous or infectious condition, the diagnosis and appropriate management are often delayed. The results of this study demonstrate the need for high awareness of HLH in patients with panniculitic lymphomas and indicate that allogeneic stem cell transplantation may be the best option for a sustained remission.
945	[Haemophagocytic syndromes: The importance of early diagnosis and treatment].	29871839	Haemophagocytic syndrome, or haemophagocytic lymphohistiocytosis (HLH), is a disorder with high mortality, typically recognised at paediatric age. Without proper treatment, HLH can be fatal. The risk of a rapid progression to multi-organ failure and central nervous system involvement leading to long-term sequelae, are the most feared consequences of a diagnostic delay. Therefore, HLH is a medical emergency that paediatricians should be able to identify in a patient with fever and progressive worsening of general condition. The application of the HLH diagnostic criteria, which include clinical and analytical data (as well as a bone marrow aspirate), and the search for a trigger (infectious, oncological, rheumatological, or metabolic). These are decisive for the establishment of a targeted treatment, which aims at neutralising the trigger and reducing the hyper-inflammation. The most relevant data for general paediatricians are presented in this review, including the physiopathology, diagnosis, and treatment of this serious disease.
946	Hemophagocytic lymphohistiocytosis with immunotherapy: brief review and case report.	29871698	Hemophagocytic Lymphohistiocytosis (HLH), a rare but potentially fatal syndrome of immune hyperactivation, may be an under-recognized immune-related adverse event (irAE). Unlike other irAEs, HLH triggered by immune checkpoint blockade is not well described; no particular diagnostic guidelines and treatment regimens exist. The HLH-2004 criteria remain as the common diagnostic guide. For the treatment of HLH, various combinations of chemotherapeutic, immunosuppressive and glucocorticoid agents are used.We report a case of HLH in a 58-year-old metastatic melanoma patient who was undergoing immune checkpoint blockade with pembrolizumab, a programmed cell death-1 (PD-1) receptor inhibitor. The patient presented with fever, upper normal sized spleen, anemia, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, reduced NK cell activity and elevated sCD163 levels, fulfilling the Histiocyte Society HLH-2004 diagnostic criteria. Our patient was successfully treated with oral prednisone (1 mg/kilogram/day), suggesting that HLH from immune checkpoint inhibitors may respond to steroids alone.Early diagnosis and treatment of HLH are critical to avoid progressive tissue damage, organ failure and possibly death. HLH should be suspected in clinical presentations with fever, cytopenias and hyperinflammatory markers. HLH in the setting of immune checkpoint blockade may be treated with steroids only but further evidence is required.
947	Hematologic Malignancies Associated With Mediastinal Germ Cell Tumors: 10 Years' Experience at Thailand's National Pediatric Tertiary Referral Center.	2986411030608488	Mediastinal germ cell tumor (MGCT), which accounts for 1% to 3% of extragonadal germ cell tumors, has unique manifestations; it is associated with several types of hematologic malignancy, particularly myeloid neoplasm. The aim of this study was to report the 10-year incidence, clinical characteristics, and outcomes of MGCT at Thailand's national pediatric tertiary referral center. This retrospective study included patients diagnosed with MGCT at the Department of Pediatrics, Siriraj Hospital during 2005 to 2014. Eight patients (all male) were diagnosed with MGCT. Five of 8 patients were found to have hematologic abnormalities. Three patients were diagnosed with acute myeloid leukemia (AML) (one patient with M1, another having M7, and the other with M0). Another patient had mixed MGCT with mediastinal myeloid sarcoma (MMS). The other patient had malignancy-associated hemophagocytic lymphohistiocytosis syndrome (M-HLH). Isochromosome 12p was detected in 3 patients (AML [2], mixed MGCT/MMS [1]). Four of 5 patients with hematologic abnormalities died of hematologic abnormalities or treatment complication (AML [3], M-HLH [1]). One patient with mixed MGCT/MMS survived with chemotherapy. All patients with AML and MMS were nonseminomatous MGCT and the onset of myeloid malignancies were within 1 year after the diagnosis of MGCT. Associated hematologic malignancies should be suspected in MGCT with abnormal blood count or hematologic symptoms. Isochromosome 12p was the most common cytogenetic finding in MGCT-associated myeloid malignancies patients. Those with nonseminomatous MGCT should have their blood count carefully monitored especially during the first year after the diagnosis of MGCT. Better treatment alternatives for MGCT with associated hematologic malignancies are warranted to ameliorate adverse outcomes.
948	A retrospective series of conditions and mortality associated with extreme hyperferritinaemia in adults.	29855115	Serum ferritin is commonly used in the diagnosis of iron deficiency anaemia. However, extreme hyperferritinaemia suggests a significant illness, including the differential diagnosis of haemophagocytic lymphohistiocytosis (HLH), which is rare and associated with a high mortality, particularly if untreated. This series aims to identify the causes and associated mortality of severe hyperferritinaemia in patients seen at a teaching hospital in London, UK.Demographic and medical data were collected for all patients over 18 years of age with extreme hyperferritinaemia (defined as serum ferritin levels of ≥4000 mcg/L). Conditions associated with hyperferritinaemia and in-hospital mortality were identified from medical records, laboratory data and discharge and death notification.One hundred and fifty-five cases of extreme hyperferritinaemia in adults were identified. Associated conditions included iron overload (35%), malignancy (24%), infectious disease (21%) and hepatocellular disease (12%). Autoimmune disease and HLH resulted in significantly higher median peak ferritin levels compared with all cases (10 616 mcg/L, P < .01 and 19 138 mcg/L, P < .05, respectively). Patients with confirmed HLH had the highest median peak ferritin. Uncommon infections were identified in this series, and included such as dengue, syphilis, HIV and murine typhus. HLH had been confirmed in seven patients (5%). In five patients (3%) no clear cause for raised ferritin was identified. Overall mortality in the whole cohort was 14% (n = 22), but there was a very high mortality of 80% in the group where no cause was found for the hyperferritinaemia, and these patients were significantly more likely to die during the index admission (P < .01).Extreme hyperferritinaemia is associated with a broad differential diagnosis of significant medical conditions, including iron overload, infections, cancer and liver disease. Rare infectious causes were also identified, and this series reports a greater proportion of cases of HLH than has previously reported. Unexplained hyperferritinaemia was associated with significant mortality.
949	Differentiation between incomplete Kawasaki disease and secondary hemophagocytic lymphohistiocytosis following Kawasaki disease using N-terminal pro-brain natriuretic peptide.	29853942	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome with many causes, including Kawasaki disease (KD). The purpose of this study was to identify the laboratory tests needed to easily differentiate KD with HLH from incomplete KD alone.We performed a retrospective study on patients diagnosed with incomplete KD and incomplete KD with HLH (HLH-KD) between January 2012 and March 2015. We compared 8 secondary HLH patients who were first diagnosed with incomplete KD with all 247 incomplete KD diagnosed patients during the study period. The complete blood count, erythrocyte sedimentation rate, platelet count, and serum total protein, albumin, triglyceride, C-reactive protein, N-terminal pro-brain natriuretic peptide (NT-proBNP), and ferritin levels were compared. Clinical characteristics and echocardiography findings were also compared between the 2 groups.The total duration of fever was longer in the HLH-KD group than in the KD group. White blood cell and platelet counts were higher in the KD group. Alanine aminotransferase, ferritin, and coronary artery diameter were increased in the HLH-KD group compared with those in the KD group. The median of NT-proBNP was significantly higher in the HLH-KD group than in the KD group at 889.0 (interquartile range [IQR], 384.5-1792.0) pg/mL vs. 233.0 (IQR, 107.0-544.0) pg/mL.The NT-proBNP level may be helpful in distinguishing incomplete KD from KD with HLH. The NT-proBNP level should be determined in KD patients with prolonged fever, in addition to the white blood cell count, platelet count, and ferritin level, to evaluate secondary HLH.
950	ALK-Negative Anaplastic Large Cell Lymphoma Presenting as Disseminated Intravascular Coagulation and Hemophagocytic Lymphohistiocytosis: A Potentially Fatal Presentation.	29850298	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder that can be familial in etiology or a result of infections, malignancy, and autoimmune or inflammatory disorders. Disseminated intravascular coagulation (DIC) is common in patients admitted to intensive care units and can confound and delay the diagnosis of HLH. We present a case of a 69-year-old female who presented with dyspnea and malaise. Her condition declined rapidly with laboratory parameters consistent with DIC. In addition, she had a ferritin of 32,522 ng/mL, low haptoglobin, and elevated LDH, and bone marrow biopsy showed hemophagocytic lymphohistiocytes. She was started on HLH-directed therapy, and later, a diagnosis of ALK-negative anaplastic large cell lymphoma was made on an excisional inguinal lymph node biopsy specimen. Our case emphasizes the importance of prompt recognition, diagnosis, and treatment of HLH while workup for a primary disorder is still being pursued.
951	Haemophagocytic lymphohistiocytosis occurred during induction chemotherapy in an acute monocytic leukemia patient with FLT3-ITD and DNMT3A mutations.	29843647	Haemophagocytic lymphohistiocytosis (HLH) is considered to be a large challenge for clinicians due to the variable overlaps of symptoms with other severe diseases and a high rate of mortality. Prompt diagnosis and treatment are crucial to avoid a fatal outcome. However, very limited reports have focused on HLH during chemotherapy (Ch-HLH) due to a low incidence and insufficient knowledge.A 22-year-old male was diagnosed with acute monocytic leukemia with FLT3-ITD and DNMT3A mutations and pulmonary infection. He received IA regimen (Idarubicin, 8 mg/m2/d for 3 days and cytarabine, 100 mg/m2/d for 7 days) chemotherapy, anti-infection drugs and blood components transfusions. During the stage of bone marrow suppression, he presented with a fever, cytopenia (WBC, 0.43 × 109/L; Hb, 73 g/L and PLT, 1 × 109/L), refractory coagulation dysfunction (APTT, 104.0 s; PT, 30.5 s and Fbg, 0.87 g/L), splenomegaly (3 cm below the costal margin), hyperferritinemia (SF > 3000 μg/L), increased soluble interleukin-II receptors (sIL-2R > 7500 u/mL) and haemophagocytosis in the bone marrow and was diagnosed with HLH. After he was treated with methylprednisolone at 500 mg/d for 3 days, 120 mg/d for 3 days and 80 mg/d for 3 days, followed by a gradually reduced dose combined with powerful anti-infection drugs, his symptoms subsided and his abnormal parameters recovered to normal levels.Patients with HLH in acute leukemia have a high rate of mortality. This case report provides helpful clinical experiences relative to the recognition and treatment of Ch-HLH for clinicians.
952	Hemophagocytic Lymphohistiocytosis Secondary to Unknown Underlying Hodgkin Lymphoma Presenting with a Cholestatic Pattern of Liver Injury.	29805352	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon disease that often presents with nonspecific findings. A high index of suspicion is necessary to make a prompt diagnosis and prevent fatal disease. A 45-year-old man presented with fever, hypotension, abdominal pain, nausea, and vomiting. Imaging showed hepatosplenomegaly and laboratory tests revealed pancytopenia, increased ferritin, and a cholestatic pattern of injury with elevated alkaline phosphatase and total bilirubin. Due to a history of Crohn disease, systemic lupus erythematous, and rheumatoid arthritis, the patient was on immunosuppressants, including infliximab. After multiple negative cultures, persistent fever, and days of empiric broad spectrum antibiotics, our differential shifted to fever of unknown origin. A liver wedge biopsy revealed areas of sinusoidal dilatation with enlarged, activated macrophages containing erythrocytes and intracytoplasmic iron, consistent with hemophagocytosis due to HLH. The portal tracts showed mixed lymphoplasmacytic inflammation, a prominent bile ductular reaction, periportal fibrosis, and scattered large cells with occasional binucleation and prominent nucleoli. These cells stained positive for Epstein-Barr virus encoding region in situ hybridization, PAX5, CD15, and CD30, and hepatic involvement by classic Hodgkin lymphoma was diagnosed and determined to be the cause of the HLH and cholestatic pattern of injury. Simultaneously, a bone marrow biopsy showed diffuse involvement by Hodgkin lymphoma with a similar staining pattern. Aggressive treatment failed and the patient succumbed to multiorgan failure. HLH is a rare, potentially fatal disease, with nonspecific signs and symptoms, and should be considered in any patient presenting with fever and pancytopenia, especially if they are immune compromised.
953	Hand anthropometry in patients with carpal tunnel syndrome: a case-control study with a matched control group of healthy volunteers.	29802717	The aim of this study was to perform anthropometrical measure- ments of patients' hands with carpal tunnel syndrome (CTS) in order to evaluate if there is a correlation between CTS occurrence and hand features regarding sexual dimorphism, age and physical activity.Study sample consisted of 48 patients (33 females) and control group included 80 healthy volunteers (58 females) with no history of CTS. The following measurements were performed: the wrist circumference, length of the hand, the hand's width, width of the wrist, thickness of the wrist, height of the hypothenar and thenar, length of the arm and forearm, circumference of the proximal phalanges and width of the digits; as well as several indexes were calculated i.e.: body mass index (BMI), shape index, digit index, wrist index, hand length/height ratio (HLH-ratio) and hand length/upper limb length ratio (HLULL-ratio).Correlation coincidences were analysed between circumferences within the hand, palm and body weight. All parameters except fingers were correlated with body weight in either gender in both groups (p < 0.05; r = 0.40-0.80); Furthermore, width of the hand was correlated with body height (p < 0.001; r = 0.56-0.71). Mean values of wrist index for CTS patients were: males: 0.8, females: 0.74 (significantly higher than in healthy individuals and indicating square shape); shape index: males 76.5, females 75.8; digit index: males 55.7, females 56.5. The calculated HLH-ratio in CTS group was: males 10.6, females 10.9; HLULL-ratio: males 23.6, females 24.9 and they did not differ significantly from healthy volunteers. Almost 90.0% of females with diagnosed CTS have BMI > 25.0 kg/m2.There are significant differences in morphometrical features of the upper limbs between CTS patients and healthy individuals. Hands of patients with CTS are more massive and with 'plumb' fingers and square shape of the wrist. Furthermore, higher BMI values were confirmed to be predisposing factors in CTS occurrence.
954	Soluble CD163, a unique biomarker to evaluate the disease activity, exhibits macrophage activation in systemic juvenile idiopathic arthritis.	29801971	This study aims to investigate the clinical significance of serum soluble CD163 (sCD163) levels as a predictor of the disease activity of systemic juvenile idiopathic arthritis (s-JIA). In this study, we examined 63 patients with s-JIA, four with Epstein-Barr virus-induced hemophagocytic lymphohistiocytosis (EBV-HLH), and seven with Kawasaki disease (KD), along with 14 healthy controls. We quantified serum cytokine levels (sCD163, neopterin, IL-18, IL-6) by enzyme-linked immunosorbent assay and compared the results with the clinical features of s-JIA. Serum sCD163 levels were significantly elevated in patients with s-JIA associated macrophage activation syndrome (MAS) and EBV-HLH compared to those in patients with acute-phase s-JIA and KD. In addition, serum sCD163 levels profoundly increased with the progress of MAS and correlated positively with the disease activity of s-JIA, even in patients receiving tocilizumab. Furthermore, serum sCD163 levels significantly decreased in the inactive phase compared to those in the active phase and normalized in remission. The correlation between macrophage activation and serum sCD163 levels might be a unique indicator of the disease activity and a potential diagnostic laboratory criterion for clinical remission in patients with s-JIA, including those receiving tocilizumab.
955	An effective diagnostic index for lymphoma-associated hemophagocytic syndrome.	2980032130192969	Lymphoma-associated hemophagocytic syndrome (LAHS) is a highly fatal immune disorder. Poor prognosis is partly attributed to under diagnosis or delayed diagnosis.Early identification of LAHS patients based on the laboratory findings could improve the outcomes.Retrospective observational cross-sectional study.From January 2011 to June 2016, 282 adult patients with hemophagocytosis in bone marrow were enrolled, and 114 hemophagocytic lymphohistiocytosis (HLH) patients with definite underlying cause were finally included for analysis. The HLH patients were further divided into LAHS (76 out of 114) and non-malignancy-associated HLH (38 out of 114) groups.Compared to non-malignancy-associated HLH, LAHS patients had significantly elevated lactate dehydrogenase (LDH) levels, increased thickness of spleen, higher proportion of patients with lymphadenopathy and significantly decreased peripheral blood cell count. In multivariate logistic regression model analysis, thickness of spleen ≥5 cm (OR = 17.9, 95%CI 1.35-236.6; P = 0.028), IL-6 level ≥55.1 pg/ml (OR = 12.01, 95%CI 1.03-138.9; P = 0.047) and IL-10 level ≥425.9 pg/ml (OR = 51.18, 95%CI 2.53-1035.1; P = 0.010) were independent predictors of LAHS diagnosis. Based on the regression parameters, we established a diagnostic index with weighted risk scores of 1 assigned to thickness of spleen and IL-6 level respectively, and a score of 3 assigned to IL-10 level. A diagnostic index ≥ 2 points had the best AUC value (0.889) with 84.2% of sensitivity and 93.7% of specificity for predicting LAHS.LAHS can be considered when HLH patients have a diagnostic index ≥2 points, so actively looking for evidence of lymphoma and effective chemotherapy may be necessary.
956	Onset of Hemophagocytic Lymphohistiocytosis during Piperacillin-Tazobactam Therapy in Three Children with Acute Focal Bacterial Nephritis.	29798969	Hemophagoytic lymphohistiocytosis (HLH) is a rare life-threatening disorder caused by overactivation of the immune system, associated with infections, autoimmune disorders, and malignancies. The pathological hallmark of HLH is phagocytosis of blood cells and platelets by activated macrophages and histiocytes. In this report, we describe the onset of HLH in three children, aged 2, 5 and 7 years old, during the treatment of acute focal bacterial nephritis (AFBN) with an antibiotic, piperacillin-tazobactam (PIPC-TAZ). AFBN is acute localized bacterial infection of the kidney without abscess formation. PIPC-TAZ was chosen for the treatment of AFBN, because it not only has indications for complicated urinary tract infections, but also covers most of the causative bacteria of urinary tract infections, including β-lactamase-producing Escherichia coli. The clinical courses of the three patients were similar, and they were treated with PIPC-TAZ and amikacin (AMK) for AFBN. Fever went down 2 to 5 days later, and AMK was discontinued by day 6. However, fever recurred on 13 to 15 days after introduction of PIPC-TAZ therapy, even though all of the patients had no signs of recurrence of AFBN. The clinical features and laboratory tests of two patients fulfilled the criteria of HLH, whereas the other patient had initiated therapy before fulfilling the criteria. Cessation of PIPC-TAZ combined with corticosteroid therapy improved clinical symptoms. HLH of our patients was probably induced by PIPC-TAZ, as judged by the timing of the onset of HLH and the positivity of the drug-lymphocyte stimulation test. In conclusion, prolonged antibiotic therapy with PIPC-TAZ could be a cause of HLH.
957	Pregnancy-related Hemophagocytic Lymphohistiocytosis Associated with Herpes Simplex Virus-2 Infection: A Diagnostic Dilemma.	29796364	Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory disorder characterized by the uncontrolled proliferation of lymphocytes and histiocytes with hemophagocytic activity in the bone marrow. To our knowledge, there have been a few reported cases of pregnancy-related HLH. This case highlights the importance of considering HLH in a pregnant woman along with other diagnoses, such as HELLP (which stands for hemolysis, elevated liver enzyme levels, and low platelet levels) syndrome and hemolytic anemias. It points to the challenges of diagnosing and managing pregnancy-related HLH due to a similarity in presentation with other conditions.
958	CD8 T Cell Memory Increases Immunopathology in the Perforin-Deficient Model of Hemophagocytic Lymphohistiocytosis Secondary to TNF-α.	29795796	Familial hemophagocytic lymphohistiocytosis 2 (FHL2) is a cytokine storm syndrome characterized by immune hyperactivation with viral infection due to a CD8 T cell cytotoxic killing defect secondary to a perforin deficiency. As most studies of FHL2 mice have used pathogen naïve animals, the effects of immune memory on FHL2 are understudied. We utilized an immunization model of the perforin-deficient mouse to study the effects of immune memory on FHL2. Prior CD8 T cell specific antigen exposure leads to enhanced HLH disease with increased morbidity and decreased time to mortality. Enhanced disease is associated with altered cytokine production and T cell proliferation. Response to IFNγ blockade is reduced and TNFα gains a pathogenic role, while blockade of the IL-33 receptor ST2 remains effective. These results suggest that pre-existing immune memory may worsen outcome and alter treatment response for FHL2 patients who may not be naïve to their immune triggers.
959	Initial Characteristics and Clinical Severity of Hemophagocytic Lymphohistiocytosis in Pediatric Patients Admitted in the Emergency Department.	29794953	The diagnosis and management of children with hemophagocytic lymphohistiocytosis (HLH) admitted in the emergency department (ED) are challenging. The present study aimed at describing the initial characteristics of pediatric patients with HLH upon admission in the ED. Moreover, the clinical severity of the condition was assessed.We performed a retrospective study of patients who visited the pediatric ED and were newly diagnosed with HLH during hospitalization between February 2012 and January 2017. The patients were classified in the clinically unstable group if at least 1 of the following conditions was observed upon admission in the ED: hypoxia requiring oxygen supplementation, hypotension requiring inotropic support, coagulopathy with prothrombin time (international normalized ratio, ≥1.5), and seizures or altered consciousness.We enrolled 31 pediatric patients with HLH, with a median age of 6.53 years (interquartile range, 1.35-13.24 years). Abdominal discomfort along with fever (74.2%) was the most common presenting symptom in patients admitted in the ED. Based on the HLH-2004 diagnostic criteria, fever (96.8%), hyperferritinemia (96.8%), splenomegaly (74.2%), hypertriglyceridemia and/or hypofibrinogenemia (67.7%), and bicytopenia (41.9%) were observed in the patients. However, only 8 patients (25.8%) met the criteria. Nineteen patients (61.3%) were included in the clinically unstable group. This group had lower albumin (2.3 vs 3.3 g/dL, P = 0.002) and fibrinogen levels and higher ferritin level and neutrophil count than the clinically stable group. Meanwhile, the number of clinical findings that met the diagnostic criteria was not different between the 2 groups. Lower albumin level was a significant risk factor in the clinically unstable group (odds ratio, 0.040; P = 0.004).Pediatric patients with HLH often have clinically unstable conditions upon admission in the ED. However, only few patients meet the HLH-2004 diagnostic criteria. Lower albumin level may be useful in assessing clinically unstable patients and preparing for possible deterioration.
960	Identification of a novel nonsense mutation in the UNC13D gene from a patient with hemophagocytic lymphohistiocytosis: a case report.	29783935	Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous and potentially fatal disease that presents symptoms of persistent fever, splenomegaly and cytopenia. Primary HLH is identified as an autosomal recessive disorder with causative genes including HPLH1, PRF1, UNC13D, STX11 and STXBP2.Here, we reported an 8-month-old female patient with compound heterozygosity in the UNC13D gene. The patient, who presented typical symptoms, was diagnosed with HLH based on HLH-2004 guidelines. High-throughput amplicon sequencing for the full-length exon, including a 5 bp padding region and 6 HLH-related genes, was performed to identify the pathogenic mutations in this patient. In all, 9 heterozygous variations were detected, namely, 7 nonpathogenic SNPs, one nonsense mutation (NM_199242.2:c.2206C > T, p.Gln736X), and one splicing mutation (NM_199242.2:c.2709 + 1G > A). These two mutations were considered pathogenic according to previous studies and functional prediction. A two-generation pedigree analysis based on Sanger sequencing was performed to confirm the result.Compound heterozygosity in the UNC13D gene was identified in trans and considered a causative mutation in a female patient with HLH. The nonsense mutation (NM_199242.2:c.2206C > T, p.Gln736X) was novel in cases of HLH. Our data expand the spectrum of HLH-related mutations in China and demonstrate the potential of high-throughput amplicon sequencing in the diagnosis of HLH.
961	Successful Treatment of Hemophagocytic Lymphohistiocytosis Associated with Low-risk Myelodysplastic Syndrome by Azacitidine.	29780114	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome that occurs as a complication in many clinical settings. Malignancy-associated HLH develops in patients with hematopoietic neoplasms, particularly in those with lymphoma, and its development in those with myelodysplastic syndrome (MDS) is uncommon. We herein report a case of HLH in a patient with low-risk MDS that was successfully treated with azacitidine. The prevalence of immune abnormalities among MDS patients and the immune effects of azacitidine have recently been elucidated, suggesting that MDS-associated HLH occurs as a result of immune impairment, and azacitidine improves this condition by restoring the immune system.
962	Nuclear Export Inhibition Enhances HLH-30/TFEB Activity, Autophagy, and Lifespan.	29768192	Transcriptional modulation of the process of autophagy involves the transcription factor HLH-30/TFEB. In order to systematically determine the regulatory network of HLH-30/TFEB, we performed a genome-wide RNAi screen in C. elegans and found that silencing the nuclear export protein XPO-1/XPO1 enhances autophagy by significantly enriching HLH-30 in the nucleus, which is accompanied by proteostatic benefits and improved longevity. Lifespan extension via xpo-1 silencing requires HLH-30 and autophagy, overlapping mechanistically with several established longevity models. Selective XPO1 inhibitors recapitulated the effect on autophagy and lifespan observed by silencing xpo-1 and protected ALS-afflicted flies from neurodegeneration. XPO1 inhibition in HeLa cells enhanced TFEB nuclear localization, autophagy, and lysosome biogenesis without affecting mTOR activity, revealing a conserved regulatory mechanism for HLH-30/TFEB. Altogether, our study demonstrates that altering the nuclear export of HLH-30/TFEB can regulate autophagy and establishes the rationale of targeting XPO1 to stimulate autophagy in order to prevent neurodegeneration.
963	How i treat primary haemophagocytic lymphohistiocytosis.	29767843	Primary haemophagocytic lymphohistiocytosis (HLH) diseases are a collection of inherited genetic disorders that cause the syndrome of HLH. Great advances have been made in the last 20 years with regard to the discovery of many of the genetic aetiologies of disease. Several advances have also been made on the clinical stage. Accurate screening diagnostics for primary HLH diseases that are superior to traditional Natural Killer cell function testing have been developed and are now available in many countries. There is now grounded clinical experience on which to base routine treatment decisions for patients with HLH. Newer approaches to allogeneic haematopoietic cell transplantation have increased overall patient survival. Despite these advances, however, there is still much work to be done to further improve patient care. This 'How I Treat' article will focus on summarizing current diagnostic, treatment and transplant strategies for patients with primary HLH diseases.
964	[Hemophagocytic lymfohistiocytosis in adults: review and case report].	29766733	Hemophagocytic lymfohistiocytosis (HLH) is rare, life-threatening condition, characterized by excessive activation of immune system with subsequent proinflammatory state resulting in multiorgan failure. Most frequently, it appears in infancy as a primary disorder caused by mutation of immune-regulatory genes. Increasingly, HLH is being diagnosed as a secondary - adult - form, which occurs as a result of aberrant immune response. Viral or bacterial systemic infections, malignancy with a predominance of lymphoproliferative disorders and autoimmune diseases are the most common triggers. Early diagnosis and initiation of therapy is crucial and increase the chance for recovery. HLH is usually presented as multisystem febrile illness, where an extensive differential diagnosis is needed. Diagnosis of HLH is defined by a combination of clinical and laboratory findings, eventually by a proof of specific mutation. The basic mechanism of therapy is an interruption of aberrant immune response by destruction and suppression of T-lymphocytes function. This is mostly achieved by corticosteroid and etoposide therapy. This review summarizes pathophysiology, diagnostics and therapy of HLH. Furthermore, a case-report of 22-years old patient with secondary HLH being manifested predominantly with acute respiratory failure is presented.Key words: acute respiratory failure - hemophagocytic lymphohistiocytosis - HLH-94 - macrophage activation syndrome - MODS.
965	Aspergillus endocarditis of the mitral valve with ventricular myocardial invasion, cerebral vasculitis, and intracranial mycotic aneurysm formation in a patient with hemophagocytic lymphohistiocytosis.	29755935	Aspergillus endocarditis is a rare infection and reported mainly in immunocompromised hosts. We report a case of mitral valve aspergillus endocarditis with ventricular myocardial invasion, cerebral vasculitis and intracranial fungal aneurysm formation in a patient with hemophagocytic lymphohistiocytosis (HLH). This case illustrates the importance of prompt investigation and treatment of masses seen on an echocardiogram for rare infections such as Aspergillus endocarditis in immunocompromised patients.
966	Cholestatic jaundice: a unique presentation leading to the diagnosis of HLH with Hodgkin lymphoma, HIV and EBV.	29754142	Haemophagocytic lymphohistiocytosis (HLH) is a syndrome of dysregulated immune activity with macrophage activation that can manifest as pancytopenia, coagulopathy and other laboratory abnormalities, usually progressing to multiorgan failure and death. This report documents the rarely reported association between HLH and Hodgkin's lymphoma (HL) with simultaneous HIV and Epstein-Barr virus (EBV) and complete resolution with chemotherapy. The patient initially presented with cholestatic jaundice. He was then found to have HL associated with HLH with coexistent HIV and EBV viraemia. A-Brentuximab-VD regimen for the lymphoma was initiated, resulting in rapid resolution of HLH and ultimately remission of HL. HLH is a syndrome known to have high mortality; thus, early diagnosis and prompt treatment are crucial. Usual presentation includes non-specific symptoms and can easily be overlooked. This case highlights the importance of diagnosing the condition in unusual settings and attempting treatment by targeting the cause of HLH, HL in our case.
967	Hemophagocytic Lymphohystiocytosis Associated With Type Ia Glycogen Storage Disease.	29750741	Hemophagocytic lymphohystiocytosis (HLH) is characterized by fever, splenomegaly, pancytopenia, and elevated levels of triglycerides and ferritin. These signs and symptoms are common to other metabolic diseases.A 5-month-old female infant, who presented with fever, respiratory distress, massive hepatomegaly, and bicytopenia, was diagnosed as having HLH and chemotherapy was initiated. The patient was negative for familial HLH gene mutations. Respiratory distress and laboratory findings improved rapidly after starting chemotherapy. However, there was no improvement in the massive hepatomegaly and she experienced hypoglycemic episodes. In addition, her family history included a cousin with glycogen storage disease (GSD). On the basis of the findings, the patient was diagnosed as having type Ia GSD. There are no previous reports of HLH secondary to GSD type Ia in the literature.Congenital metabolic diseases should be considered in the differential diagnosis of children with HLH.
968	Hemophagocytic Lymphohistiocytosis Associated With Visceral Leishmaniasis.	29746438	This is the report of a 2-year-old boy who presented with fever, cytopenia, and splenomegaly. The patient was diagnosed with hemophagocytic lymphohistiocytosis (HLH) and treated with HLH-2004 protocol. Repeated bone marrow aspiration showed amastigotes on follow-up. In endemic countries, visceral leishmaniasis should be considered in the differential diagnosis to avoid chemotherapy toxicity.
969	Early onset lysosomal acid lipase deficiency presenting as secondary hemophagocytic lymphohistiocytosis: Two infants treated with sebelipase alfa.	29705274	Two unrelated infants were diagnosed with and initially treated for hemophagocytic lymphohistiocytosis (HLH), but progressed to cholestasis and liver failure. Early onset lysosomal acid lipase deficiency (EO-LAL-D) was suspected due to lymphocytes with cytoplasmic vacuolation and/or adrenal calcifications and confirmed by enzymatic and genetic analysis. Enzyme replacement therapy with sebelipase alfa was implemented, but both children died, despite initial improvement. Since this inborn error of metabolism progresses rapidly in infants, early diagnosis is crucial, and appropriate treatment should be started as soon as possible. The authors suggest that the diagnosis of EO-LAL-D should be considered in infants with symptoms of HLH.
970	Deletion of Inflammasome Components Is Not Sufficient To Prevent Fatal Inflammation in Models of Familial Hemophagocytic Lymphohistiocytosis.	29695416	Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition that occurs in patients with genetic defects of cytotoxicity (familial HLH [FHL]) or secondary to other immunological disorders such as juvenile idiopathic arthritis. HLH is characterized by elevated levels of serum IL-18 and other cytokines. Moreover, a novel clinical entity has been recently identified in which constitutive NLRC4 inflammasome activation leads to severe HLH. Altogether, these clinical observations suggest that inflammasome activation is a central event in the development of all HLH forms and that inflammasome blockade could alleviate inflammation in FHL patients. To formally address this question, we invalidated genes encoding for Caspase-1 or the inflammasome adapter ASC in perforin-deficient mice that were subsequently infected with lymphocytic or mouse choriomeningitis virus as models of FHL. These deletions nearly abrogated IL-18 production occurring during HLH in all models. However, they did not reduce serum IFN-γ levels at the peak of the inflammatory reaction nor did they modulate inflammatory parameters at mid and late stages or fatal outcome. These data show that inflammasome blockade is not sufficient to prevent cytokine storm and lethality in mouse models of FHL and suggest that different pathophysiological mechanisms underlie HLH in genetic defects of cytotoxicity and genetic forms of inflammasome activation.
971	Oxidative Stress Induces Neuronal Apoptosis Through Suppressing Transcription Factor EB Phosphorylation at Ser467.	29689560	This study determined the role and mechanism of action of transcription factor EB (TFEB) in H2O2-induced neuronal apoptosis.SH-SY5Y cells were treated with Akt inhibitor/activator and different concentrations of H2O2. Cell apoptosis was detected by flow cytometric analysis. Akt and TFEB phosphorylation and PARP cleavage were determined by Western blotting. HEK293T cells were transfected with different truncated TFEB mutants and HA-Akt-WT; SH-SY5Y cells were transfected with Flag-vector, Flag-TFEB, Flag-TFEB-S467A or Flag-TFEB-S467D; and TFEB interaction with Akt was determined by co-immunoprecipitation and GST pull-down assays.A low concentration of H2O2 induces TFEB phosphorylation at Ser467 and nuclear translocation, facilitating neuronal survival, whereas a high concentration of H2O2 promotes SH-SY5Y cell apoptosis via suppressing TFEB Ser467 phosphorylation and nuclear translocation. The TFEB-S467D mutant is more easily translocated into the nucleus than the non-phosphorylated TFEB-S467A mutant. Further, Akt physically binds to TFEB via its C-terminal tail interaction with the HLH domain of TFEB and phosphorylates TFEB at Ser467. Mutation of TFEB-Ser467 can prevent the phosphorylation of TFEB by Akt, preventing inhibition of oxidative stress-induced apoptosis.Oxidative stress induces neuronal apoptosis through suppressing TFEB phosphorylation at Ser467 by Akt, providing a novel therapeutic strategy for neurodegenerative diseases.
972	Lymphocyte subsets in children with hemophagocytic lymphohistiocytosis (HLH) and its clinical significance.	29687855	To study the peripheral blood lymphocyte subsets in children with hemophagocytic lymphohistiocytosis (HLH) in acute period as well as remission period, and compare them with healthy children to investigate the significance of lymphocyte subsets in the diagnosis, treatment, and prognosis in children with HLH.From January 2009 to March 2014, 30 HLH patients were enrolled in this study. Among them, 20 were placed in the remission group, while 10 cases were placed in the death group. 6 cases died within 8 weeks due to the illness, and 4 cases died within 9 to 34 weeks. 30 children who were confirmed healthy after physical check-ups in the same period were enrolled in the control group. Peripheral blood was collected from both groups and lymphocyte subsets were studied using flow cytometry.The ratios of CD3+ T and CD8+ T cells increased in the HLH remission group and the death group, while CD4+, CD4+/CD8+ and CD3-CD16+CD56+NK ratios decreased. Difference detected in the proportion of CD19+ B cells was not statistically significant. By comparing lymphocyte subsets in the HLH acute period and the remission period in HLH patients we discovered that the differences in CD3+, CD8+, CD4+ and CD4+/CD8+, CD19+ B cells ratios were not statistically significant (p > 0.05). CD3-CD16+CD56+NK cells ratios in the remission period increased significantly.The lymphocyte subsets in children with HLH underwent obvious changes and there was an imbalance in cellular immunity. We believe that dynamic detection of changes may help us to evaluate the prognosis and the effect of the treatment.
973	Dual Threat of Epstein-Barr Virus: an Autopsy Case Report of HIV-Positive Plasmablastic Lymphoma Complicating EBV-Associated Hemophagocytic Lymphohistiocytosis.	29687211	Epstein-Barr virus (EBV) reactivation causes serious diseases in immunocompromised hosts, such as acquired immunodeficiency syndrome (AIDS). We report on a case of plasmablastic lymphoma (PBL) with hemophagocytic lymphohistiocytosis (HLH).A-53-year-old Japanese man was diagnosed with PBL and AIDS. In addition to combined antiretroviral therapy, HyperCVAD (cyclophosphamide, doxorubicin, vincristine, prednisone)/high-dose methotrexate + cytarabine was initiated immediately. Partial remission was attained with chemotherapy. However, the patient developed HLH and died despite intensive therapy. Autopsy findings suggested that PBL was controlled, and immunosuppression appeared to cause fatal infection. The patient showed high titers of EBV viral-capsid antigen (VCA)-IgG (1:2560) on PBL diagnosis and high EBV-DNA levels throughout the clinical course. Moreover, EBV-DNA was detected in the fraction of CD8-positive cells, which strongly supports the pathogenesis of EBV-associated HLH.Our report highlights the importance of EBV control in patients with EBV-positive AIDS lymphoma. EBV not only behaves as the etiologic pathogen of PBL but also can be a trigger of HLH, the fatal complication. Careful follow-up of the EBV status should be performed, and if needed, preemptive anti-EBV therapy should also be considered to prevent EBV-associated complications such as HLH.
974	Secondary HLH Case Report Highlighting Clinical Challenges.	29686911	A 19-year-old patient with relapsed acute myeloid leukemia (AML) developed severe and prolonged cytopenia and unexplained jaundice and fever after salvage chemotherapy. His workup revealed hemophagocytosis on the bone marrow biopsy. He was treated for HLH (hemophagocytic lymphohistiocytosis) secondary to AML and chemotherapy. The patient died on day 56 after starting his salvage chemotherapy. Unexpectedly, after his death, the microbiology laboratory reported positive mycobacterial growth from a bronchoalveolar lavage (BAL) sample taken during the workup of his fever. This case illustrates the difficulties in the diagnostic workup of HLH to identify triggers in a timely manner so that a targeted and specific therapy can be administered quickly, given the rapid and deadly evolution of the HLH process.
975	A Rare Case of Hemophagocytic Lymphohistiocytosis Associated With Myelodysplastic Syndrome and Trisomy 8 in a Pediatric Patient.	29683949	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease resulting in clinical and biochemical manifestations of extreme inflammation. Myelodysplastic syndrome (MDS) represents a heterogenous group of clonal hematopoietic disorders. The development of MDS is common in children with trisomy of chromosome 8. Here, we report a fatal case of 8-year-old girl who was admitted to the emergency department with status epilepticus, and later diagnosed with HLH associated with MDS and trisomy of chromosome 8. We believe this is the first reported case of HLH associated with MDS and trisomy 8 in a pediatric patient.
976	A Case of Spontaneously Improving Secondary Hemophagocytic Lymphohistiocytosis in an Adult Associated with T-Cell Histiocyte-Rich Large B-Cell Lymphoma.	29682376	Secondary hemophagocytic lymphohistiocytosis (HLH) in adults is a rare, often fatal syndrome characterized by widespread immune dysregulation. It is seen as a complication of infections, autoimmune diseases, and malignancies. Among the malignancy-related causes, aggressive T-cell or NK-cell neoplasms are most notable, while B-cell lymphomas are less commonly implicated. We present the case of a 32-year-old male transferred to our facility with concern for HLH. During the first week of his hospitalization, his diagnosis was confirmed and the patient demonstrated spontaneous improvement in his symptoms prompting us to delay therapy while searching for a primary cause. In the second week, the patient deteriorated, leading us to initiate steroid monotherapy in the absence of a cause for his HLH. Meanwhile, pathology results from an excisional lymph node biopsy confirmed a diagnosis of T-cell/histiocyte-rich large B-cell lymphoma (TCHRLBCL). Subsequently, we initiated therapy with dose-adjusted R-EPOCH. The patient achieved a complete remission of both HLH and TCHRLBCL as well as a complete return to his prior functional status. In our review of the literature, this represents only the second documented case of HLH associated with TCHRLBCL and the only documented case of an adult experiencing significant spontaneous recovery in this context.
977	Hemophagocytic lymphohistiocytosis presenting with acute liver failure and central nervous system involvement in early infancy.	29676379	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal and likely underdiagnosed disease characterized by unregulated histiocyte proliferation, hypercytokinemia and hemophagocytosis, causing life-threatening tissue damage and organ failure. We report a case of a 56-day-old infant presenting with fever, acute liver failure, and neurological manifestations as presenting features that succumbed to rapidly progressive HLH. Our objective is to emphasize the importance of early diagnosis by high suspicion in varied initial presentation of HLH so that life-saving therapy may be instituted in time.
978	Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity.	29672507	Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor-encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an "ur-" bHLH gene.
979	ANCA Vasculitis and Hemophagocytic Lymphohistiocytosis following a Fecal Microbiota Transplant.	29670798	A 69-year-old female with antisynthetase syndrome, a history of multiple recurrent infections, and documented previous negative titres for anti-neutrophil cystoplasmic antibody (ANCA) suddenly developed a de novo MPO-ANCA-associated glomerulonephritis three weeks after a fecal microbiota transplantation (FMT) for recurrent Clostridium difficile infections. Six months following her FMT and less than two weeks following treatment for urosepsis, she developed severe cholestasis, a markedly elevated ferritin and hypertriglyceridemia. An initial liver biopsy was suggestive of drug-induced liver injury and thus she was treated with supportive care. After she failed to improve, a second liver biopsy supported the diagnosis of hemophagocytic lymphohistiocytosis (HLH). This case highlights difficulties surrounding the early diagnosis of HLH and also questions the role of FMT and/or recurrent infections as a trigger for ANCA-associated vasculitis.
980	X-Linked Lymphoproliferative Disease Type 1: A Clinical and Molecular Perspective.	29670631	X-linked lymphoproliferative disease (XLP) was first described in the 1970s as a fatal lymphoproliferative syndrome associated with infection with Epstein-Barr virus (EBV). Features include hemophagocytic lymphohistiocytosis (HLH), lymphomas, and dysgammaglobulinemias. Molecular cloning of the causative gene, SH2D1A, has provided insight into the nature of disease, as well as helped characterize multiple features of normal immune cell function. Although XLP type 1 (XLP1) provides an example of a primary immunodeficiency in which patients have problems clearing primarily one infectious agent, it is clear that XLP1 is also a disease of severe immune dysregulation, even independent of EBV infection. Here, we describe clinical features of XLP1, how molecular and biological studies of the gene product, SAP, and the associated signaling lymphocyte activation molecule family receptors have provided insight into disease pathogenesis including specific immune cell defects, and current therapeutic approaches including the potential use of gene therapy. Together, these studies have helped change the outcome of this once almost uniformly fatal disease.
981	Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: Molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP.	29665027	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory disease. This study aimed to investigate the frequencies and distributions of inherited variants in PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP genes in Chinese patients with HLH. A total of 265 patients diagnosed with HLH from January, 2010 to December, 2016 were recruited and analyzed for the 6 genes. Genetic variants were observed in 87 (32.83%) patients. 36 (13.58%) exhibited variants in UNC13D, 18 (6.79%) exhibited PRF1 variants, 10 (3.77%) had variants in XIAP, 9 (3.40%) exhibited variants in STXBP2, 6 (2.26%) carried variants in SH2D1A, 1 (0.38%) had STX11 variant, and 7 (2.64%) exhibited digenic variants. Monoallelic variants were the most common, which accounted for 49.43% of all cases with variants. All variants were confirmed to be germline-derived. The present study describes a distinct variant spectrum in Chinese patients with HLH, whereby UNC13D is the most frequently mutated gene with missense variants that are the most common molecular defects. The variant profile of Chinese HLH patients is quite different from that of Western cohorts but similar to that of Korean patients, yet showing its own uniqueness. This racial difference shows the role of genetic background in the occurrence of HLH.
982	[Clinical features and prognosis of malignancy-associated hemophagocytic lymphohistiocytosis in children: a clinical analysis of 24 cases].	29658454	To investigate the clinical features and prognosis of malignancy-associated hemophagocytic lymphohistiocytosis (MAHS) in children.A retrospective analysis was performed for the primary diseases, clinical features, and prognosis of 24 children with MAHS.Among the 24 children, 11 (46%) had MAHS induced by tumor and 13 (54%) had chemotherapy-associated MAHS. As for primary diseases, 17 children had acute leukemia, 6 had lymphoma, and 1 had neuroblastoma. The most common clinical manifestations were pyrexia, respiratory symptoms, and hepatosplenomegaly. The most common laboratory abnormalities were hemocytopenia, elevated serum ferritin, and elevated lactate dehydrogenase. Of the 24 children, 22 were treated according to the HLH-2004 protocol and 2 gave up treatment; 18 children died, 1 was lost to follow-up, and 5 survived. The survival time ranged from 3 days to 2 years and 4 months (median 28 days).Children with MAHS have various clinical features and extremely poor treatment outcomes.
983	Hemophagocytic lymphohistiocytosis (HLH) secondary to disseminated histoplasmosis in the setting of Acquired Immunodeficiency Syndrome (AIDS).	29657917	Hemophagocytic lymphohistiocytosis (HLH) is a rare and aggressive disease involving immune system over-activation leading to hemophagocytosis. HLH requires early diagnosis and prompt treatment initiation, especially in patients with Acquired Immunodeficiency Syndrome (AIDS). We present a case of a middle-aged male with AIDS and renal failure, who developed HLH secondary to disseminated histoplasmosis. Etoposide chemotherapy as recommended by the HLH 2004 Guidelines was deferred and treatment focused instead on anti-fungal therapy. Anti-retroviral therapy followed thereafter.
984	Recombinant luteinizing hormone supplementation in assisted reproductive technology: a systematic review.	2965371729653710	To assess the role of recombinant human LH (r-hLH) supplementation in ovarian stimulation for ART in specific subgroups of patients.Systematic review.Centers for reproductive care.Six populations were investigated: 1) women with a hyporesponse to recombinant human FSH (r-hFSH) monotherapy; 2) women at an advanced reproductive age; 3) women cotreated with the use of a GnRH antagonist; 4) women with profoundly suppressed LH levels after the administration of GnRH agonists; 5) normoresponder women to prevent ovarian hyperstimulation syndrome; and 6) women with a "poor response" to ovarian stimulation, including those who met the European Society for Human Reproduction and Embryology Bologna criteria.Systematic review.Implantation rate, number of oocytes retrieved, live birth rate, ongoing pregnancy rate, fertilization rate, and number of metaphase II oocytes.Recombinant hLH supplementation appears to be beneficial in two subgroups of patients: 1) women with adequate prestimulation ovarian reserve parameters and an unexpected hyporesponse to r-hFSH monotherapy; and 2) women 36-39 years of age. Indeed, there is no evidence that r-hLH is beneficial in young (<35 y) normoresponders cotreated with the use of a GnRH antagonist. The use of r-hLH supplementation in women with suppressed endogenous LH levels caused by GnRH analogues and in poor responders remains controversial, whereas the use of r-hLH supplementation to prevent the development of ovarian hyperstimulation syndrome warrants further investigation.Recombinant hLH can be proposed for hyporesponders and women 36-39 years of age.
985	Identification of a novel nonsense mutation in SH2D1A in a patient with X-linked lymphoproliferative syndrome type 1: a case report.	29649976	X-linked lymphoproliferative syndrome type 1 (XLP1) is an X-linked recessive genetic disorder with a strong resemblance to hemophagocytic lymphohistiocytosis (HLH). Causative mutations for XLP1 have been identified in SH2D1A, located on chromosome Xq25.We report a case of an 18-month-old male with a novel nonsense mutation in SH2D1A. The patient presented the typical phenotype of HLH, including splenomegaly and hemophagocytosis in the bone marrow. Thus, he was initially diagnosed with HLH based on HLH-2004 guidelines. High-throughput amplicon sequencing was performed to detect mutations in the most commonly reported causative genes of HLH, i.e., PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP. A likely pathogenic nonsense mutation was detected in SH2D1A (NM_002351.4:c.300T>A). The mutation was inherited from the patient's mother, and an X-linked recessive mode of inheritance was confirmed by a two-generation pedigree analysis based on Sanger sequencing results.The nonsense mutation in SH2D1A (NM_002351.4:c.300T>A) was reported for the first time in a case of XLP1 and was considered to be likely pathogenic based on the truncation of the mRNA sequence. This finding expands the spectrum of known XLP-related mutations in Chinese patients and indicates the utility of amplicon sequencing for XLP and HLH diagnosis.
986	The Recombinant Inhibitor of DNA Binding Id2 Forms Multimeric Structures via the Helix-Loop-Helix Domain and the Nuclear Export Signal.	29642431	The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to β-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.
987	Genetic and mechanistic diversity in pediatric hemophagocytic lymphohistiocytosis.	2963202429976779	The HLH-2004 criteria are used to diagnose hemophagocytic lymphohistiocytosis (HLH), yet concern exists for their misapplication, resulting in suboptimal treatment of some patients. We sought to define the genomic spectrum and associated outcomes of a diverse cohort of children who met the HLH-2004 criteria. Genetic testing was performed clinically or through research-based whole-exome sequencing. Clinical metrics were analyzed with respect to genomic results. Of 122 subjects enrolled over the course of 17 years, 101 subjects received genetic testing. Biallelic familial HLH (fHLH) gene defects were identified in only 19 (19%) and correlated with presentation at younger than 1 year of age (P < .0001). Digenic fHLH variants were observed but lacked statistical support for disease association. In 28 (58%) of 48 subjects, research whole-exome sequencing analyses successfully identified likely molecular explanations, including underlying primary immunodeficiency diseases, dysregulated immune activation and proliferation disorders, and potentially novel genetic conditions. Two-thirds of patients identified by the HLH-2004 criteria had underlying etiologies for HLH, including genetic defects, autoimmunity, and malignancy. Overall survival was 45%, and increased mortality correlated with HLH triggered by infection or malignancy (P < .05). Differences in survival did not correlate with genetic profile or extent of therapy. HLH should be conceptualized as a phenotype of critical illness characterized by toxic activation of immune cells from different underlying mechanisms. In most patients with HLH, targeted sequencing of fHLH genes remains insufficient for identifying pathogenic mechanisms. Whole-exome sequencing, however, may identify specific therapeutic opportunities and affect hematopoietic stem cell transplantation options for these patients.
988	[Eperythrozoonosis complicated with hemophagocytic syndrome: report of four cases and review of literature].	29614573	Objective: To analyze the clinical characteristics of eperythrozoonosis complicated with hemophagocytic syndrome (HPS) in 4 children. Methods: Four patients diagnosed with eperythrozoonosis complicated with HPS in the Children's Hospital Affiliated Capital Institute of Pediatrics during the period from June 2014 to July 2016 were enrolled. The clinical manifestations, laboratory examination data and therapeutic strategies were analyzed. A literature search (search terms included 'eperythrozoonosis' and 'hemophagocytic syndrome') was conducted using CNKI, Wanfang database, Chinese biomedical literature database and PubMed to include recently published studies (searched from the database establishment to January 2017). Results: Four patients were included in the study. One was boy and the other three were girls. The age range of the 4 patients was between 9 months and 17 years (9 months, 2 years and 17 years, 11 months respectively). All the patients presented with recurrent high fever. During the course of fever, 3 patients presented with rash, and 2 patients presented with joint pain and swelling, which mimicked systemic juvenile idiopathic arthritis. Only 1 patient had the contact history of infectious disease. All patients had normal or decreased white blood cell count ((0.80-13.12)×109/L), suffered from varied degrees of anemia and showed the increased C reactive protein (13.0-84.7 mg/L) anderythrocyte sedimentation rate (13-72 mm/1 h). Examination of peripheral blood smears confirmed eperythrozoonosis. After fever continued about 1 month, all the 4 patients rapidly progressed. Among the 4 patients, 1 patient died for giving up further therapy, and the other 3 patients completely recovered after treatment, including azithromycin for the treatment of eperythrozoonosis, and high-dose intravenous methylprednisolone pulse therapy and human immunoglobulin for the treatment of HPS. For the disease not satisfactory, the hemophagocytic lymphohistiocytosis-2004 (HLH-2004) protocol is given. After the hospitalization of 1 to 2 months, the conditions improved and the children were discharged from hospital. Three patients were followed up for 8 months to 2 years, and their conditions were stable. In the PubMed database, no report was found. Nine cases of children with eperythrozoonosis were found in CNKI, Wanfang database and Chinese biomedical literature database, and 1 case was complicated with HPS. These findings, taken together our report, provided the data of 5 children with eperythrozoonosis complicated with HPS (4 cases were younger than 2 years old). A patient had contact history of infectious disease. Five patientss showed fever of unknown origin. All the patients had severe eperythrozoonosis, and 2 cases at younger age died. Conclusions: Children with eperythrozoonosis often present with the protracted fever of unknown origin, and clinical manifestations mimic those of juvenile idiopathic arthritis (systemic type). The patients with eperythrozoonosis of mild-to-moderate disease severity may have a good prognosis. Children with severe eperythrozoonosis, especially those HPS cases with early onset before 2 years old, may have high risk of mortality. Once the patient's condition aggravates in the course of fever, HPS should be highly suspected. For the patients with eperythrozoonosis complicated with HPS, early diagnosis and the combination of anti-infection with the treatment of HPS are crucial for a good prognosis. For the treatment of HPS, HLH-2004 protocol is recommended.目的： 总结4例附红细胞体病合并噬血细胞综合征（HPS）儿童的临床特点。 方法： 回顾性分析2014年6月至2016年7月首都儿科研究所附属儿童医院风湿免疫科诊治的4例附红细胞体病合并HPS患儿的临床资料。以"附红细胞体病""儿童"和"eperythrozoonosis""hemophagocytic syndrome"为主题词分别检索中国知网、万方数据库、中国生物医学文献数据库及PubMed（建库至2017年1月）的文献资料。 结果： 4例患儿中男1例，女3例，年龄分别为9月龄、2岁、17岁、11月龄。4例患儿均以长期发热为主要表现，3例有皮疹，2例有关节肿痛，1例有传染病接触史；血常规示白细胞（0.80~13.12）×109/L，均有不同程度贫血，C反应蛋白（CRP）13.0~84.7 mg/L，红细胞沉降率（ESR）13~72 mm/1 h；外周血涂片检查确诊附红细胞体病。4例患儿均在发热1个月左右病情骤然加重合并HPS；3例给予阿奇霉素抗感染，甲泼尼龙冲击、人免疫球蛋白等治疗，病情控制不满意，给予噬血细胞性淋巴组织细胞增多症2004（HLH-2004）方案治疗，住院1~2个月，病情好转出院，随访8个月~2年，病情稳定；1例放弃治疗出院后死亡。在PubMed数据库中未检索到相关报道，在中国知网、万方数据库、中国生物医学文献数据库中检索到9例儿童附红细胞体病，1例合并HPS，加上本组报道的4例，共5例附红细胞体病合并HPS病例，2岁内者4例，1例有传染病接触史，均表现为不明原因发热，均为重度附红细胞体病，其中年龄最小的2例死亡。 结论： 儿童附红细胞体病多以长期不明原因发热为首发症状，临床有类似幼年特发性关节炎（全身型）的表现；轻中度感染的附红细胞体病预后好，而重度感染尤其是年龄小于2岁者易合并HPS，病情凶险，死亡风险越高；儿童附红细胞体病一旦合并HPS，应积极抗感染，按照HLH-2004治疗方案进行治疗方能改善预后。.
989	Neonatal Adenovirus Infection Complicated by Hemophagocytic Lymphohistiocytosis Syndrome.	29610175	Two infants with disseminated adenoviral infections are described. Both these infants had a similar clinical course and were also diagnosed with secondary hemophagocytic lymphohistiocytosis (HLH). Previous reports of immunocompromised adults with adenovirus-associated HLH are in the literature; however, this is the first report that we are aware of with this pathology occurring in infants. These cases are used to demonstrate the importance of thinking about HLH in patients who are diagnosed with adenovirus and exhibit prolonged fevers that are unresponsive to antimicrobial agents with hepatosplenomegaly and cytopenias.
990	Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB.	29600279	Acinetobacter baumannii is a significant human pathogen associated with hospital-acquired infections. While adhesion, an initial and important step in A. baumannii infection, is well characterized, the intracellular trafficking of this pathogen inside host cells remains poorly studied. Here, we demonstrate that transcription factor EB (TFEB) is activated after A. baumannii infection of human lung epithelial cells (A549). We also show that TFEB is required for the invasion and persistence inside A549 cells. Consequently, lysosomal biogenesis and autophagy activation were observed after TFEB activation which could increase the death of A549 cells. In addition, using the Caenorhabditis elegans infection model by A. baumannii, the TFEB orthologue HLH-30 was required for survival of the nematode to infection, although nuclear translocation of HLH-30 was not required. These results identify TFEB as a conserved key factor in the pathogenesis of A. baumannii. IMPORTANCE Adhesion is an initial and important step in Acinetobacter baumannii infections. However, the mechanism of entrance and persistence inside host cells is unclear and remains to be understood. In this study, we report that, in addition to its known role in host defense against Gram-positive bacterial infection, TFEB also plays an important role in the intracellular trafficking of A. baumannii in host cells. TFEB was activated shortly after A. baumannii infection and is required for its persistence within host cells. Additionally, using the C. elegans infection model by A. baumannii, the TFEB orthologue HLH-30 was required for survival of the nematode to infection, although nuclear translocation of HLH-30 was not required.
991	PIL5 represses floral transition in Arabidopsis under long day conditions.	29588173	PHYTOCHROME INTERACING FACTOR 3 LIKE 5 (PIL5), also named PHYTOCHROME INTERACTING FACTOR 1 (PIF1) is an important b-HLH transcription factor in Arabidopsis thaliana. Here we show that mutant of pil5-1 displays early flowering phenotype. We demonstrate that the expressions of the major flowering promoter genes [FLOWERING LOCUS T (FT), SUPPRESOR OF OVEREXPRESSION OF CO 1 (SOC1), and LEAFY (LFY)] are upregulated in the mutant of pil5-1. There is a significant increase of the mRNA of PIL5 in the mutants of co2-1, ft-10, soc1-2, and lfy-4. These changes provide the molecular evidence that PIL5 interacts with the flowering regulators to control flowering time. Moreover, it is shown in our results that PIL5 mutation mediates the increased contents of gibberellic acid (GA). Which is further supported by the qRT-PCR analysis, an increased transcriptome level of the GA biosynthesis genes (GA3ox1, GA3ox2, GA20ox1, GA20ox2, and GA20ox3) has been observed in the pil5-1 mutants as compared to the wild type. Collectively, PIL5 is involved in floral transition interacting with flowering integrators and GA.
992	Central Nervous System Involvement in Hemophagocytic Lymphohistiocytosis in Adults: A Retrospective Analysis of 96 Patients in a Single Center.	29578120	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening clinical syndrome. Central nervous system (CNS) involvement is a severe complication, which can lead to rapid disease development and higher morality. However, this has not been given enough attention in adult HLH. Therefore, we carried out this study to analyze the clinical features, laboratory findings, treatment outcomes, and other characteristics of adult HLH with CNS involvement.A retrospective analysis of 96 adult patients with HLH combined with CNS involvement between June 2003 and December 2016 was conducted. Clinical features, cerebrospinal fluid (CSF) features, image changes, and therapeutic outcomes were analyzed.Among the 96 patients, 86 had various CNS symptoms and 33 (38.4%) had already presented symptoms before the HLH diagnosis was confirmed. A total of 59 patients received CSF examinations and showed abnormalities in 23 patients (39.0%). Seventy patients received imaging examinations and the results showed fifty patients with imaging changes (71.4%). Fifty-seven patients received multiple rounds of repeated intrathecal injection therapy and 35 patients improved (61.4%). As for the multiple analyses of effective factors on survival time, the results showed that the effects of combined Epstein-Barr virus (EBV) infection (P = 0.026, Exp(B) = 2.309, 95% confidence interval [CI] [1.108, 4.823) and intrathecal injection therapy (P = 0.013, Exp(B) = 0.422, 95% CI [0.214, 0.831]) on the survival time of the CNS-HLH patients were significant.Complication with EBV infection is a risk factor, and intrathecal injection is a protective factor. CNS involvement in HLH is not rare, which can result in a poor prognosis. Multiple rounds of repeated intrathecal injection therapy can improve the prognosis of CNS-HLH patients.成人噬血细胞淋巴组织增多症伴中枢神经系统受累：单中心96例回顾性分析摘要背景：噬血细胞淋巴组织增多症（hemophagocytic lymphohistiocytosis，HLH）是一类威胁生命的临床综合征。中枢神经系统（central nervous system，CNS）受累是其一种严重的并发症，进展较快，死亡率增加，但在成人HLH中未得到广泛重视。因此，我们通过观察这类患者，来分析成人HLH伴CNS受累的临床表现、实验室检查、治疗预后等特点。 方法：回顾性分析2003年6月至2016年12月我中心96例HLH伴CNS受累成人患者的临床资料、脑脊液变化、影像学异常及治疗、转归。 结果：在收录的96名患者中，86名存在神经系统症状，其中33名（38.4%）在HLH确诊前即出现神经系统症状。59名患者接受脑脊液检查，其中23名（39.0%）存在异常。70名患者接受影像学检查，其中50名（71.4%）出现影像学改变。共57名患者接受多次鞘内注射治疗，其中35名（61.4%）患者情况改善。对于影响预后的多因素分析发现，合并EB病毒（Epstein-Barr virus，EBV）感染（P=0.026，Exp(B)=2.309，95% CI [1.108，4.823]）及接受鞘内注射治疗（P=0.013, Exp(B)=0.422，95% CI [0.214,0.831]）对生存时间的影响有统计学意义。 结论：对于成人HLH合并CNS受累的患者，存在EB病毒感染提示不良预后，而接受鞘内注射治疗则可改善预后。成人当中的HLH合并CNS受累并不少见，并且一旦出现CNS受累提示预后较差。多疗程的鞘内注射治疗可以有效改善这类CNS-HLH患者的预后。.
993	Post-hematopoietic stem cell transplant hemophagocytic lymphohistiocytosis or an impostor: Case report and review of literature.	29577525	HLH occurring after HSCT is a relatively rare disease. Many conditions may mimic or trigger HLH in post-HSCT period (eg, cytokine release syndrome, engraftment syndrome, graft rejection/failure, acute graft-vs-host disease, infections systemic inflammatory response syndrome/sepsis, and thrombotic microangiopathy). Moreover, this period is usually marked by febrile illness, cytopenia, and a "cytokine storm" leading to elevation of inflammatory biomarkers like ferritin and sCD25. These parameters overlap with the diagnostic criteria for HLH. Such confounding factors make the management of post-HSCT HLH quite challenging. We illustrate this critical issue with case report of a patient who was diagnosed with HLH after allogeneic HSCT for tAML. He received MP and CsA for HLH but VP-16 was not administered due to fear of severe myelosuppression. Fortunately, he responded well to treatment and remains in remission to date. We recommend caution while using HLH-94/HLH-2004 guidelines for the diagnosis and management of post-HSCT HLH. In this article, we pinpoint these issues with a brief review of all the pediatric cases and clinical studies of post-HSCT HLH along with a critical evaluation of its various diagnostic criteria. Finally, based on the limitations of current diagnostic criteria, we suggest a need for formulating disease-specific diagnostic criteria for post-HSCT HLH.
994	Hemophagocytic lymphohistiocytosis in an infant with multiple triggers!!	29563683	Hemophagocytic lymphohistiocytosis (HLH) is a disorder of immune dysregulation secondary to a massive unregulated cytokine storm and its downstream consequences. HLH is being increasingly recognized as a cause of pyrexia of unknown origin, unexplained cytopenias, and hepatic dysfunction. However, this potentially treatable condition is often missed due to lack of suspicion, variable, and nonspecific presentations, inability to fulfil all the diagnostic criteria and availability of diagnostic tests in resource limited settings. Both familial and acquired forms of HLH can be triggered by multiple factors in a susceptible patient. We report a 9-month old infant who developed HLH in association with Stevens-Johnson syndrome following massive blood transfusion.
995	Approach to pancytopenia: Diagnostic algorithm for clinical hematologists.	29555368	Pancytopenia is a relatively common phenomenon encountered in clinical practice. The evaluation of a patient with pancytopenia requires a comprehensive approach and identifying the underlying cause can be challenging given the wide range of etiologies including drugs, autoimmune conditions, malignancies, infections, hemophagocytosis, and inheritable conditions. Recent advances in molecular hematology which include genomic profiling and next-generation sequencing have helped gain major insights into various hematological conditions and can guide diagnosing specific diseases in a shorter time at lower costs. However the approach to manage patients with pancytopenia in the current era of genomics is not well defined in the literature and is widely variable in practice. Herein, we conducted a systematic review to help devise an algorithm and management approach for pancytopenia, which serves as a general consultative approach.
996	Identification of IgG3-specific epitope that remedies problem in diagnostic IgG subclass determination due to human genetic variation.	29550761	There are four subtypes of human IgG with different effector functions. Quantifying the relative amount of each IgG subtype is important for laboratory diagnosis in multiple settings. However, in an evolving landscape of the appreciation of human variability and the need for precision/personalised laboratory diagnosis, it has also been shown that there are numerous natural variants of IgG subtypes, with at least 29 having been described. It has recently been reported that commercially available polyclonal antisera to IgG3 cross react with variants of other IgG subtypes, while available monoclonal anti-IgG3 have a blind-spot for the IgG3-04 variant. Herein, we report that IgG3-04 contains an epitope in common with all known IgG3 variants and absent in other subtypes. A novel monoclonal anti-IgG3 is described that is specific to IgG3 but without any blind-spots for known IgG3 variants, providing a remedy to the problem of genetic variability of IgG3.
997	Human CTL-based functional analysis shows the reliability of a munc13-4 protein expression assay for FHL3 diagnosis.	29549174	Familial hemophagocytic lymphohistiocytosis (FHL) is the major form of hereditary hemophagocytic lymphohistiocytosis (HLH); as such, it requires prompt and accurate diagnosis. We previously reported that FHL type 3 (FHL3) can be rapidly screened by detecting munc13-4 expression in platelets using flow cytometry; however, the reliability of the munc13-4 expression assay for FHL3 diagnosis is unclear. Regardless of the type of UNC13D mutation, all reported FHL3 cases examined for the munc13-4 protein showed significantly reduced expression. However, the translated munc13-4 protein of some reportedly disease-causing UNC13D missense variants has not been assessed in terms of expression or function; therefore, their clinical significance remains unclear. The aim of this study was to determine the reliability of a munc13-4 expression assay for screening FHL3. Between 2011 and 2016, 108 HLH patients were screened by this method in our laboratory, and all 15 FHL3 patients were diagnosed accurately. To further elucidate whether munc13-4 expression analysis can reliably identify FHL3 patients harboring missense mutations in UNC13D, we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by Herpesvirus saimiri derived from FHL3 patients. We then performed a comprehensive functional analysis of UNC13D variants. Transient expression of UNC13D complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported UNC13D missense variants according to expression levels of their translated munc13-4 proteins. Taken together with previous findings, the results presented herein show that the munc13-4 protein expression assay is a reliable tool for FHL3 screening.
998	A reminder of the place of morphology and the H-score in the diagnosis of hemophagocytic lymphohistiocytosis (HLH).	29531733	This case report reminds the reader of the place of hemophagocytosis and the H-Score in the diagnosis of secondary hemophagocytic lymphohistiocytosis.
999	Severe complication during remission of Crohn's disease: hemophagocytic lymphohistiocytosis due to acute cytomegalovirus infection.	29529681	Immunosuppressive therapy is today's standard treatment of patients with moderate to severe inflammatory bowel disease (IBD). The risk for opportunistic infections is increased due to this therapy and is a concern in the management of patient with IBD undergoing such a treatment.In this paper, we describe a case of an acute cytomegalovirus (CMV) infection in a 35-year-old male patient with Crohn's disease being in remission while receiving azathioprine therapy. His clinical presentation was high-grade fever, night sweats, skin rash, and abdominal pain.Laboratory findings showed pancytopenia, elevated liver enzymes, and high ferritin levels. Sonographic examination revealed splenomegaly and serological analysis proved an acute CMV infection. The severity of the acute illness and these results in the setting of immunosuppressive treatment with azathioprine were highly suspicious of hemophagocytic lymphohistiocytosis (HLH).Further investigations including bone marrow biopsy, analysis of natural killer cell function, and measurement of T-cell activity confirmed the suspected diagnosis. Treatment consisted of antiviral and symptomatic therapy.HLH is a rare and severe condition triggered by uncontrolled stimulation of histiocytes and lymphocytes, resulting in abnormal cytokine production. The causes can be primary (genetic) or secondary due to acquired immunodeficiency or viral infections such as CMV. Several symptoms of this condition are unspecific, but the summary of clinical symptoms and signs are diagnostic. Treatment consists of specific intervention if possible and application of immunosuppressive drugs such as corticosteroids.Die immunsuppressive Therapie gilt als heutiger Standard in der Behandlung mittel bis schwer verlaufender chronisch entzündlicher Darmerkrankungen (IBD). Die Unterdrückung des Immunsystems erhöht das Risiko opportunistischer Infektionen und stellt eine besondere Herausforderung in der Behandlung von Patienten mit IBD dar.In dieser Arbeit beschreiben wir einen Fall einer akuten Cytomegalievirus (CMV)-Infektion bei einem 35-jährigen männlichen Patienten mit Morbus Crohn in Remission unter laufender Azathioprin-Therapie. Klinisch präsentierte sich der Patient mit hochgradigem Fieber, Nachtschweiß, Hautausschlag und Bauchschmerzen. In den Laborbefunden fielen eine Panzytopenie, erhöhte Leberenzyme als auch hohe Ferritinspiegel auf. Sonografisch imponierte eine Splenomegalie und serologische Analysen bestätigten eine akute CMV-Infektion.Die Schwere des Erkrankungsbildes als auch die auffälligen Befunde in Zusammenhang mit der immunsuppressiven Behandlung mit Azathioprin führten zur Verdachtsdiagnose einer hämophagozytischen Lymphohistiozytose (HLH).Weitere Untersuchungen einschließlich Knochenmarkbiopsie, Analyse der natürlichen Killerzellfunktion und Messung der T -Zell-Aktivität bestätigten die vermutete Diagnose.Die Behandlung unseres Patienten bestand aus antiviralen und symptomatischen Maßnahmen.HLH ist zwar ein seltenes aber schweres Erkrankungsbild, welches durch unkontrollierte Stimulation des Immunsystems ausgelöst wird. Eine kontinuierliche Aktivierung von Histiozyten und Lymphozytenführt hierbei zu einer überschießenden Zytokinproduktion. Die Ursachen für HLH können primär (genetisch) oder sekundär aufgrund erworbener Immunschwächenoder Virusinfektionen, wie z. B. CMV, sein. Die Symptome dieses Erkrankungsbildes sind unspezifisch, die Diagnosefindung erfolgt an Hand der Klinik und der laborchemischen Untersuchungen. Die Behandlung besteht einerseits aus der Behebung der zu Grunde liegenden Erkrankung und andererseits aus der Gabe immunsupprimierender Medikamente, wie z. B. Kortikosteroiden.
1000	Comparison of Clinical Characteristics among Subtypes of Visual Symptoms in Patients with Transient Ischemic Attack: Analysis of the PROspective Multicenter registry to Identify Subsequent cardiovascular Events after TIA (PROMISE-TIA) Registry.	29525079	A transient visual symptom (TVS) is a clinical manifestation of transient ischemic attack (TIA). The aim of this study was to investigate differences in clinical characteristics among subtypes of TVS using multicenter TIA registry data.Patients with TIA visiting within 7 days of onset were prospectively enrolled from 57 hospitals between June 2011 and December 2013. Clinical characteristics were compared between patients with 3 major subtypes of TVS (transient monocular blindness [TMB], homonymous lateral hemianopia [HLH], and diplopia).Of 1365 patients, 106 (7.8%) had TVS, including 40 TMB (38%), 34 HLH (32%), 17 diplopia (16%), and 15 others/unknown (14%). Ninety-one patients with 1 of the 3 major subtypes of TVS were included. Symptoms persisted on arrival in 12 (13%) patients. Isolated TVS was significantly more common in TMB than in HLH and diplopia (88%, 62%, and 0%, respectively; P < .001). Duration of symptoms was shorter in patients with TMB than those with HLH (P = .004). The ABCD2 score was significantly lower in patients with TMB compared with those with HLH and diplopia (median 2 [interquartile range 2-3] versus 3 [2-4] and 4 [2-5], respectively; P = .005). Symptomatic extracranial internal carotid artery stenosis or occlusion was seen in 14 (16%) patients, and was more frequent in TMB than in HLH and diplopia (28%, 9%, and 0%, respectively; P = .015).TVS was an uncommon symptom in our TIA multicenter cohort. Some differences in clinical characteristics were found among subtypes of TVS.
1001	NHR-49/HNF4 integrates regulation of fatty acid metabolism with a protective transcriptional response to oxidative stress and fasting.	29508513	Endogenous and exogenous stresses elicit transcriptional responses that limit damage and promote cell/organismal survival. Like its mammalian counterparts, hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor α (PPARα), Caenorhabditis elegans NHR-49 is a well-established regulator of lipid metabolism. Here, we reveal that NHR-49 is essential to activate a transcriptional response common to organic peroxide and fasting, which includes the pro-longevity gene fmo-2/flavin-containing monooxygenase. These NHR-49-dependent, stress-responsive genes are also upregulated in long-lived glp-1/notch receptor mutants, with two of them making critical contributions to the oxidative stress resistance of wild-type and long-lived glp-1 mutants worms. Similar to its role in lipid metabolism, NHR-49 requires the mediator subunit mdt-15 to promote stress-induced gene expression. However, NHR-49 acts independently from the transcription factor hlh-30/TFEB that also promotes fmo-2 expression. We show that activation of the p38 MAPK, PMK-1, which is important for adaptation to a variety of stresses, is also important for peroxide-induced expression of a subset of NHR-49-dependent genes that includes fmo-2. However, organic peroxide increases NHR-49 protein levels, by a posttranscriptional mechanism that does not require PMK-1 activation. Together, these findings establish a new role for the HNF4/PPARα-related NHR-49 as a stress-activated regulator of cytoprotective gene expression.
1002	Macrophage activation syndrome in adults: recent advances in pathophysiology, diagnosis and treatment.	29481673	Haemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome, which if not promptly treated, can lead rapidly to critical illness and death. HLH is termed macrophage activation syndrome (MAS) when associated with rheumatic disease (where it is best characterized in systemic JIA) and secondary HLH (sHLH) when associated with other triggers including malignancy and infection. MAS/sHLH is rare and coupled with its mimicry of other conditions, is underrecognized. These inherent challenges can lead to diagnostic and management challenges in multiple medical specialties including haematology, infectious diseases, critical care and rheumatology. In this review we highlight the pathogenesis of MAS/sHLH including its underlying triggers, key clinical features and diagnostic challenges, prognostic factors and current treatments in adults.
1003	Spatial and historical distribution of organic phosphorus driven by environment conditions in lake sediments.	29478654	The chemistry of sedimentary organic phosphorus (OP) and its fraction distribution in sediments are greatly influenced by environmental conditions such as terrestrial inputs and runoffs. The linkage of OP with environmental conditions was analyzed on the basis of OP spatial and historical distributions in lake sediments. The redundancy analysis and OP spatial distribution results suggested that both NaOH-OP (OP extracted by NaOH) and Re-OP (residual OP) in surface sediments from the selected 13 lakes reflected the gradient effects of environmental conditions and the autochthonous and/or allochthonous inputs driven by latitude zonality in China. The lake level and salinity of Lake Hulun and the runoff and precipitation of its drainage basin were reconstructed on the basis of the geochemistry index. This work showed that a gradient in weather conditions presented by the latitude zonality in China impacts the OP accumulation through multiple drivers and in many ways. The drivers are mainly precipitation and temperature, governing organic matter (OM) production, degradation rate and transportation in the watershed. Over a long temporal dimension (4000years), the vertical distributions of Re-OP and NaOH-OP based on a dated sediment profile from HLH were largely regulated by the autochthonous and/or allochthonous inputs, which depended on the environmental and climate conditions and anthropogenic activities in the drainage basin. This work provides useful environmental geochemistry information to understand the inherent linkage of OP fractionation with environmental conditions and lake evolution.
1004	Severe Human Granulocytic Anaplasmosis With Significantly Elevated Ferritin Levels in an Immunocompetent Host in Pennsylvania: A Case Report.	29468169	Human granulocytic anaplasmosis (HGA) is a tick-borne, infectious disease caused by Anaplasma phagocytophilum that generally presents with nonspecific symptoms such as fever, chills, headache, malaise, and myalgia. If not treated immediately, HGA can cause hemophagocytic lymphohistiocytosis (HLH), a well-documented but underrecognized sequela of severe HGA. In this article, we report a case of severe HGA with hyperferritinemia in a 74-year-old male from Central Pennsylvania who initially presented with recurrent fevers, nausea, and malaise to our emergency department and was subsequently discharged home that same day. Ten days later, the patient returned with acute kidney injury, elevated liver transaminases, and profound hyperferritinemia to 5130 ng/mL. Empiric doxycycline was administered for suspected tick-borne disease and serologies eventually came back positive for anti-Anaplasma phagocytophilum antibodies. The patient returned to baseline status 15 days after discharge. Our case shows the challenges in the timely diagnosis of HGA and highlights the role of serum ferritin in aiding this diagnosis. Although our patient did not fulfill the HLH diagnostic criteria, our report demonstrates the importance of recognizing HGA as a reversible cause of HLH.
1005	Secondary Hemophagocytic Syndrome Associated with COG6 Gene Defect: Report and Review.	29445937	Hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal disease that is characterized by proliferation and infiltration of hyperactivated macrophages and T-lymphocytes. Clinically, it is characterized by prolonged fever, hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, pancytopenia, and hemophagocytosis in the bone marrow, spleen, or lymph nodes. It can be classified as primary if it is due to a genetic defect, or secondary if it is due to a different etiology such as severe infection, immune deficiency syndrome, rheumatological disorder, malignancy, and inborn errors of metabolism such as galactosemia, multiple sulfatase deficiency, lysinuric protein intolerance, Gaucher disease, Niemann-Pick disease, Wolman disease, propionic acidemia, methylmalonic acidemia, biotinidase deficiency, cobalamin C defect, galactosialidosis, Pearson syndrome, and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. For the first time in the literature, we report on a 5-year-old girl diagnosed with a Component of Oligomeric Golgi Complex 6 (COG6) gene defect complicated by HLH. Finally, we review the literature on inborn errors of metabolism associated with HLH and compare the previously reported patients of COG6 gene defect with our patient.
1006	Neural Decoding of Bistable Sounds Reveals an Effect of Intention on Perceptual Organization.	29440556	Auditory signals arrive at the ear as a mixture that the brain must decompose into distinct sources based to a large extent on acoustic properties of the sounds. An important question concerns whether listeners have voluntary control over how many sources they perceive. This has been studied using pure high (H) and low (L) tones presented in the repeating pattern HLH-HLH-, which can form a bistable percept heard either as an integrated whole (HLH-) or as segregated into high (H-H-) and low (-L-) sequences. Although instructing listeners to try to integrate or segregate sounds affects reports of what they hear, this could reflect a response bias rather than a perceptual effect. We had human listeners (15 males, 12 females) continuously report their perception of such sequences and recorded neural activity using MEG. During neutral listening, a classifier trained on patterns of neural activity distinguished between periods of integrated and segregated perception. In other conditions, participants tried to influence their perception by allocating attention either to the whole sequence or to a subset of the sounds. They reported hearing the desired percept for a greater proportion of time than when listening neutrally. Critically, neural activity supported these reports; stimulus-locked brain responses in auditory cortex were more likely to resemble the signature of segregation when participants tried to hear segregation than when attempting to perceive integration. These results indicate that listeners can influence how many sound sources they perceive, as reflected in neural responses that track both the input and its perceptual organization.SIGNIFICANCE STATEMENT Can we consciously influence our perception of the external world? We address this question using sound sequences that can be heard either as coming from a single source or as two distinct auditory streams. Listeners reported spontaneous changes in their perception between these two interpretations while we recorded neural activity to identify signatures of such integration and segregation. They also indicated that they could, to some extent, choose between these alternatives. This claim was supported by corresponding changes in responses in auditory cortex. By linking neural and behavioral correlates of perception, we demonstrate that the number of objects that we perceive can depend not only on the physical attributes of our environment, but also on how we intend to experience it.
1007	Atypical presentation of atypical haemolytic uraemic syndrome.	29440240	A 17-year-old girl presented with fever, myalgia, vomiting for 1 month and oliguria and dyspnoea for 4 days. She was tachycardic,hypertensive, with pedal oedema and decreased breath sounds. She had high serum creatinine (3 mg/dL), anaemia, thrombocytopenia, leucocytosis and eosinophilia with schistocytes. Lactate dehydrogenase, transaminases were high , with low haptoglobin and high ferritin (5269 ng/mL). Complement C3/C4 and fibrinogen were normal. Urinalysis showed large blood and protein and stool studies were negative. Her ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) was normal. Kidney biopsy showed acute interstitial nephritis (AIN) in addition to thrombotic angiopathy. The differentials - haemolytic uraemic syndrome (HUS), thrombotic thrombocytopenia (TTP) and haemophagocytic lymphohistiocytosis (HLH) were ruled out. Her genetic testing was abnormal for large CFHR1-CFHR3 homozygous deletion and heterozygous missense variant in exon 2 of DGKE making the diagnosis of atypical HUS. She received eculizumab and was discharged on oral steroids for AIN and biweekly eculizumab infusions with excellent recovery.
1008	Leuconostoc pseudomesenteroides-associated hemophagocytic syndrome: A case report.	29434707	Hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome characterized by fever, pancytopenia and splenomegaly. The underlying hemophagocytosis occurs primarily in the bone marrow, liver and lymph nodes. Multiple microbiological agents, including cytomegalovirus, Epstein-Barr virus and Mycobacterium tuberculosis, have been implicated in the pathogenesis of HLH. The present study presents a case of HLH associated with Leuconostoc pseudomesenteroides infection treated successfully with clindamycin. A 33-year-old man presented with recurrent episodes of fever and diarrhea. Upon initial treatment at another hospital (the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China), blood chemistry analysis demonstrated moderate anemia (hemoglobin 88 g/l; reference range, 120.0-160.0), elevated ferritin (1,068.47 mg/l; reference range, 21.81-274.66), total bilirubin (392.4 mmol/l; reference range, 5.1-28.0), conjugated bilirubin (335.7 mmol/l; reference range, 0-10.0), and γ-glutamyl transpeptidase (150 U/l; reference range, 10-60). The patient was treated with antibiotics for suspected pneumonia and cholecystitis, but new symptoms (including diarrhea and inflammatory colitis) started to emerge. The patient was subsequently treated with ganciclovir (5 mg/kg/day for 1 month), but body temperature increased to 41.0°C. Upon transferring to our hospital, the patient had severe anemia (hemoglobin, 39 g/l; red blood cell, 1.61×1012/l; reference range, 4.0-5.5×1012/l). Jaundice was apparent: Total bilirubin, 299.5 mmol/l; conjugated bilirubin, 215.7 mmol/l. The patient was treated with clindamycin (150 mg, taken orally every 12 h for 1 week) and supportive care that included parenteral nutrition. Symptoms rapidly dissipated after the treatment. Blood chemistry analysis 5 days after the first dose of clindamycin revealed substantial improvement in anemia and jaundice. The patient requested discharge for financial reasons, but continued treatment (details not available) at a local hospital (Pengpai Memorial Hospital, Shanwei, China). Upon a visit to our hospital 8 months later, the patient has no notable complaints, with the exception of moderate anemia. The present case suggests that HLH may be associated with L. pseudomesenteroides infection.
1009	Hemophagocytic lymphohistiocytosis in an HIV-positive patient with concomitant disseminated histoplasmosis.	29433397	A 46-year-old Dominican man, known to have HIV, presented with constitutional symptoms of two week's duration. The patient was found to have cytopenias, significantly elevated ferritin level and lymphadenopathy. Biopsies and laboratory studies met the criteria for hemophagocytic lymphohistiocytosis (HLH). A concomitant diagnosis of histoplasmosis was confirmed as the trigger for HLH and treatment resulted in clinical improvement and resolution of symptoms.
1010	Parvovirus B19-associated Hemophagocytic Lymphohistiocytosis in a Patient With Glucose-6-phosphate Dehydrogenase Deficiency.	29432306	We report a case of a 12-year-old male with glucose-6-phosphate dehydrogenase deficiency presenting with clinical signs of sepsis and pancytopenia. Investigations revealed parvovirus B19 (PVB19)-associated hemophagocytic lymphohistiocytosis (HLH). The patient recovered fully and quickly with symptomatic treatment. Current evidence suggests that PVB19-associated HLH has a favorable prognosis. Mild undiagnosed cases of HLH may be the cause of pancytopenia in PVB19 infections.
1011	Epstein-Barr Virus-associated Hemophagocytic Lymphohistiocytosis in Adults: A Retrospective Analysis of 23 Patients in China.	29431300	Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease with poor prognosis despite intensive therapy.To discuss the ideal therapy of EBV-associated HLH for adults.We retrospectively studied 23 adult patients with EBV-associated HLH at our institution between January 2000 and June 2015. The clinical characteristics, treatment, and prognosis of adult EBV-associated HLH were analyzed. The median age was 38 years (range 18-72).All patients were found to have high fever, thrombocytopenia, abnormal liver function, elevated ferritin, and lactate dehydrogenase. Leukopenia, anemia, coagulopathy, hypofibrinogenemia, and splenomegaly were found in more than 80% of patients. Ten patients were treated with HLH-2004 protocol. Eventually, 95.7% of patients died of EBV-associated HLH. Non-HLH-2004 treatment and bone marrow suppression may predict early relapse independently, and the poor performance status and high lactate dehydrogenase level can be poor prognostic factors. It was also validated in comprehensive analysis of published articles.Adult EBV-associated HLH occurs most often in people of Asian descent who are older than 35 years. These patients had a disappointing outcome despite intensive treatment, especially with high lactate dehydrogenase levels, poor performance status, and bone marrow suppression. HLH-2004 protocol has shown a glimmer of hope in the adult populations.
1012	Convergent pathways of the hyperferritinemic syndromes.	29420734	Hyperferritinemia and pronounced hemophagocytosis help distinguish a subset of patients with a particularly inflammatory and deadly systemic inflammatory response syndrome. Two clinically similar disorders typify these hyperferritinemic syndromes: hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS). HLH is canonically associated with a complete disturbance of perforin/granzyme-mediated cytotoxicity, whereas MAS occurs in the context of the related rheumatic diseases systemic juvenile idiopathic arthritis and adult-onset Still's disease, with associated IL-1 family cytokine activation. In practice, however, there are accumulating lines of evidence for innate immune dysregulation in HLH as well as partial impairments of cytotoxicity in MAS, and these mechanisms likely represent only a fraction of the host and environmental factors driving hyperferritinemic inflammation. Herein, we present new findings that highlight the pathogenic differences between HLH and MAS, two conditions that present with life-threatening hyperinflammation, hyperferritinemia and hemophagocytosis.
1013	Malignancy and chemotherapy induced haemophagocytic lymphohistiocytosis in children and adolescents-a single centre experience of 20 years.	29411124	Haemophagocytic lymphohistiocytosis (HLH) is a possibly life-threatening syndrome of immune dysregulation and can be divided into primary (hereditary) and secondary forms (including malignancy-associated HLH (M-HLH)). We retrospectively analysed epidemiological, clinical, virological and laboratory data from patients with M-HLH treated at our department between 1995 and 2014. Out of 1.706 haemato-/oncologic patients treated at our department between 1995 and 2014, we identified 22 (1.29%) patients with secondary HLH (1.3-18.0, median 10.1 years; malignancy induced n = 2; chemotherapy induced n = 20). Patients with acute myeloblastic leukaemia (AML) developed HLH significantly more often than patients with acute lymphoblastic leukaemia (ALL) (10/55, 18.2% vs. 6/148, 4.1%, p = 0.0021). As possible viral triggers, we detected BKV (53.8% of the tested patients), HHV-6 (33.3%), EBV (27.8%), CMV (23.5%), ADV (16.7%) and PVB19 (16.7%) significantly more frequently than in haemato-/oncologic patients without HLH. Despite lacking evidence of concurrent bacterial infection, C-reactive protein (CRP) and procalcitotnin (PCT) were elevated in 94.7 and 77.7% of the patients, respectively. Ferritin and sIL2R were markedly elevated in all patients. HLH-associated mortality significantly (p = 0.0276) decreased from 66.6% (1995-2004) to 6.25% (2005-2014), suggesting improved diagnostic and therapeutic management. Awareness of HLH is important, and fever refractory to antibiotics should prompt to consider this diagnosis. Elevated ferritin and sIL2R seem to be good markers, while inflammatory markers like CRP and PCT are not useful to discriminate viral triggered HLH from severe bacterial infection. Re-/activation of several viruses may play a role as possible trigger.
1014	Outcomes of Children with Hemophagocytic Lymphohistiocytosis Given Allogeneic Hematopoietic Stem Cell Transplantation in Italy.	29410181	We report on 109 patients with hemophagocytic lymphohistiocytosis (HLH) undergoing 126 procedures of allogeneic hematopoietic stem cell transplantation (HSCT) between 2000 and 2014 in centers associated with the Italian Pediatric Hematology Oncology Association. Genetic diagnosis was FHL2 (32%), FHL3 (33%), or other defined disorders known to cause HLH (15%); in the remaining patients no genetic abnormality was found. Donor for first transplant was an HLA-matched sibling for 25 patients (23%), an unrelated donor for 73 (67%), and an HLA-partially matched family donor for 11 children (10%). Conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 (20%), and fludarabine-based for 26 children (24%). The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 71% and 60%, respectively. Twenty-six patients (24%) died due to transplant-related causes, whereas 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively. Twelve of 14 children given a second HSCT after graft failure/relapse are alive and disease-free. Use of HLA-partially matched family donors was associated with higher risk of graft failure and thus with lower EFS (but not with lower OS) in multivariable analysis. Active disease at transplantation did not significantly affect prognosis. These data confirm that HSCT can cure most HLH patients, active disease not precluding successful transplantation. Because in HLH patients HLA-haploidentical HSCT performed through CD34+ cell positive selection was found to be associated with poor sustained engraftment of donor cells, innovative approaches able to guarantee a more robust engraftment are warranted in patients given this type of allograft.
1015	Brief Report: Novel UNC13D Intronic Variant Disrupting an NF-κB Enhancer in a Patient With Recurrent Macrophage Activation Syndrome and Systemic Juvenile Idiopathic Arthritis.	29409136	Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (JIA) and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH type 3 (FHLH3), but the role of noncoding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS.A custom enrichment library was constructed to sequence a genomic region of ~1 Mb flanking UNC13D in 24 patients with systemic JIA, recurrent MAS, and negative results of prior genetic (exon/coding) testing. The functional consequences of intronic variants were assessed using quantitative polymerase chain reaction in patient-derived peripheral blood mononuclear cells (PBMCs), electromobility shift assay, in vitro transcriptional enhancer assays, and natural killer (NK) cell degranulation assays.We evaluated a patient with systemic JIA and recurrent MAS in whom a novel functional intronic variant in UNC13D, c.117+143A>G, was observed. This variant occurred in a proposed regulatory region that drives lymphocyte-specific UNC13D expression and is associated with reduced transcript levels in patient PBMCs. This variant also disrupted NF-κB binding to a functional transcriptional enhancer, leading to reduced enhancer activity in vitro. Partial knockdown of UNC13D expression also led to impaired NK cell degranulation. An additional patient was identified with a previously described UNC13D intronic variant, for a total noncoding variant hit rate of 8.3% (2 of 24).These findings highlight the notion that intronic variants in key regulatory regions may be associated with MAS in patients with systemic JIA and support deep sequencing approaches when causative coding variants are not identified.
1016	Role of Lh polymorphisms and r-hLh supplementation in GnRh agonist treated ART cycles: A cross sectional study.	29408742	To investigate the effect of N312S polymorphism in the LHCGR gene as a predictive pharmacogenetic marker on clinical and embryological parameters and determining the need of r-hLH supplementation combine with r-hFSH in patients undergoing ART treatment.In a cross-sectional study, a retrospective analysis of women (n = 553), who underwent controlled ovarian stimulation treatment protocol was conducted during the years 2012-2014. R-hFSH (Gonal-F, Merck Serono) was administered to all patients undergoing ART cycle after initiating long luteal gonadotrophin-releasing hormone (GnRH) agonist down-regulation. R-hLH was supplemented based on P.C. Wong criteria. N312S genotype was determined using sequencing methodology. The mean r-hFSH, r-hLH doses, total number of oocytes, cleavage rates of embryos and clinical pregnancy were recorded. The association between the r-hLH supplementation and LHCGR N312S polymorphism and clinical pregnancy rates was determined using regression analysis by SPSS.19.7% of women were homozygous for A allele encoding asparagine (N/N), 45.7% were heterozygous (N/S) and 34.6% were homozygous (S/S) for G allele encoding serine. Women heterozygous (N/S) or homozygous (S/S) for serine showed a higher requirement for r-hLH (OR, 95% p-trend = <0.0001) compared to those homozygous for asparagine (N/N). Homozygous G allele was also associated with higher daily and total r-hLH dose per treatment cycle p-trend = <0.0001. Though, the total no of oocytes (14.87 ± 4.95 vs 12.98 ± 5.39 and 13.58 ± 5.45), Gr-I quality embryos (2.61 ± 1.81 vs 2.18 ± 1.96 and 1.98 ± 2.05) were significantly higher in women homozygous for A allele compared to women with heterozygous and homozygous for G allele, clinical pregnancy rates were significantly more in women with for G allele after excluding patients with PCOS and endometriosis conditions (P < 0.04).The present findings reveal that women heterozygous and homozygous for G allele required higher doses of r-hLH supplementation and these women were shown to have higher clinical pregnancy rates.
1017	Modulatory upregulation of an insulin peptide gene by different pathogens in C. elegans.	29405821	When an animal is infected, its innate immune response needs to be tightly regulated across tissues and coordinated with other aspects of organismal physiology. Previous studies with Caenorhabditis elegans have demonstrated that insulin-like peptide genes are differentially expressed in response to different pathogens. They represent prime candidates for conveying signals between tissues upon infection. Here, we focused on one such gene, ins-11 and its potential role in mediating cross-tissue regulation of innate immune genes. While diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine, we show that epidermal infection with the fungus Drechmeria coniospora triggers an upregulation of ins-11 in the epidermis. Using the Shigella virulence factor OpsF, a MAP kinase inhibitor, we found that in both cases, ins-11 expression is controlled cell autonomously by p38 MAPK, but via distinct transcription factors, STA-2/STAT in the epidermis and HLH-30/TFEB in the intestine. We established that ins-11, and the insulin signaling pathway more generally, are not involved in the regulation of antimicrobial peptide gene expression in the epidermis. The up-regulation of ins-11 in the epidermis does, however, affect intestinal gene expression in a complex manner, and has a deleterious effect on longevity. These results support a model in which insulin signaling, via ins-11, contributes to the coordination of the organismal response to infection, influencing the allocation of resources in an infected animal.
1018	Synthesis and characterization of Ciprofloxacin-containing divinyl oligomers and assessment of their biodegradation in simulated salivary esterase.	29402541	Two leading causes contributing to dental restoration replacement are the marginal breakdown at the composite/dentin interface and secondary caries mediated by bacteria. The objective of the present study was to synthesize oligomers which incorporated enhanced bio-stability but would also be able to generate antimicrobial function if they underwent degradation.Stability was incorporated into the oligomers by generating structural features that would physically hinder the availability of hydrolytically sensitive groups in the oligomers. As a proof-of concept for the antibacterial feature, antimicrobial function was achieved by covalently incorporating Ciprofloxacin (CF) into the backbone of cross-linking divinyl oligomers (referred to as EDV and HLH-CFPEG). The hydrolytic stability of the oligomers was studied in simulated human salivary esterase and compared to the commercial monomer 2,2-bis[4(2-hydroxy-3-methacryloxypropoxy)-phenyl]propane (BisGMA).Both drug oligomers were found to be significantly more stable than BisGMA. Upon degradation, both drug oligomers released CF differentially in free form. Polymer synthesis from resin formulations containing 15wt% HLH-CFPEG showed a high degree of vinyl group conversion and gel content, and under hydrolytic conditions showed the release of CF during a 28-day monitoring study period.HLH-CFPEG can be used in dental resin adhesive systems for local delivery of CF to the marginal interface. Minimizing the growth of Streptococcus mutans at the marginal site can improve longevity by reducing esterase activity derived specifically from S. mutans.
1019	Successful management of a complicated clinical crisis: A patient with left-sided endocarditis and secondary hemophagocytic lymphohistiocytosis: a rare case report and literature review.	29390580	Hemophagocytic lymphohistiocytosis (HLH) secondary to methicillin-resistant Staphylococcus epidermidis (MRSE)-related left-sided infectious endocarditis had never been reported before. In the last decade, daptomycin, a novel lipopeptide antibiotic, showed its excellent role in anti-Gram-positive bacteria, including soft tissue infection, bloodstream and deep tissueinfection.An Asian women under sever condition due to the cooccurrence of HLH and MRSE-related endocarditis while also be allergic to vancomycin. The patient was cured by high-dose daptomycin monotheraphy, HLH-2004 protocol and cardiothoracic surgery to remove the valve at last, and was obviously benefit from the endeavor of a multidisciplinary team (MDT) strategy.IE was made on March 27according to the modified Duke criteria. HLH was diagnosed too.The patient was cured by high-dose daptomycin monotheraphy, HLH-2004 protocol and cardiothoracic surgery to remove the valve at last, and was obviously benefit from the endeavor of a multidisciplinary team (MDT) strategy.The patient was healthy andstable when we published this case.This case proves high-dose daptomycin monotheraphy could be used as an effective alternative regimen for vancomycin in treating MRSE-related left-sided endocarditis and highlight the importance of early diagnosis and appropriate management for HLH. Furthermore, our work suggests an MDT model as a practical strategy in managing similar clinical situation.
1020	Hemophagocytic lymphohistiocytosis in an adult kidney transplant recipient successfully treated by plasmapheresis: A case report and review of the literature.	29390386	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease entity primarily described in children, but not less relevant in adults. It is characterized by a misdirected activation of the immune system, resulting in an uncontrolled cytokine release from macrophages and cytotoxic T-cells (CTLs). Primary HLH relies on a genetic predisposition, whereas secondary HLH develops in the context of infections, malignancies or autoimmune diseases. However, the awareness and therapeutic knowledge for HLH in adulthood is limited. Most therapy protocols are almost exclusively validated in pediatric cohorts and for primary HLH. Their transferability to adult individuals with mostly secondary HLH is doubtful. Especially the high liver and bone marrow toxicity of applied etoposide-based protocols is discussed controversially and connected to overwhelming infections and death.A 51-year old, male, kidney transplant recipient was admitted to our center suffering from diarrhea, fever, nausea, hyponatremia, kidney graft failure, disorientation, progressive hemodynamic instability, and multiorgan failure.Clinical and laboratory findings resembled those of a septic shock. Ferritin and soluble interleukin-2 receptor (sCD25) levels were disproportionally elevated. Only a mild hepatosplenomegaly was diagnosed in a CT scan. A T2-weighted, fluid-attenuated inversion recovery MRI showed marked, bilateral and periventricular white matter hyperintensities. The cerebrospinal fluid (CSF) analysis showed a moderately elevated protein content and cell count. There was no evidence of any bacterial, viral, or parasitic infection. The diagnosis of HLH was made.The patient was successfully treated by a combined approach consisting of plasma exchange (PE), corticosteroids, anakinra, and cyclosporine (CsA).HLH is an important differential diagnosis in critically ill patients. Its unspecific clinical picture complicates an early diagnosis and may be misclassified as sepsis. A combination of plasma exchange (PE), corticosteroids, anakinra, and cyclosporine (CsA) may be a promising and less toxic approach for HLH therapy in adults.
1021	Hemophagocytic lymphohistiocytosis complicated by multiorgan failure: A case report.	29390336	We present a case of hemophagocytic lymphohistiocytosis (HLH) with severe pulmonary complication and acute respiratory distress syndrome (ARDS) hospitalized in our intensive care unit (ICU) in 2014; distinctive trait of this case has been the challenging diagnosis, with a bone marrow biopsy always negative, the severe pulmonary complication with ARDS and severe pulmonary hypertension, and the ferritin temporal kinetics that precisely followed the clinical course of disease.A 32-year-old woman from the Philippines first diagnosed with upper airway infection, was subsequently hospitalized in infectious disease department and treated for community acquired pneumonia.After clinical picture worsened with a profound respiratory insufficiency, the patient was intubated and transferred to our ICU. During this hospitalization, the clinical picture of fever, cutaneous rashes, lymphadenitis, hepatitis, leukopenia, anemia, hyperferritinemia, hypertriglyceridemia, high level of auto-antibodies, and low NK activity suggested an hemophagocytic lymphohistiocytosis syndrome, even if bone marrow biopsy was negative for hemophagocytosis.Immunosuppressive therapy with dexamethasone and etoposide was started, and the patient was discharged from ICU 4 months after admission.HLH is a rare disorder of the mononuclear phagocytic system, characterized by systemic proliferation of non- neoplastic histiocytes. The diagnosis is often challenging and not all of the diagnostic criteria may be present at the same time; this case shows how complex the diagnosis could be, how hematic ferritin levels could help in following the course of the disease, and the possibility of severe pulmonary complication either due to the disease itself and to possible sovra infections.
1022	Acute appendicitis complicated with necrotizing fasciitis in a patient with adult-onset Still's disease: A case report.	29384877	Adult-onset Still disease (AOSD) is a rare systemic inflammatory disease of unknown etiology characterized by evanescent salmon-pink rash, spiking fever, arthralgia/ arthritis, and lymphadenopathy. AOSD sometimes was fatal when it is complicated by macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH). Nonetheless, the literature provides no recommendations for treatment of AOSD patients with severe sepsis.A previously healthy 65-year-old man with history of AOSD was referred to our hospital for persistent right lower quadrant abdominal pain for 2 days. One week later, an abdominal wall abscess and hematoma developed by extravasation from the inferior epigastric vessels, complicated by necrotizing fasciitis of the right thigh and groin region. To our best knowledge, this case was the first reported case of a perforated appendix complicated with necrotizing fasciitis in a patient with AOSD.The patient was diagnosed as acute appendicitis complicated with necrotizing fasciitis and abdominal wall abscess.This case received intravenous tigecycline injection and daily 10 mg prednisolone initially, and shifted to daily intravenous hydrocortisone 200 mg for suspected MAS or HLH. This patient underwent surgical intervention and debridement for necrotizing fasciitis.The patient's symptoms progressed worse rapidly. He died from cytomegalovirus viremia and bacterial necrotizing fasciitis complicated by septic shock.(1) The steroid dose was difficult to titrate when AOSD complicated by sepsis. The differential diagnosis from MAS/HLH with bacterial/viral infection related severe sepsis was difficult but critical for decision making from clinicians and rheumatologists. (2) The conservative treatment with antibiotics for perforated appendix is safe but has a higher failure rate in immunocomprised patients such as systemic lupus erythematosus and AOSD. Early surgical intervention might contribute to better outcome. (3) The abdominal wall abscess can be spread from intra-abdominal lesion through the inferior epigastric vessels which were as weak points of abdominal wall. Imaging examinations contribute to acute diagnosis and help surgeons perform surgical interventions to prevent morbidity and mortality.
1023	Pregnancy-associated hemophagocytic lymphohistiocytosis secondary to NK/T cells lymphoma: A case report and literature review.	29381934	Hemophagocytic lymphohistiocytosis (HLH) occurs primarily in pediatric population, or secondary to malignancy, infection, or autoimmune disease. This disease is rare and prognosis is generally poor. Only a small number of cases during pregnancy have been reported in literature.We report a case of pregnancy-associated HLH secondary to natural killer (NK)/T cells lymphoma. She was admitted at 30 weeks and 3 days of pregnancy with complaints of abdominal pain and fever as high as 39.2°C. The patient was found to have splenomegaly, pancytopenia, and acute hepatic failure.A subsequent bone marrow biopsy revealed focal hemophagocytosis and atypical lymphoid cells. The splenic pulp also contained a large number of tissue cells proliferating and devouring mature red blood cells, lymphocytes, and cell debris. On the basis of these findings, we diagnosed the case as pregnancy-associated hemophagocytic lymphohistiocytosis secondary to NK/T cells lymphoma.Treatment consisted with dexamethasone and etoposide in combination with rituximab.Due to timely termination of pregnancy, the neonate was in good condition. However, the patient died on the 18th day postoperation due to multiorgan failure.We recommend that HLH be considered as differential diagnosis in a pregnant patient complaining of persistent fever, cytopenia, or declining clinical condition despite delivery of the baby. Prompt diagnosis and treatment is essential and fetal outcomes should also be considered. The decision to terminate a pregnancy and initiate chemotherapy during pregnancy with malignancy-associated HLH (M-HLH) needs to be further investigated in a larger cohort.
1024	Genetic Loci Controlling Carotenoid Biosynthesis in Diverse Tropical Maize Lines.	29378820	The discovery and use of genetic markers associated with carotenoid levels can help to exploit the genetic potential of maize for provitamin A accumulation more effectively. Provitamin A carotenoids are classes of carotenoids that are precursors of vitamin A, an essential micronutrient in humans. Vitamin A deficiency is a global public health problem affecting millions of people, especially in developing countries. Maize is one of the most important staple crops targeted for provitamin A biofortification to help alleviate vitamin A deficiency in developing countries. A genome-wide association study (GWAS) of maize endosperm carotenoids was conducted using a panel of 130 diverse yellow maize tropical inbred lines genotyped with Genotyping by Sequencing (GBS) SNP markers. Numerous significant association signals co-localizing with the known carotenoid biosynthesis genes crtRB1, lcyE and ZEP1 were identified. The GWAS confirmed previously reported large effects of the two major carotenoid biosynthesis genes lcyE and crtRB1 In addition, significant novel associations were detected for several transcription factors (e.g., RING zinc finger domain and HLH DNA-binding domain super family proteins) that may be involved in regulation of carotenoid biosynthesis in maize. When the GWAS was re-conducted by including the major effects of lcyE and crtRB1 genes as covariates, a SNP in a gene coding for an auxin response factor 20 transcription factor was identified which displayed an association with β-carotene and provitamin A levels. Our study provides a foundation for design and implementation of genomics-assisted selection strategies for provitamin A maize breeding in tropical regions, and advances efforts toward identification of additional genes (and allelic variants) involved in the regulation of carotenoid biosynthesis in plants.
1025	Open-Label, Randomized, Two-Way, Crossover Study Assessing the Bioequivalence of the Liquid Formulation versus the Freeze-Dried Formulation of Recombinant Human FSH and Recombinant Human LH in a Fixed 2:1 Combination (Pergoveris®) in Pituitary-Suppressed Healthy Women.	29375477	This was a Phase I, open-label, randomized, two-period, two-sequence crossover study [ClinicalTrials.gov NCT02317809 (https://www.clinicaltrials.gov/ct2/show/NCT02317809); EudraCT 2014-003506-32] assessing the bioequivalence of the liquid and freeze-dried formulations of fixed-dose, fixed-ratio (2:1) combination recombinant human follicle-stimulating hormone plus recombinant human luteinizing hormone (r-hFSH/r-hLH). The safety and tolerability of the two formulations were also assessed. Healthy premenopausal women were randomized to one of two crossover dosing schedules. Subjects in Treatment Sequence 1 received a single subcutaneous dose (900/450 IU r-hFSH/r-hLH) of the liquid formulation of r-hFSH/r-hLH on Day 1 of Dose Period 1 and, after a washout period of at least 14 days, a single subcutaneous dose (900/450 IU r-hFSH/r-hLH) of the freeze-dried formulation of r-hFSH/r-hLH (reconstituted in water for injection prior to administration) on Day 1 of Dose Period 2. Subjects in Treatment Sequence 2 received the treatments in reverse order. The primary endpoints were AUC0-t (area under the serum concentration-time curve from time 0 to the time of the last quantifiable concentration) and Cmax (maximum serum concentration) for FSH and LH, both baseline corrected. A total of 34 subjects were randomized, and 22 subjects were included in the bioequivalence evaluation. Overall, the mean observed PK profiles and individual PK parameters were comparable for the liquid and freeze-dried formulations, although a median difference in the tmax (time to reach maximum observed concentration) of FSH of ~4.5 h was observed between the formulations. The calculated 90% confidence intervals of the mean liquid formulation/freeze-dried formulation ratios for Cmax and AUC0-t were within the bioequivalence range (80-125%) for both LH and FSH, confirming bioequivalence between the two formulations. The safety and tolerability profiles of the two formulations were similar. The liquid formulation can, therefore, be expected to provide the same efficacy as the freeze-dried formulation, with no differences in tolerability.
1026	Hyperferritinemia in Hemophagocytic Lymphohistiocytosis: A Single Institution Experience in Pediatric Patients.	29371779	Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory condition that may run a rapid fatal course and calls for prompt diagnosis. Early intervention with steroids and other immunosuppressive drugs can contain the disease process and favours positive outcome. Ferritin ≥500 ng/ml is a HLH diagnostic criterion. We evaluated the diagnostic potential of admission ferritin, in children with HLH. Pediatric patients of a referral teaching hospital from Feb 2010-Oct 2013 having been investigated for ferritin on admission were included. HLH was confirmed when patients had clinical features and met 5/8 diagnostic criteria of the revised 2004 HLH guidelines. Ferritin was estimated on Cobas e411 by electrochemiluminiscence, with a measuring range of 0.5-2000 ng/ml. Dilutions were made when linearity exceeded and absolute values were reported. 905 on-admission ferritin investigations were reviewed out of which 346 values ≥500 ng/ml. Hyperferritinemia was seen in HLH/MAS (macrophage activation syndrome) [HLH group, median age 4 year 4 month, 59% male] and in systemic lupus erythematosus, sepsis, juvenile idiopathic arthritis, impending HLH, haemolytic anemias and malignancy [non-HLH group, median age 4 year 6 month, 60% male]. Of 346, 72 cases of hyperferritinemia were diagnosed with secondary HLH while one patient had primary HLH. 13/73 patients expired. The median ferritin level of the HLH group was significantly higher [6556 (2402-11,734) ng/ml] compared to non-HLH group [median 1175 (943-2000) ng/ml] (p < 0.0001). Receiver operator characteristics curve analysis revealed optimal admission ferritin of 3120 ng/ml as the cut-off with sensitivity of 70% and specificity of 88.9% for HLH diagnosis, exceeding the currently prescribed cut-off of 500 ng/ml. Hyperferritinemia below 3120 ng/ml has higher negative predictive value to rule out secondary HLH on admission in the study population of children predominantly diagnosed with infection associated HLH than the prescribed cut-off as per the 2004 guidelines. This may prove to be beneficial to alert physicians for prompt intervention which considerably decreases mortality in this often fatal condition.
1027	Haemophagocytic lymphohistiocytosis in a preterm infant: A case report.	29371734	Haemophagocytic lymphohistiocytosis (HLH) is a rare disease with a sepsis-like progression that leads to multiple organ dysfunction syndrome, especially in preterm infants. We present herein a case of HLH in a premature infant presenting with disseminated intravascular coagulopathy (DIC) and liver failure. A male infant, with weight 810g and delivered at the gestational age of 25 weeks and 2 days, was misdiagnosed with tyrosinaemia for several weeks. He presented with anaemia, thrombocytopaenia, persistent DIC, and elevated liver enzymes despite continuous transfusion and broad-spectrum antibiotics. A bone marrow puncture biopsy revealed haemophagocytosis, leading to HLH diagnosis. It is important for paediatricians to consider the possibility of HLH when liver function test results are abnormal in such patients.
1028	Autophagy and Longevity.	29370695	Autophagy is an evolutionally conserved cytoplasmic degradation system in which varieties of materials are sequestered by a double membrane structure, autophagosome, and delivered to the lysosomes for the degradation. Due to the wide varieties of targets, autophagic activity is essential for cellular homeostasis. Recent genetic evidence indicates that autophagy has a crucial role in the regulation of animal lifespan. Basal level of autophagic activity is elevated in many longevity paradigms and the activity is required for lifespan extension. In most cases, genes involved in autophagy and lysosomal function are induced by several transcription factors including HLH-30/TFEB, PHA-4/FOXA and MML-1/Mondo in long-lived animals. Pharmacological treatments have been shown to extend lifespan through activation of autophagy, indicating autophagy could be a potential and promising target to modulate animal lifespan. Here we summarize recent progress regarding the role of autophagy in lifespan regulation.
1029	[Clinical significance of serum calcium and albumin in patients with secondary hemophagocytic lymphohistiocytosis].	29365395	Objective: To investigate the clinical significance of serum calcium concentrations and albumin levels in patients with secondary hemophagocytic lymphohistiocytosis (sHLH) . Methods: The clinical and laboratory data of 68 patients with newly diagnosed sHLH from April 2013 to April 2016, and 20 healthy controls were retrospectively analyzed. The patients were classified as the clinical remission group and the progression group according to the response criteria. To explore the changes of serum calcium concentrations and albumin levels in the patients, its correlations with other laboratory indexes and the impact on survival. Results: ①The serum calcium concentrations and albumin levels in lymphoma-related HLH group and non-tumor HLH group were lower than control group (P<0.05) ; The serum calcium concentrations and albumin levels in the clinical remission group after treatment was higher than that before treatment (P<0.01) , while in the progression group the serum calcium concentrations after treatment decreased (P=0.002) , the albumin levels did not changed significantly (P=0.086) .②The serum calcium concentrations in newly diagnosed sHLH patients was positively correlated with ANC, PLT, HGB (P<0.05) , and negatively with ferritin (P<0.05) ; The overall survival (OS) was significantly longer in patients with serum calcium concentration≥1.92 mmol/L than that<1.92 mmol/L (266 d vs 95 d, P=0.006) . ③The serum albumin levels in newly diagnosed sHLH patients was positively correlated with ANC, HGB (P<0.05) , and negatively with ferritin (P<0.05) ; The overall survival was longer in patients with serum albumin level ≥ 26.7 g/L than that<26.7 g/L (263 d vs 95 d, P=0.023) . ④The serum calcium concentrations in patients before treatment was positively correlated with serum albumin levels (P<0.001) , and no significant correlation was found in control group (P=0.079) . Conclusion: The serum calcium concentrations and albumin levels have clinical significance for disease diagnosis, decision-making therapy and treatment outcomes in the patients with sHLH.目的： 探讨血清钙离子浓度及白蛋白水平在继发性噬血细胞性淋巴组织细胞增多症（sHLH）患者中的临床意义。 方法： 回顾性分析2013年4月至2016年4月收治的68例初诊sHLH患者的临床和实验室资料。将患者按疗效标准分为好转组和进展组，探讨血清钙离子浓度和白蛋白水平在患者中的变化、与其他实验室指标的相关性以及对患者生存的影响，以20例正常体检者为正常对照。 结果： ①与正常对照组比较，淋巴瘤相关性和非肿瘤相关性HLH组患者的血清钙离子浓度、血清白蛋白水平均降低（P值均<0.05）；与治疗前比较，好转组患者治疗后血清钙离子浓度、血清白蛋白水平均升高（P值均<0.01），进展组患者治疗后血清钙离子浓度降低（P=0.002），血清白蛋白水平差异无统计学意义（P=0.086）。②初诊时患者血清钙离子浓度与ANC、PLT、HGB呈正相关（P值均<0.05），与铁蛋白呈负相关（P<0.05）；初诊时血清钙离子浓度≥1.92 mmol/L组患者的总生存期较<1.92 mmol/L组延长（266 d对95 d，P=0.006）。③初诊患者血清白蛋白水平与ANC、HGB呈正相关（P值均<0.05），与铁蛋白呈负相关（P<0.05）；初诊时血清白蛋白水平≥26.7 g/L组患者的总生存期较<26.7 g/L组延长（263 d对95 d，P=0.023）。④患者组治疗前血清钙离子浓度与血清白蛋白水平呈正相关（P<0.001），正常对照组无明显相关性（P=0.079）。 结论： 血清钙离子浓度及白蛋白水平在sHLH患者的诊断、指导治疗及疗效监测等方面具有重要意义。.
1030	Epstein-Barr Virus and Hemophagocytic Lymphohistiocytosis.	29358936	Epstein-Barr virus (EBV) is a ubiquitous virus that infects nearly all people worldwide without serious sequela. However, for patients who have genetic diseases which predispose them to the development of hemophagocytic lymphohistiocytosis (HLH), EBV infection is a life-threatening problem. As a part of a themed collection of articles on EBV infection and human primary immune deficiencies, we will review key concepts related to the understanding and treatment of HLH.
1031	Primary hemophagocytic lymphohistiocytosis in adults: the utility of family surveys in a single-center study from China.	29357941	This study investigated the clinical characteristics of primary hemophagocytic lymphohistiocytosis (HLH) in adults, including immunological markers, pedigree findings, and conditions of allogeneic hematopoietic stem cell transplantation (Allo-HSCT).The study included clinical data of 18 adult patients with primary HLH treated in our center from June 2010 to January 2017.Of these 18 cases, pathogenic variants were found in the following genes: PRF1 (n = 11), UNC13D (n = 5), SH2D1A (n = 2), RAB27a (n = 1), and LYST (n = 2). One patient had pathogenic variants in both PRF1 and UNC13D genes, one patient had pathogenic variants in both LYST and UNC13D genes and another patient had pathogenic variants in both PRF1 and SH2D1A genes. Additionally, 3 of the 18 cases involved homozygous pathogenic variants, while 2 cases involved hemizygous pathogenic variants. The remaining 13 cases involved compound heterozygous pathogenic variants. The natural killer (NK) cell activity test was conducted in all 18 cases where 14(77.8%)patients showed reduction in NK cell activity. Furthermore, this article presents 3 representative results of the pedigree findings from 12 patients who underwent family surveys. The 8 patients who underwent Allo-HSCT had a median survival of 27.2 months, as compared with the median survival of 7 months for the10 patients who did not undergo Allo-HSCT, a significant difference between the two groups of patients (p = 0.006).PRF1 was one of the most commonly mutated gene in adult patients with primary HLH. Family surveys and immunological markers were important for the HLH diagnosis and the selection of an appropriate donor. Allo-HSCT was an effective therapy for adult primary HLH.
1032	T-cell gene therapy for perforin deficiency corrects cytotoxicity defects and prevents hemophagocytic lymphohistiocytosis manifestations.	29355678	Mutations in the perforin 1 (PRF1) gene account for up to 58% of familial hemophagocytic lymphohistiocytosis syndromes. The resulting defects in effector cell cytotoxicity lead to hypercytokinemia and hyperactivation with inflammation in various organs.We sought to determine whether autologous gene-corrected T cells can restore cytotoxic function, reduce disease activity, and prevent hemophagocytic lymphohistiocytosis (HLH) symptoms in in vivo models.We developed a gammaretroviral vector to transduce murine CD8 T cells in the Prf-/- mouse model. To verify functional correction of Prf-/- CD8 T cells in vivo, we used a lymphocytic choriomeningitis virus (LCMV) epitope-transfected murine lung carcinoma cell tumor model. Furthermore, we challenged gene-corrected and uncorrected mice with LCMV. One patient sample was transduced with a PRF1-encoding lentiviral vector to study restoration of cytotoxicity in human cells.We demonstrated efficient engraftment and functional reconstitution of cytotoxicity after intravenous administration of gene-corrected Prf-/- CD8 T cells into Prf-/- mice. In the tumor model infusion of Prf-/- gene-corrected CD8 T cells eliminated the tumor as efficiently as transplantation of wild-type CD8 T cells. Similarly, mice reconstituted with gene-corrected Prf-/- CD8 T cells displayed complete protection from the HLH phenotype after infection with LCMV. Patients' cells showed correction of cytotoxicity in human CD8 T cells after transduction.These data demonstrate the potential application of T-cell gene therapy in reconstituting cytotoxic function and protection against HLH in the setting of perforin deficiency.
1033	Comparative effects of sub-stimulating concentrations of non-human versus human Luteinizing Hormones (LH) or chorionic gonadotropins (CG) on adenylate cyclase activation by forskolin in MLTC cells.	29355532	We have compared various Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) preparations from non-human and human species in their ability to synergize with 10 µM forskolin (FSK) for cyclic AMP intracellular accumulation, in MLTC cells. LH from rat pituitary as well as various isoforms of pituitary ovine, bovine, porcine, equine and human LHs and equine and human CG were studied. In addition, recombinant human LH and CG were also compared with the natural human and non-human hormones. Sub-stimulating concentrations of all LHs and CGs (2-100 pM) were found to stimulate cyclic AMP accumulation in MLTC cells in the presence of an also non-stimulating FSK concentration (10 µM). Like rat LH, the most homologous available hormone for mouse MLTC cells, all non-human LHs and CG exhibit a strong potentiating effect on FSK response. The human, natural and recombinant hLH and hCG also do so but in addition, they were found to elicit a permissive effect on FSK stimulation. Indeed, when incubated alone with MLTC cells at non-stimulating concentrations (2-70 pM) hLH and hCG permit, after being removed, a dose-dependent cyclic AMP accumulation with 10 µM FSK. Our data show a clearcut difference between human LH and CG compared to their non-human counterparts on MLTC cells adenylate cyclase activity control. This points out the risk of using hCG as a reference ligand for LHR in studies using non-human cells.
1034	Hemophagocytic Lymphohistiocytosis in Adults with Intraocular Involvement: Clinicopathologic Features of 3 Cases.	29344491	Hemophagocytic lymphohistiocytosis (HLH) is an infrequent inflammatory multisystemic syndrome. Only rare cases with ophthalmic involvement describing their pathologic features have been previously reported.We report 3 cases of adult-onset HLH with bilateral ocular involvement and describe their clinicopathologic features.Three adult males - 2 with a history of viral infection - developed persistent fever, fatigue, bone marrow abnormalities, and irreversible multiorgan failure. Visual impairment was also documented in 2 cases. Complete autopsies were performed. Ophthalmic pathology demonstrated a bilateral histiocytic infiltrate with scant lymphocytes affecting the uvea. Focal extension to the retina, optic nerve, and trabecular meshwork were also identified, as well as hemophagocytosis in 1 case. Macrophages showed strong immunoreactivity for CD163 antibody and lacked BRAF p.V600E mutant protein.HLH is an unusual disorder associated with several systemic conditions. Histologic features in the eye are poorly documented, with prior reports restricted to children. Our 3 adult cases are reported using updated criteria and, despite the difference in age, show changes similar to those observed in the pediatric population.
1035	[X-linked inhibitor of apoptosis deficiency manifested as Crohn's disease: a case report and literature review].	29342997	Objective: To analyze the clinical characteristics of X-linked inhibitor of apoptosis (XIAP) deficient patients with clinical manifestation of Crohn's disease. Methods: Clinical manifestations, laboratory investigations, genetic testing and therapeutic interventions of one case of XIAP deficiency who was admitted to Department of Gastroenterology in Children's Hospital, Zhejiang University School of Medicine in May 2016 were summarized. PubMed and Chinese database for articles published from January 2016 to June 2017 were searched using the key words of'Crohn's disease'and'XIAP', and the relevant literature was reviewed. Results: The case we reported was a 6-year-1-month-old boy with recurrent bloody stool for 2 months, and abdominal pain with fever for 2 weeks. The patient had a past history of hemophagocytic lymphohistiocytosis (HLH) and epilepsy in the past one year. Complete blood cell count showed mild anemia (Hb108 g/L). The patient had an elevated high-sensitivity C reactive protein (86 mg/L) and erythrocyte sedimentation rate (46 mm/1h) . White blood cells, pus cells and red blood cells were found on routine stool examination. Biochemical panel showed hypoalbuminemia (25.2 g/L) , elevated transaminase (alanine aminotransferase 175 U/L, aspartate transaminase 229 U/L) , hypertriglyceridemia (4.41 mmol/L) , and hyperferritinemia (>1 650.0 μg/L) . Magnetic resonance enterography revealed the intestinal wall thickening and increased enhancement in parts of illeum and colon. Capsule endoscopy revealed multiple ulcers in jejunum. Colonoscopy showed multiple ulcers in colon and the pathological examination revealed chronic inflammation in mucosa of terminal ileum and colon, which was combined with partial necrosis and ulceration. Some phagocytes were seen in bone marrow smears. The patient was given multiple diagnoses, including hemophagocytic lymphohistiocytosis, Crohn's disease, sepsis, epilepsy, severe malnutrition, and hypoproteinemia. The pediatric Crohn's disease activity index (PCDAI) was 37.5. Genetic testing identified a hemizygotic mutation of c.910G>T chrX:123022501 p.G304X in XIAP. The parents had no such mutation. The patient showed response to infliximab with oral intake of mercaptopurine and corticosteroids, and had remission with PCDAI of 0. There were 9 relevant articles (Chinese 0 English 9), which showed 33.3% XIAP deficient patients manifested with inflammatory bowel disease(IBD), who might have other manifestations such as hemophagocytic lymphohistiocytosis or splenomegaly simultaneously or sequentially. Those patients showed poor response to monotherapy. Conclusion: XIAP deficient patients have various clinical manifestations. Genetic testing is important to those male pediatric IBD patients who have the complicated symptoms or little response to standard therapy.目的： 总结以克罗恩病为主要临床表现的X连锁凋亡抑制因子(XIAP)缺陷病例的临床特点。 方法： 报道2016年5月浙江大学医学院附属儿童医院消化科收治的1例XIAP缺陷患儿的临床表现、治疗、转归和基因测序结果，并以"克罗恩病""XIAP"为检索词，检索2006年1月至2017年6月中文数据库及PUBMED数据库进行文献复习。 结果： 患儿男，6岁1月龄，因反复便血2个月余，加重伴发热腹痛2周入院。既往1年余前曾有"噬血细胞综合征、癫痫"病史。入院后完善相关检查，血常规提示轻度贫血（血红蛋白108 g/L）；超敏C反应蛋白（86 mg/L）及红细胞沉降率（46 mm/1h）均升高；粪常规白或脓细胞及红细胞阳性；血生化提示低白蛋白血症（25.2 g/L），转氨酶（丙氨酸转氨酶175 U/L、天冬氨酸转氨酶229 U/L）升高，甘油三酯（4.41 mmol/L）升高，铁蛋白（>1 650.0 μg/L）升高；小肠磁共振水成像：部分结肠、回肠肠壁增厚、强化。胶囊内镜：空肠黏膜多发小溃疡。肠镜提示结肠多发溃疡。肠镜病理：回肠末端及全结肠黏膜慢性活动性炎，部分伴小片坏死溃疡。骨髓细胞学检查可见少量吞噬细胞。诊断为"噬血细胞综合征、克罗恩病、败血症、癫痫、重度营养不良、低蛋白血症"，儿童克罗恩病活动指数（PCDAI）评分37.5分。基因检测发现XIAP基因存在半合子突变c.910G>T chrX:123022501 p.G304X。父母均未检测出突变。该患儿经英夫利昔单抗治疗及巯嘌呤、糖皮质激素口服，病情缓解，PCDAI评分0分。文献检索共9篇，其中中文0篇，英文9篇，共有111例患者，炎症性肠病样表现约占33.3%，可相继或同时存在噬血细胞综合征、脾肿大等临床表现，单一治疗措施效果欠佳。 结论： XIAP缺陷患儿表现多样，在临床表现复杂、对治疗不敏感的男性炎症性肠病患儿中行基因检测，对指导治疗及判断预后有重要意义。.
1036	Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome.	2932609929599143	Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.
1037	Hemophagocytic syndrome masquerading as septic shock: An approach to such dilemma.	29318013	Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis is a rare condition characterized by excessive inflammation that is thought to be caused by the absence of normal downregulation of activated macrophages and lymphocytes. The treatment of hemophagocytic lymphohistiocytosis can depend on whether it is primary or secondary. In secondary hemophagocytic lymphohistiocytosis, the treatment can be directed according to the cause. In general, protocol HLH-94 (which consists of dexamethasone and etoposide in induction and maintenance) has been widely used as it has good outcomes. Hemophagocytic lymphohistiocytosis and septic shock largely overlap which can lead to refractory septic shock and death if not treated. Unfortunately, there is no clear approach for such dilemma. Thereby, we would like to present our case as it has a valuable approach to hemophagocytic lymphohistiocytosis in the setting of sepsis.A 60-year-old female, with history of hypertension, came with fever, productive cough, and dyspnea; she was admitted for acute exacerbation of chronic obstructive pulmonary disease and was transferred to intensive care unit for septic shock. The patient progressed to refractory septic shock with no focus of infection. After further investigations, detailed history raised the suspicion of hemophagocytic lymphohistiocytosis; a bone marrow biopsy was collected and confirmed the diagnosis. The patient was on methylprednisolone while waiting for other investigation results and improved markedly. After ruling out secondary causes of hemophagocytic lymphohistiocytosis, she was switched to protocol-94 and continued to improve.It should be emphasized that septic shock, with or without focus of infection, overlaps with hemophagocytic lymphohistiocytosis and can consequently lead to refractory septic shock and death. Thus, our aim of this case is to encourage further investigations, specifically for hemophagocytic lymphohistiocytosis in the setting of septic shock of unknown origin, to decrease mortality rate. More importantly, early initiation of immunosuppression therapy may be a crucial step before switching to hemophagocytic lymphohistiocytosis-specific treatment.
1038	Rare causes of hyperbilirubinemia after lung transplantation: our experience at a single center.	29312707	Lung transplantation is the last treatment option for end-stage lung disease, and the number of lung transplantations has been steadily increasing. Hyperbilirubinemia is a rare complication after lung transplantation. The aim of this study was to review rare causes of hyperbilirubinemia after lung transplantation at our center.In this single-center study, we retrospectively reviewed the records of 116 consecutive lung transplantation patients who underwent lung transplantation at Severance Hospital and Gangnam Severance Hospital of Yonsei University College of Medicine in South Korea between December 22, 2010 and January 1, 2016. Hyperbilirubinemia was defined as a total bilirubin level exceeding 5 mg/dL for at least 3 days after lung transplantation.Hyperbilirubinemia occurred in 33 patients (28.4%) who received lung transplants at our institution. Twenty-four cases involved common causes such as drug toxicity, biliary tract stone, sepsis, and bleeding. However, rare causes of hyperbilirubinemia including hemophagocytic lymphohistiocytosis (HLH), thrombotic microangiopathy (TMA), and ischemic cholangiopathy were observed in 9 (7.8%) patients during the study period. All patients with hyperbilirubinemia due to a rare cause died despite aggressive treatment.Causes of hyperbilirubinemia after lung transplantation are varied, and the prognosis of patients with hyperbilirubinemia arising from rare causes was poor. Therefore, early evaluation and management of hyperbilirubinemia after lung transplantation is important to improve patient outcomes.
1039	Two Brothers with Atypical UNC13D-Related Hemophagocytic Lymphohistiocytosis Characterized by Massive Lung and Brain Involvement.	29312353	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyperinflammatory condition. Variants in different genes have been associated with the familial forms of the syndrome (FHL), usually presenting within the first 2 years of life. Due to increasing awareness of the signs and symptoms of HLH and a better understanding of the genetic basis of the disease, FHL has been increasingly diagnosed in patients presenting beyond infancy. Here, we report on two brothers with atypical, late-onset HLH in which whole exome sequencing revealed a homozygous pathogenic UNC13D variant. In the first brother, the clinical phenotype was dominated by a massive lung involvement. In the second brother a progressive neurological deterioration was observed. In both cases, the clinical manifestations at symptom onset were misleading, making the diagnosis difficult to achieve. This report expands the spectrum of clinical presentations of FLH3. Moreover, it highlights the importance to warn clinicians to keep a high level of suspicion in patients presenting with fever, cytopenia, splenomegaly of unknown origin, and unresponsiveness to conventional treatment even beyond early childhood. Moreover, this report emphasizes that insidious neurologic symptoms may represent the initial or sole presenting sign of FHL, even in the absence of peripheral signs of activation.
1040	Streptococcus pneumoniae-Related Hemophagocytic Lymphohistiocytosis Treated with IVIG and Steroids.	29307884	BACKGROUND Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening condition that has a poor prognosis due to the ensuing cytokine storm leading to severe organ damage. Current treatment guidelines suggest using a combination of steroid- and etoposide-based chemotherapy. CASE REPORT The authors present a case of a 41-year-old African-American female who presented with symptoms of foodborne illness and who developed multi-organ dysfunction. HLH was suspected because of poor response to broad-spectrum antibiotics with a constellation of findings, including cytopenia, hypofibrinogenemia, hypertriglyceridemia, and hyperferritinemia. Clinical improvement was noted after administration of intravenous immunoglobulin and dexamethasone while waiting for the soluble interleukin-2 receptor levels; therefore, chemotherapy was not administered.  CONCLUSIONS Despite the variable and poor prognosis of HLH, early treatment with steroids and immunosuppressive therapy is crucial to improving the survival rate. The inclusion of immunoglobulin therapy should be considered a treatment option for HLH.
1041	Gene transfer into hematopoietic stem cells reduces HLH manifestations in a murine model of Munc13-4 deficiency.	29296930	Patients with mutations in the UNC13D gene (coding for Munc13-4 protein) suffer from familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a life-threatening immune and hyperinflammatory disorder. The only curative treatment is allogeneic hematopoietic stem cell (HSC) transplantation, although the posttreatment survival rate is not satisfactory. Here, we demonstrate the curative potential of UNC13D gene correction of HSCs in a murine model of FHL3. We generated a self-inactivating lentiviral vector, used it to complement HSCs from Unc13d-deficient (Jinx) mice, and transplanted the cells back into the irradiated Jinx recipients. This procedure led to complete reconstitution of the immune system (ie, to wild-type levels). The recipients were then challenged with lymphocytic choriomeningitis virus to induce hemophagocytic lymphohistiocytosis (HLH)-like manifestations. All the clinical and biological signs of HLH were significantly reduced in mice having undergone HSC UNC13D gene correction than in nontreated animals. This beneficial effect was evidenced by the correction of blood cytopenia, body weight gain, normalization of the body temperature, decreased serum interferon-γ level, recovery of liver damage, and decreased viral load. These improvements can be explained by the restoration of the CD8+ T lymphocytes' cytotoxic function (as demonstrated here in an in vitro degranulation assay). Overall, our results demonstrate the efficacy of HSC gene therapy in an FHL-like setting of immune dysregulation.
1042	Soluble interleukin-2 receptor is a sensitive diagnostic test in adult HLH.	29296904	Serum soluble interleukin-2 receptor (sIL-2r) is an important disease marker in hemophagocytic lymphohistiocytosis (HLH), but there are no published data on its diagnostic value in adults. We conducted a single-center retrospective study of 78 consecutive adults who had sIL-2r measured for suspected HLH. Serum sIL-2r levels were measured by enzyme-linked immunosorbent assay (adult reference range, 241-846 U/mL). There were 38 patients with HLH and 40 with a non-HLH diagnosis (such as sepsis, liver disease, histiocyte disorders, autoimmune disease, leukemia, or lymphoma). The receiver operating characteristic curve demonstrated that sIL-2r is a good to excellent diagnostic test for adult HLH, with an area under the curve (AUC) of 0.90 (95% confidence interval, 0.83-0.97) compared with AUC 0.78 (95% confidence interval, 0.67-0.88) for ferritin. The optimal threshold for sIL-2r was 2515 U/mL (sensitivity, 100%; specificity, 72.5%). Although there was a large indeterminate range for sIL-2r, a level of 2400 U/mL or less was helpful for ruling out HLH (sensitivity, 100%), and more than 10 000 U/mL was helpful for ruling in HLH (specificity, 93%). Higher mean sIL-2r levels were seen in malignancy-associated HLH (20 241 U/mL) compared with infection-associated HLH and macrophage activation syndrome (9720 and 5008 U/mL, respectively; P < .05). Levels above 10 000 U/mL were not associated with worse prognosis in patients with HLH. Serum sIL-2r is a sensitive test for diagnosis of adult HLH, but is not as specific as previously reported in children. Additional studies enriched with patients without HLH who have conditions associated with T-cell activation, such as lymphoma and autoimmune lymphoproliferative syndrome, are needed.
1043	Ruxolitinib for treatment of refractory hemophagocytic lymphohistiocytosis.	29296794	Optimal salvage therapy for refractory HLH is unknown.In our patient, ruxolitinib treatment led to clinical remission of refractory HLH.
1044	Proliferation through activation: hemophagocytic lymphohistiocytosis in hematologic malignancy.	29296722	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of cytokine-driven immune activation. Cardinal features include fever, hemophagocytosis, hepatosplenomegaly, lymphocytic infiltration, and hypercytokinemia that result in multisystem organ dysfunction and failure. Familial HLH is genetically driven, whereas secondary HLH (SHL) is caused by drugs, autoimmune disease, infection, or cancer. SHL is associated with worse outcomes, with a median overall survival typically of less than 1 year. This reflects difficulty in both diagnostic accuracy and in establishing reliable treatments, especially in cases of malignancy-induced SHL, which have significantly worse outcomes. Malignancy-induced HLH is seen almost exclusively with hematologic malignancies, constituting 97% of cases in the literature over the past 2 years. In these situations, the native immune response driven by CD8 T cells produces an overabundance of T helper 1 cytokines, notably interferon-γ, tumor necrosis factor-α, and interleukin-6, which establish a positive feedback loop of inflammation, enhancing replication of hematologic malignancies while leaving the host immune system in disarray. In this paper, we present 2 case studies of secondary HLH driven by HM, followed by a review of the literature discussing the cytokines driving HLH, diagnostic criteria, and current treatments used or undergoing investigation.
1045	Clinical characteristics, therapy response, and outcome of 51 adult patients with hematological malignancy-associated hemophagocytic lymphohistiocytosis: a single institution experience.	29295642	Hemophagocytic lymphohistiocytosis (HLH) is an underdiagnosed but life-threatening syndrome of hyperinflammation often occurring in adults with hematological malignancies (hM-HLH). The aim of the study was to describe clinical characteristics, therapy response, and outcome of adults with hM-HLH. The study included 51 adults with hM-HLH aged 23-84 years. Hyperferritinemia ≥500 µg/L was present in 96% of patients. The serum concentration of sIL-2Rα ≥ 2400 U/mL was revealed in 94% of patients. Twenty-three patients (45%) responded to therapy and achieved remission of HLH. The probability of overall survival (OS) at 6, 12, 24, and 60 months after HLH diagnosis were 42, 20, 15, and 15%, respectively. Patients with HLH during chemotherapy showed longer OS (median 124 days) than the patients who had HLH solely attributed to malignancy (median 65 days), but this difference was not statistically significant. Awareness of HLH in lymphoid and myeloid malignancies is crucial for improved survival.
1046	Sustained hyperferritinemia in a child with macrophage activation syndrome secondary to systemic juvenile idiopathic arthritis - perforinopathy: case based review.	30968645	Çakan M, Aktay-Ayaz N, Gemici H, Annayev A, Çıtak A, Akçay A, Öztürk G. Sustained hyperferritinemia in a child with macrophage activation syndrome secondary to systemic juvenile idiopathic arthritis - perforinopathy: case based review. Turk J Pediatr 2018; 60: 598-603. Systemic juvenile idiopathic arthritis is a subtype of juvenile idiopathic arthritis and characterized by arthritis and many systemic features like fever, rash, hepatosplenomegaly, lymphadenopathy and serositis. Macrophage activation syndrome is the most dreadful complication of systemic juvenile idiopathic arthritis and can cause mortality and morbidity if not recognized and treated early and aggressively. Hemophagocytic lymphohistiocytosis (HLH) is characterized by diminished or absent activities of natural killer cells and cytotoxic T lymphocytes leading to cytokine storm and uncontrolled activation of T cells and macrophages. Primary (familial) HLH is a group of autosomal recessive disorders caused by mutations in the perforin and other related genes and distinctive for onset during early infancy and high rate of mortality. Secondary HLH may be caused by infectious, oncologic and rheumatologic disorders. The term Perforinopathy is used to describe cases with classical familial HLH and also for cases with familial HLH gene mutations but not following a classical familial HLH course. Herein we report a case of chronic perforinopathy in which clinical symptoms started with systemic juvenile idiopathic arthritis and severe macrophage activation syndrome that needed plasma exchange and extracorporeal membrane oxygenation during acute period and ongoing interleukin-1 blockage for sustained hyperferritinemia.
1047	Minimal Change Disease as Initial Presentation of ALK-Positive Anaplastic Large-Cell Lymphoma in a Pediatric Patient.	29279700	The association between nephrotic syndrome (NS), hemophagocytic lymphohistiocytosis (HLH), and certain paraneoplastic syndromes has been documented in the literature. However, nephrotic changes as part of paraneoplastic syndromes are rare in lymphoid malignancies, particularly in non-Hodgkin lymphoma. We report the sudden onset of acute renal failure and NS in a 14-year-old male who initially presented with HLH and was subsequently diagnosed with ALK-positive anaplastic large-cell lymphoma (ALCL). The treatment of ALCL reversed both the HLH and NS findings. This case demonstrates the importance of considering lymphoma in pediatric patients presenting with NS and HLH.
1048	HHV-8-associated haemophagocytic lymphohistiocytosis in a patient with advanced AIDS.	29275390	We present a patient with advanced AIDS admitted with recurrent shock of unclear aetiology, fevers, altered mental status and refractory cytopenias. His case posed a diagnostic challenge because evaluation of septic shock in the setting of advanced AIDS requires a time-consuming work-up for broad infectious aetiologies that can delay consideration of other diagnoses, including primary or secondary haemophagocytic lymphohistiocytosis (HLH). After this patient did not improve with supportive care and empiric antimicrobials, there was concern for HLH given that he met ≥5 of the HLH consortium criteria. He underwent bone marrow biopsy, which was non-diagnostic. Empiric therapy for HLH was initiated, but unfortunately, the patient died. Autopsy revealed extensive haemophagocytosis in the spleen, bone marrow and liver, confirming the diagnosis of HLH. Postmortem, his soluble CD-25 returned 18 890 pg/mL (<1033 pg/mL), and his serum HHV-8 PCR resulted positive. The diagnosis was HLH secondary to Human Herpes Virus 8 (HHV-8) in a patient with advanced AIDS.
1049	Results of the HLH-2004 study.	29269537	NaN
1050	Etoposide for HLH: the limits of efficacy.	2926952928935695	NaN
1051	[Familial Haemophagocytic Lymphohistiocytosis Occurs in A Fetus at His Third Trimester-A Case Report].	29262924	To analyze the clinical features and pathogenetic gene mutation in a fetus at his third trimester with familial haemophagocytic lymphohistiocytosis (FHL).Target region sequencing and high-throughput sequencing were used to detect pathogenetic gene mutations for familial haemophagocytic lymphohistiocytosis in a late onset HLH fetus. Pathogenetic gene mutations of the patient and his parents were verified by Sanger dideoxy sequencing.A male neonate, who had right pleural effusion, hepatomegaly and splenomegaly previously revealed by fetus ultrasound, was delivered at full-term by cesarean section. His clinical presentation showed recurrent fever, tachypenea, decreased breath sounds on right side, hepatosplenomegaly etc., which were gradually aggravating Lab.tests results were as follows: WBC 9.88×109/L, Hb 91 g/L, Plt 13×109/L, ALT 18 U/L,AST 69 U/L,TBIL 207.2 µmol/L, DBIL 183.5 µmol/L, TG 3.05 mmol/L, Fib 0.88 g/L, Serum ferritin 3 120 ng/ml and sIL-2R 57 420 U/ml. FCM showed that CD3-CD16+CD56+ cells reached to 3.60% in the pripheral blood. Haemophagocytes were occasionally found in the bone marrow. NK/NKT stimulation test showed a severe damage of degranulation of NK cells. Sequence analysis of genomic DNA from his peripheral blood demonstrated the compound heterozygous mutations of UNC13D gene: c.2448-13 G>A in exon26 and c.1055+1 G>A in exon12, both were pathogenetic mutations. In detailed family survey, it was confirmed that the mutation c.2448-13 G>A in exon26 was inherited from his mother and c.1055+1 G>A in exon12 from his father.A rare case of familial haemophagocytic lymphohistiocytosis type 3 (FHL3) with late fetus onset who carried pathogenetic compound heterozygous mutations of UNC13D gene. Those neonates with recurrent fever, serous effusions and multiple organ failure should be screened for FHL. Identifying the pathogenic gene mutations laid the foundation of conceiving disease-free newborns.
1052	Lytic viral replication and immunopathology in a cytomegalovirus-induced mouse model of secondary hemophagocytic lymphohistiocytosis.	29258535	Hemophagocytic lymphohistiocytosis (HLH) is a rare immunological disorder caused by unbridled activation of T cells and macrophages, culminating in a life-threatening cytokine storm. A genetic and acquired subtype are distinguished, termed primary and secondary HLH, respectively. Clinical manifestations of both forms are frequently preceded by a viral infection, predominantly with herpesviruses. The exact role of the viral infection in the development of the hemophagocytic syndrome remains to be further elucidated.We utilized a recently developed murine model of cytomegalovirus-associated secondary HLH and dissected the respective contributions of lytic viral replication and immunopathology in its pathogenesis.HLH-like disease only developed in cytomegalovirus-susceptible mouse strains unable to clear the virus, but the severity of symptoms was not correlated to the infectious viral titer. Lytic viral replication and sustained viremia played an essential part in the pathogenesis since abortive viral infection was insufficient to induce a full-blown HLH-like syndrome. Nonetheless, a limited set of symptoms, in particular anemia, thrombocytopenia and elevated levels of soluble CD25, appeared less dependent of the viral replication but rather mediated by the host's immune response, as corroborated by immunosuppressive treatment of infected mice with dexamethasone.Both virus-mediated pathology and immunopathology cooperate in the pathogenesis of full-blown virus-associated secondary HLH and are closely entangled. A certain level of viremia appears necessary to elicit the characteristic HLH-like symptoms in the model.
1053	AetMYC1, the Candidate Gene Controlling the Red Coleoptile Trait in Aegilops tauschii Coss. Accession As77.	29258257	The red coleoptile trait can help monocotyledonous plants withstand stresses, and key genes responsible for the trait have been isolated from Triticum aestivum, Triticum urartu, and Triticum monococcum, but no corresponding research has been reported for Aegilops tauschii. In this research, transcriptome analysis was performed to isolate the candidate gene controlling the white coleoptile trait in Ae. tauschii. There were 5348 upregulated, differentially-expressed genes (DEGs) and 4761 downregulated DEGs in red coleoptile vs. white coleoptile plants. Among these DEGs, 12 structural genes and two transcription factors involved in anthocyanin biosynthesis were identified. The majority of structural genes showed lower transcript abundance in the white coleoptile of accession 'As77' than in the red coleoptile of accession 'As60', which implied that transcription factors related to anthocyanin biosynthesis could be the candidate genes. The MYB and MYC transcription factors AetMYB7D and AetMYC1 were both isolated from Ae. tauschii accessions 'As60' and 'As77', and their transcript levels analyzed. The coding sequence and transcript level of AetMYB7D showed no difference between 'As60' and 'As77'. AetMYC1p encoded a 567-amino acid polypeptide in 'As60' containing the entire characteristic domains, bHLH-MYC_N, HLH, and ACT-like, belonging to the gene family involved in regulating anthocyanin biosynthesis. AetMYC1w encoded a 436-amino acid polypeptide in 'As77' without the ACT-like domain because a single nucleotide mutation at 1310 bp caused premature termination. Transient expression of AetMYC1p induced anthocyanin biosynthesis in 'As77' with the co-expression of AetMYB7D, while AetMYC1w could not cause induced anthocyanin biosynthesis under the same circumstances. Moreover, the transcript abundance of AetMYC1w was lower than that of AetMYC1p. AetMYC1 appears to be the candidate gene controlling the white coleoptile trait in Ae. tauschii, which can be used for potential biotech applications, such as producing new synthetic hexaploid wheat lines with different coleoptile colors.
1054	Hepatitis during pregnancy: A case of hemophagocytic lymphohistiocytosis.	29239849	Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe and potentially fatal syndrome that can occur during pregnancy. A 36 years-old woman, at 29 weeks of gestation, presented with itchiness and jaundice since a week. On clinical examination she was apyrexial and frankly icteric. Laboratory data showed evidence of acute hepatitis. A complete work-up was made excluding viral hepatitis (HAV, HEV, HBV, HCV, HHV6, CMV, EBV) and autoimmune liver disease. Liver diseases related to pregnancy were not completely excluded. A liver biopsy was performed and firstly interpreted as showing features of acute hepatitis. The clinical situation worsened, she developed fever with signs of fetal distress and emergent delivery was done. A second look at the liver biopsy showed features compatible with HLH, which was also confirmed on bone marrow biopsy. Extensive work-up with exclusion of infectious and malignant diseases, lead us to the diagnosis of HLH secondary to pregnancy and short term steroid therapy was started. She then completely recovered and didn't present any relapse after 4 months of follow up. HLH during pregnancy is very rare and this is the first case of HLH presenting as acute hepatitis and diagnosed on a liver biopsy.
1055	Clinical outcomes of unrelated cord blood transplantation in children with malignant and non-malignant diseases: Multicenter experience in China.	29239497	This multicenter retrospective study included 184 children with malignant and non-malignant diseases who underwent UCBT between January 1998 and August 2012. The malignant disease group included 101 children with ALL, AML, CML, JMML, and MDS, and the non-malignant disease group included 83 children with PID, β-thalassemia, IMD BMF, and HLH. The median duration to neutrophil and platelet engraftment was 16 and 35 days in the malignant disease group vs 15 and 38 days in the non-malignant disease group. The cumulative incidence of grade II-IV aGVHD and cGVHD was 25.6% and 13.5% in the malignant disease group vs 19.7% and 11.1% in the non-malignant disease group, respectively. The median duration and cumulative incidence of neutrophil and platelet engraftment, and the cumulative incidence of grade II-IV aGVHD and cGVHD were similar between the two groups. Of the 184 pediatric patients, 114 patients survived during a median follow-up period of 14 months (range 4-138). The 5-year OS and DFS were not statistically different between the two groups (56.3% and 46.1% in malignant disease group vs 68.5% and 52.8% in non-malignant disease group). The above results indicate that UCB is a viable source for HSCT for children with malignant or non-malignant diseases, especially in urgent cases.
1056	Allogeneic hematopoietic stem cell transplantation is associated with cure and durable remission of late-onset primary isolated central nervous system hemophagocytic lymphohistiocytosis.	29239076	Primary isolated CNS presentation of HLH is exceedingly rare and typically associated with significant morbidity and mortality. We describe an adolescent patient with late-onset, primary isolated CNS HLH and a compound heterozygous PRF1 mutation (c50delT (p.L17 fs); c.1229G>C (p.R410P)), not previously reported with this phenotype. He was successfully treated with allogeneic HSCT following a reduced-intensity conditioning regimen, despite a high pre-HSCT comorbidity index. Two years after transplant, he is alive and in disease remission. While patients with systemic HLH and active CNS disease have relatively poorer outcomes, a high index of suspicion may aid with early diagnosis of primary isolated CNS HLH; prompt treatment with HSCT may be associated with improved cure and durable remission of this rare disease.
1057	Multiple Nonleukemic Myeloid Sarcoma Associated Hemophagocytic Lymphohistiocytosis in an Adult.	29226650	Nonleukemic myeloid sarcoma (MS) occurs rarely. Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal condition. We report a rare case of nonleukemic MS associated with HLH.Hematologic investigation, 18F-FDG PET/CT, bone marrow aspirate and biopsy, and lymph node biopsy were performed in a 25-year-old male patient.The patient was given a short-term treatment of etoposide and dexamethasone to control HLH. Then he received chemotherapy and responded well.It is important to find the underlying cause of HLH in high-risk patients. HLH can occur secondary to nonleukemic MS. Early diagnosis and treatment can improve survival.
1058	Reactivation of Hemophagocytic Lymphohistiocytosis Triggered by Antithymocyte Globulin.	29225252	A 16-year-old boy with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (HLH) underwent allogeneic hematopoietic stem cell transplantation after conditioning with fludarabine, melphalan, total body irradiation, and rabbit antithymocyte globulin (ATG). A severe, persistent infusion reaction occurred after the initial administration of ATG. Investigations showed a rapid increase in the levels of liver enzymes and ferritin, and the reactivation of HLH was confirmed by marked hemophagocytosis in the bone marrow. Treatment with pulse glucocorticoid therapy resulted in the improvement of HLH. This is the first case of HLH reactivation triggered by ATG. Physicians should therefore be cautious of HLH reactivation, especially when a severe and prolonged infusion reaction occurs.
1059	Direct HPV E6/Myc interactions induce histone modifications, Pol II phosphorylation, and hTERT promoter activation.	29221209	Human Papillomavirus Viruses (HPVs) are associated with the majority of human cervical and anal cancers and 10-30% of head and neck squamous carcinomas. E6 oncoprotein from high risk HPVs interacts with the p53 tumor suppressor protein to facilitate its degradation and increases telomerase activity for extending the life span of host cells. We published previously that the Myc cellular transcription factor associates with the high-risk HPV E6 protein in vivo and participates in the transactivation of the hTERT promoter. In the present study, we further analyzed the role of E6 and the Myc-Max-Mad network in regulating the hTERT promoter. We confirmed that E6 and Myc interact independently and that Max can also form a complex with E6. However, the E6/Max complex is observed only in the presence of Myc, suggesting that E6 associates with Myc/Max dimers. Consistent with the hypothesis that Myc is required for E6 induction of the hTERT promoter, Myc antagonists (Mad or Mnt) significantly blocked E6-mediated transactivation of the hTERT promoter. Analysis of Myc mutants demonstrated that both the transactivation domain and HLH domain of Myc protein were required for binding E6 and for the consequent transactivation of the hTERT promoter, by either Myc or E6. We also showed that E6 increased phosphorylation of Pol II on the hTERT promoter and induced epigenetic histone modifications of the hTERT promoter. More important, knockdown of Myc expression dramatically decreased engagement of acetyl-histones and Pol II at the hTERT promoter in E6-expressing cells. Thus, E6/Myc interaction triggers the transactivation of the hTERT promoter by modulating both histone modifications, Pol II phosphorylation and promoter engagement, suggesting a novel mechanism for telomerase activation and a new target for HPV- associated human cancer.
1060	Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis Mimicking Lymphoma on FDG PET/CT.	29215415	A 15-year-old boy with fever, pancytopenia, and hepatosplenomegaly was diagnosed as hemophagocytic lymphohistiocytosis (HLH). F-FDG PET/CT showed hypermetabolic foci in the liver, spleen, and bone marrow, as well as multiple FDG-avid lymph nodes, which were highly suggestive of lymphoma. Specimens from splenectomy depicted a large number of macrophages/histiocytes with hemophagocytosis of erythrocytes, without evidence of malignancy. Considering increased Epstein-Barr virus (EBV) DNA in peripheral blood and positive staining for EBV-encoded RNA in the spleen, EBV-associated HLH was confirmed. This case indicates that FDG-avid foci in the liver, spleen, and bone marrow may also be seen in EBV-associated HLH.
1061	Macrophage Activation Syndrome: A Report of Two Cases and a Literature Review.	29209549	Macrophage activation syndrome (MAS) is a severe, potentially fatal condition that may complicate autoimmune diseases, and it belongs to hemophagocytic lymphohistiocytosis (HLH) disorders. MAS occurs in adults and children. However, it is rare in juvenile systemic lupus erythematosus (jSLE), and it is extremely rare to be the initial presentation of jSLE. Here, we report two patients with juvenile SLE who initially presented with MAS. One of the two patients is 4 years old. This is the youngest reported patient to our knowledge.
1062	Effect and mechanism of RNAi targeting WWTR1 on biological activity of gastric cancer cells SGC7901.	29207147	Gastric cancer (GC) is one of the most common malignancies in the world. It is essential to develop novel targets and therapeutic approaches for GC, which requires identification of novel functional molecules. WW‑domain containing transcription regulator 1 (WWTR1) may activate many transcriptional factors and exhibit an important role in the development of various tissues in mammals. The results of the present study demonstrated that mRNA and protein levels of WWTR1 are increased in GC tissues and cell lines. The SGC7901 cell line was selected to perform RNA interference (RNAi) targeting WWTR1, and for subsequent study. Compared with control groups (cells without any treatment) and mock groups (cells treated with nonspecific siRNA), cell proliferation of siWWTR1 cells (cells treated with WWTR1 siRNA) was detected using a Cell Counting Kit‑8 assay at 12, 24 and 48 h, and decreased in a time‑dependent manner. Cell cycle and apoptosis status were determined by flow cytometry, and it was demonstrated that G1/S transition was blocked in the cell cycle and apoptosis promoted in siWWTR1 cells, compared with control and mock cells. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect the mRNA and protein levels of cell cycle and apoptosis‑associated factors. The expression of Cyclin D1, cancer Myc and B cell lymphoma/leukemia‑2 (Bcl‑2) decreased and Bcl‑2 associated X protein increased significantly in siWWRT1 cells, at the mRNA and protein level, compared with control and mock cells. With the exception of the Hippo pathway, siWWTR1 regulated downstream factors, including mothers against decapentaplegic homolog family member 3 (SMAD3) and inhibitor of DNA binding 1, HLH protein (ID1), HLH protein in the transforming growth factor (TGF)‑β pathway. The expression of asparagine synthetase was decreased whereas ID1, SMAD3 (proteins that participate in intracellular TGF‑β transduction) and betacellulin increased notably in siWWRT1 cells. In conclusion, WWTR1 promotes cell proliferation and inhibits apoptosis of GC cells by regulating cell cycle/apoptosis‑associated factors, and effectors in the TGF‑β pathway.
1063	Dengue-associated Hemophagocytic Lymphohistiocytosis: A Rare Complication of a Common Infection in Singapore.	29200153	Hemophagocytic lymphohistiocytosis (HLH) can progress rapidly, often leading to multisystem organ failure and death. Prompt recognition of the syndrome and institution of appropriate treatment are crucial steps in improving the outcome. Dengue virus infection is not commonly known to be associated with secondary HLH. We present a case of a child with dengue fever who subsequently developed classical features of HLH. He was treated successfully with 4 weeks of steroid monotherapy instead of the multidrug therapy proposed in the HLH 2004 protocol. There was prompt response to the treatment with resolution of clinical and biochemical features. He remains in complete remission 3 years from the diagnosis.
1064	Expression and regulation of inhibitor of DNA binding proteins ID1, ID2, ID3, and ID4 at the maternal-conceptus interface in pigs.	29197292	Inhibitor of DNA binding (ID) proteins, ID1, ID2, ID3, and ID4 are transcriptional regulators that have a helix-loop-helix (HLH) domain but not a basic DNA binding domain. ID proteins inhibit the functions of basic HLH transcription factors and regulate cell proliferation and differentiation. However, the expression and function of ID1, ID2, ID3, and ID4 at the maternal-conceptus interface are not fully understood in pigs. Therefore, we determined the expressions of ID1, ID2, ID3, and ID4 in porcine endometrium, conceptus, and chorioallantoic tissues. ID1, ID2, ID3, and ID4 mRNAs were expressed in the endometrium, with lower levels of ID1, ID2, and ID4 on Day 12 of pregnancy than during the estrous cycle. ID1, ID2, ID3, and ID4 mRNAs were also detected in conceptus and chorioallantoic tissues during pregnancy. ID2 protein was mainly localized to luminal epithelia and weakly to vascular smooth muscle cells in the endometrium and conceptus trophectoderm. Increasing doses of interleukin-1β decreased levels of ID2 mRNA, while estradiol-17β increased levels of ID3 mRNA in endometrial explants. The expressions of ID2 and ID4 mRNAs were higher in endometria from gilts with somatic cell nuclear transfer-derived conceptuses compared with endometria from gilts carrying conceptuses derived from natural mating on Day 12. These results indicate that the expressions of ID family genes are dynamically regulated at the maternal-conceptus interface, suggesting that ID proteins may play critical roles in the regulation of endometrial epithelial cell function and conceptus development to support the establishment and maintenance of pregnancy in pigs.
1065	Fetal Splenomegaly: A Review.	29194292	Enlargement of the fetal spleen is usually found secondary to systemic diseases and is frequently associated with hepatomegaly. By far, the most common causes of fetal splenomegaly are infectious. Other etiologies responsible for this sign are hemolytic anemia, congestive cardiac failure, metabolic disorders, and rarely, leukemia, lymphoma, and histiocytosis.We report a case of prenatal splenomegaly diagnosed at 35 weeks, confirmed in the postnatal period. The postnatal workup showed the newborn had a familial type 3 form of hemophagocytic lymphohistiocytosis (HLH).Hemophagocytic lymphohistiocytosis is an aggressive and life-threatening syndrome of excessive immune activation. The genes implicated in the hereditary forms of the disease act in an autosomal recessive fashion.
1066	Hemophagocytic Lymphohistiocytosis Secondary to T Cell/Histiocyte-rich Large B Cell Lymphoma in an Adolescent Male.	29187030	Hemophagocytic Lymphohistiocytosis (HLH) is a hyperinflammatory disorder that may be encountered as a primary or secondary phenomenon. HLH secondary to lymphoma has been described, more frequently in adults than in children. T-cell/Histiocyte-rich B-cell lymphoma (THRLBCL) is a large B-cell lymphoma that resides in a microenvironment of robust host immune response and has previously been associated with HLH in adults. Here, we describe the first case of HLH secondary to THRLBCL in an adolescent patient.
1067	A nine-month-old-boy with Atypical Hemophagocytic Lymphohistiocytosis.	29181134	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and histiocytes. Often, HLH is an acquired syndrome. We report a case of a nine month-old-boy presented with hepatosplenomegaly, severe anemia, thrombocytopenia, hypertriglyceridemia and high hyperferritinemia. These clinical features of HLH prompted a wide range of infectious and auto-immune tests to be performed. After an extensive diagnostic workup, he was referred to the immune-hematologic unit for HLH suspicion with an unknown cause. Primary HLH due to familial lymphohistiocytosis (FLH) was first evoked in front of consanguinity, probable HLH in the family, early onset, and in the absence of a causative pathology like infection or cancer. However, functional tests were normal. Atypical features like the: absence of fever, hypotonia, recurrent diarrhea since diversification, hematuria, and proteinuria suggested an inborn metabolism error with gastrointestinal involvement. Specific tests were performed to reach a final diagnosis.
1068	Lymphocyte-independent pathways underlie the pathogenesis of murine cytomegalovirus-associated secondary haemophagocytic lymphohistiocytosis.	29178470	Haemophagocytic lymphohistiocytosis (HLH) constitutes a spectrum of immunological disorders characterized by uncontrolled immune activation and key symptoms such as fever, splenomegaly, pancytopenia, haemophagocytosis, hyperferritinaemia and hepatitis. In genetic or primary HLH, hyperactivated CD8+ T cells are the main drivers of pathology. However, in acquired secondary HLH, the role of lymphocytes remains vague. In the present study the involvement of lymphocytes in the pathogenesis of a cytomegalovirus-induced model of secondary HLH was explored. We have previously reported CD8+ T cells to be redundant in this model, and therefore focused on CD4+ helper and regulatory T cells. CD4+ T cells were activated markedly and skewed towards a proinflammatory T helper type 1 transcription profile in mice displaying a severe and complete HLH phenotype. Counter to expectations, regulatory T cells were not reduced in numbers and were, in fact, more activated. Therapeutic strategies targeting CD25high hyperactivated T cells were ineffective to alleviate disease, indicating that T cell hyperactivation is not a pathogenic factor in cytomegalovirus-induced murine HLH. Moreover, even though T cells were essential in controlling viral proliferation, CD4+ T cells, in addition to CD8+ T cells, were dispensable in the development of the HLH-like syndrome. In fact, no T or B cells were required for induction and propagation of HLH disease, as evidenced by the occurrence of cytomegalovirus-associated HLH in severe combined immunodeficient (SCID) mice. These data suggest that lymphocyte-independent mechanisms can underlie virus-associated secondary HLH, accentuating a clear distinction with primary HLH.
1069	Elevated luteinizing hormone despite normal testosterone levels in older men-natural history, risk factors and clinical features.	2917835929677870	Elevated luteinizing hormone (LH) with normal testosterone (T) suggests compensated dysregulation of the gonadal axis. We describe the natural history, risk factors and clinical parameters associated with the development of high LH (HLH, LH >9.4 U/L) in ageing men with normal T (T ≥ 10.5 nmol/L).We conducted a 4.3-year prospective observational study of 3369 community-dwelling European men aged 40-79 years. Participants were classified as follows: incident (i) HLH (n = 101, 5.2%); persistent (p) HLH (n = 128, 6.6%); reverted (r) HLH (n = 46, 2.4%); or persistent normal LH (pNLH, n = 1667, 85.8%). Potential predictors and changes in clinical features associated with iHLH and rHLH were analysed using regression models.Age >70 years (OR = 4.12 [2.07-8.20]), diabetes (OR = 2.86 [1.42-5.77]), chronic pain (OR = 2.53 [1.34-4.77]), predegree education (OR = 1.79 [1.01-3.20]) and low physical activity (PASE ≤ 78, OR = 2.37 [1.24-4.50]) predicted development of HLH. Younger age (40-49 years, OR = 8.14 [1.35-49.13]) and nonsmoking (OR = 5.39 [1.48-19.65]) predicted recovery from HLH. Men with iHLH developed erectile dysfunction, poor health, cardiovascular disease (CVD) and cancer more frequently than pNLH men. In pHLH men, comorbidities, including CVD, developed more frequently, and cognitive and physical function deteriorated more, than in pNLH men. Men with HLH developed primary hypogonadism more frequently (OR = 15.97 [5.85-43.60]) than NLH men. Men with rHLH experienced a small rise in BMI.Elevation of LH with normal T is predicted by multiple factors, reverts frequently and is not associated with unequivocal evidence of androgen deficiency. High LH is a biomarker for deteriorating health in aged men who tend to develop primary hypogonadism.
1070	Atypical Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis simulating lymphadenitis on 18F-FDG PET/CT and its differential diagnosis.	29177266	Diagnosis of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is challenging as its clinical presentation is atypical. Here we present a case of atypical EBV-HLH simulating lymphadenitis on fluorrine-18-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT), with a view to consider this kind of EBV-HLH as a possible differential diagnosis in lymphadenitis. A 68 years old male who had episodic fever accompanied by weight loss and weakness for two weeks was studied. Finally, biopsies in bone marrow and spleen revealed hemophagocytic cells. He was diagnosed with EBV-HLH and treated with etoposide and prednisone. His condition started improving soon, and his abnormal laboratory findings were normalized at day 15. He remained in good clinical condition at 3 months follow-up after hospital discharge.
1071	The relationship between circulating concentration of AMH and LH content in the follicle stimulating hormone (FSH) preparations on follicular growth and ovulatory response to superovulation in water buffaloes.	29175175	The relationship between circulating concentration of anti-mullerian hormone (AMH) and the LH content of follicle stimulating hormone (FSH) preparation on follicular growth and ovulatory response in water buffaloes was evaluated. A single blood sample was taken from cows (N=31; age: 9.06±0.98years) to determine systemic AMH. Animals with concentrations higher or lower than 194±30pg/ml were placed into LOW and HIGH AMH groups and were assigned randomly to be superovulated FSH containing either a high (FSHp, HLH) or low (FolltropinV, LLH) LH content. Follicular growth and ovulation were monitored using transrectal ultrasonography. In animals with HIGH systemic AMH, treatment with FSH with a high LH content was associated with more small follicles (AMH X FSH; P=0.02). AMH had no effect on small follicles in animals treated with LLH. Females with a HIGH AMH had greater numbers of small follicles (P=0.01) and total follicles (P=0.005) than LOW AMH cows. Animals treated with HLH had more small follicles (P=0.001) but fewer large (P<0.001) and total follicles (P=0.0005) than those treated with LLH. Among animals with HIGH AMH, those treated with LLH, ovulated more follicles than those treated with HLH. (AMH X FSH; P=0.03). In conclusion, selecting animals with high AMH concentration and the use of FSH preparations with a lower LH content may improve the superovulatory response in water buffaloes.
1072	Arabidopsis RSS1 Mediates Cross-Talk Between Glucose and Light Signaling During Hypocotyl Elongation Growth.	29170398	Plants possess exuberant plasticity that facilitates its ability to adapt and survive under challenging environmental conditions. The developmental plasticity largely depends upon cellular elongation which is governed by a complex network of environmental and phytohormonal signals. Here, we report role of glucose (Glc) and Glc-regulated factors in controlling elongation growth and shade response in Arabidopsis. Glc controls shade induced hypocotyl elongation in a dose dependent manner. We have identified a Glc repressed factor REGULATED BY SUGAR AND SHADE1 (RSS1) encoding for an atypical basic helix-loop-helix (bHLH) protein of unknown biological function that is required for normal Glc actions. Phenotype analysis of mutant and overexpression lines suggested RSS1 to be a negative regulator of elongation growth. RSS1 affects overall auxin homeostasis. RSS1 interacts with the elongation growth-promoting proteins HOMOLOG OF BEE2 INTERACTING WITH IBH 1 (HBI1) and BR ENHANCED EXPRESSION2 (BEE2) and negatively affects the transcription of their downstream targets such as YUCs, INDOLE-3-ACETIC ACID INDUCIBLE (IAAs), LONG HYPOCOTYL IN FAR-RED1 (HFR1), HOMEOBOX PROTEIN 2 (ATHB2), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASES (XTHs) and EXPANSINS. We propose, Glc signals might maintain optimal hypocotyl elongation under multiple signals such as light, shade and phytohormones through the central growth regulatory bHLH/HLH module.
1073	[Clinical analysis of patients with hemophagocytic lymphohistiocytosis complicated with gastrointestinal bleeding].	29166737	Objective: To explore the clinical features of patients with hemophagocytic lymphohistiocytosis (HLH) complicated with gastrointestinal bleeding. Methods: The clinical data of 52 patients with HLH diagnosed from January 2015 to February 2017 were analyzed retrospectively. Results: ①Of the 52 patients[including 36 cases of EBV related (69.2%) , 5 cases of lymphoma related (9.6%) , 3 cases of primary HLH (5.8%) , 3 cases of rheumatism related (5.8%) , 2 cases of tuberculosis related (3.8%) , 3 cases of unknown causes (3.8%) ], 32 cases were male (61.5%) and 20 cases female (38.5%) with a median age of 26 (6-64) years old. ②The overall survival rates at 1, 3, 6 and 12 months were 74.7%, 53.8%, 32.9% and 23.3% respectively. ③Multivariate analysis (logistic regression) showed thrombocytopenia (P=0.036) and other sites of hemorrhage (P=0.030) were risk factors of poor prognosis, and allogeneic hematopoietic stem cell transplantation (allo-HSCT, P=0.026) was the influence of good prognostic factor. Conclusion: HLH combined with gastrointestinal bleeding was an entity of disease with poor prognosis. Thrombocytopenia and other sites of bleeding had a significant negative impact on patients, allo-HSCT produced a significant positive impact on patients.目的： 探究噬血细胞综合征（HLH）合并消化道出血患者的临床特征。 方法： 对2015年1月至2017年2月确诊为HLH且合并消化道出血的52例患者的临床资料进行回顾性分析。 结果： ①52例患者中，男32例（61.5%），女20例（38.5%），中位年龄为26（6~64）岁，其中EBV相关者36例（69.2%），淋巴瘤相关者5例（9.6%），原发性HLH者3例（5.8%），风湿免疫相关者3例（5.8%），结核相关者2例（3.8%），原因不明者3例（3.8%）。②52例患者的1、3、6、12个月总体生存率分别为74.7%、53.8%、32.9%和23.3%。③多因素分析（Logistic回归）结果显示血小板减少（P=0.036）、合并其他部位出血（P=0.030）是影响患者预后的不良因素，行异基因造血干细胞移植（P=0.026）是影响患者预后的良好因素。 结论： HLH合并消化道出血患者预后差。血小板减少及合并其他部位出血的患者预后不良，而异基因造血干细胞移植有助于改善患者预后。.
1074	[Prognosis of the central nervous system involvement in patients with hemophagocytic lymphohistiocytosis].	29166736	Objective: To investigate the characteristics and prognostic factor of central nervous system (CNS) involvement in patients with hemophagocytic lymphohistiocytosis (HLH) . Methods: From January 2006 to October 2015, 152 patients with HLH, 88 patients had CNS involvement, their clinical data were collected, and survival was analyzed using the Kaplan-Meier life table method, univariate and multivariate Cox regression model analyses were applied to identify the risk factors of prognosis. Results: ①57.9% patients complicated with neurological symptoms, cerebrospinal fluid abnormalities were observed in 37.0% patients, 57.5% patients had abnormal neuroradiology. ②36 patients survived well, 3 patients lost to follow-up, 49 dead, 1 survival patient had epilepsy. ③The 3-year overall survival rate of 88 patients was 44%. ④abnormal CSF and unreceived IT bore a significant, independent adverse prognostic value (P<0.05) . Conclusion: CNS involvement in HLH has a high frequency and poor prognosis, few patients remained neurologic sequelae; abnormal CSF related to poor prognosis, positive intrathecal injections could improve the prognosis.目的： 了解中枢神经系统（CNS）受累的儿童噬血细胞淋巴组织细胞增生症（HLH）的临床特征及预后影响因素。 方法： 回顾性分析2006年1月至2015年10月诊治的88例有CNS受累HLH患儿临床资料，采用Kaplan-Meier法绘制生存曲线，建立Cox回归模型对可能影响其预后的危险因素进行单因素及多因素分析。 结果： ①88例CNS受累的HLH患儿中，男38例，女50例，中位年龄4岁1个月（6个月~15岁10个月），占同期诊治HLH患儿比例的57.9%（88/152）。②88例患儿中有54例进行脑脊液检查，其中20例（37.0%）脑脊液异常；有73例患儿进行头颅影像学检查，其中42例（57.5%）影像学异常。③88例患儿中3例失访，36例存活（1例继发癫痫），49例死亡。中位随访时间为37（6~122）个月，3年总体生存率为44%。④多因素分析结果显示脑脊液异常（HR=3.467，95%CI 1.276~9.423，P=0.015）、未给予鞘注（HR=0.231，95%CI 0.081~0.657，P=0.006）是影响CNS-HLH患儿预后的独立危险因素。 结论： HLH患儿CNS受累率较高，预后差，少数还会遗留神经系统后遗症；脑脊液异常的患儿预后不良，积极的鞘注治疗可以改善预后。.
1075	Genomic analysis of NF-κB signaling pathway reveals its complexity in Crassostrea gigas.	29162540	NF-κB signaling pathway is an evolutionarily conserved pathway that plays highly important roles in several developmental, cellular and immune response processes. With the recent release of the draft Pacific oyster (Crassostra gigas) genome sequence, we have sought to identify the various components of the NF-κB signaling pathway in these mollusks and investigate their gene structure. We further constructed phylogenetic trees to establish the evolutionary relationship of the oyster proteins with their homologues in vertebrates and invertebrates using BLASTX and neighbor-joining method. We report the presence of two classic NF-κB/Rel homologues in the pacific oyster namely Cgp100 and CgRel, which possess characteristic RHD domain and a consensus nuclear localization signal, similar to mammalian homologues and an additional CgRel-like protein, unique to C. gigas. Further, in addition to two classical IκB homologues, CgIκB1 and CgIκB2, we have identified three atypical IκB family members namely CgIκB3, CgIκB4 and CgBCL3 which lack the IκB degradation motif and consist of only one exon that might have arisen by retrotransposition from CgIκB1. Finally, we report the presence of three IKKs and one NEMO genes in oyster genome, named CgIKK1, CgIKK2, CgIKK3 and CgNEMO, respectively. While CgIKK1 and CgIKK3 domain structure is similar to their mammalian homologues, CgIKK2 was found to lack the HLH and NBD domains. Overall, the high conservation of the NF-κB/Rel, IκB and IKK family components in the pacific oyster and their structural similarity to the vertebrate and invertebrate homologues underline the functional importance of this pathway in regulation of critical cellular processes across species.
1076	Characterization of a novel splicing mutation in UNC13D gene through amplicon sequencing: a case report on HLH.	29157204	Hemophagocytic lymphohistiocytosis (HLH) is a rare but fatal disease caused by uncontrolled proliferation of activated lymphocytes and macrophages. Six genes including SH2D1A, PRF1, UNC13D, STX11, STXBP2 and XIAP were reported as causative genes in most cases.Here we report a novel splicing mutation in UNC13D gene, which was identified in an 18-year-old female. Patient was diagnosed as HLH base on HLH-2004 guidelines, no history of inherited diseases was revealed in this family, parents were healthy and non-consanguineous. Splenomegaly and hemophagocytosis in bone marrow were observed in clinical examination. Amplicon sequencing for the whole coding region of 6 HLH-related genes was performed on Ion S5XL genetic analyzer. In all, four heterozygous mutations were detected, including 2 nonpathogenic SNPs (PRF1:c.900C > T, STX11:c.*70G > A) and 2 splicing mutations in UNC13D gene (UNC13D:c.1299 + 1G > A and UNC13D:c.2709 + 1G > A), both of which were predicted to be potentially pathogenic by human splicing finder (HSF3) tool. The result was confirmed by two-generation pedigree analysis base on sanger sequencing.Two compound heterozygous splicing mutations in UNC13D gene were identified and considered to be potential pathogenesis in a female patient of HLH. The mutation UNC13D:c.1299 + 1G > A was reported in HLH for the first time. The inheritance mode and source of the mutation in the proband was examined by family analysis. Our data suggest that further studies of the spectrum of HLH-related mutations in China are warranted.
1077	Cardanol Groups Grafted on Poly(vinyl chloride)-Synthesis, Performance and Plasticization Mechanism.	30965920	Internally plasticized poly(vinyl chloride) (PVC) materials are investigated via grafting of propargyl ether cardanol (PEC). The chemical structure of the materials was studied by FT-IR and ¹H NMR. The performace of the obtained internally plasticized PVC materials was also investigated with TGA, DSC and leaching tests. The results showed that grafting of propargyl ether cardanol (PEC) on PVC increased the free volume and distance of PVC chains, which efficiently decreased the glass transition temperature (Tg). No migration was found in the leaching tests for internally plasticized PVC films compared with plasticized PVC materials with commercial plasticizer dioctyl phthalate (DOP). The internal plasticization mechanism was also disscussed according to lubrication theory and free volume theory. This work provides a meaningful strategy for designing no-migration PVC materials by introducing cardanol groups as branched chains.
1078	Autophagy and innate immunity: Insights from invertebrate model organisms.	29130360	Macroautophagy/autophagy is a fundamental intracellular degradation process with multiple roles in immunity, including direct elimination of intracellular microorganisms via 'xenophagy.' In this review, we summarize studies from the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans that highlight the roles of autophagy in innate immune responses to viral, bacterial, and fungal pathogens. Research from these genetically tractable invertebrates has uncovered several conserved immunological paradigms, such as direct targeting of intracellular pathogens by xenophagy and regulation of autophagy by pattern recognition receptors in D. melanogaster. Although C. elegans has no known pattern recognition receptors, this organism has been particularly useful in understanding many aspects of innate immunity. Indeed, work in C. elegans was the first to show xenophagic targeting of microsporidia, a fungal pathogen that infects all animals, and to identify TFEB/HLH-30, a helix-loop-helix transcription factor, as an evolutionarily conserved regulator of autophagy gene expression and host tolerance. Studies in C. elegans have also highlighted the more recently appreciated relationship between autophagy and tolerance to extracellular pathogens. Studies of simple, short-lived invertebrates such as flies and worms will continue to provide valuable insights into the molecular mechanisms by which autophagy and immunity pathways intersect and their contribution to organismal survival. Abbreviations Atg autophagy related BECN1 Beclin 1 CALCOCO2 calcium binding and coiled-coil domain 2 Cry5B crystal toxin 5B Daf abnormal dauer formation DKF-1 D kinase family-1 EPG-7 Ectopic P Granules-7 FuDR fluorodeoxyuridine GFP green fluorescent protein HLH-30 Helix Loop Helix-30 Imd immune deficiency ins-18 INSulin related-18; LET-363, LEThal-363 lgg-1 LC3, GABARAP and GATE-16 family-1 MAPK mitogen-activated protein kinase MATH the meprin and TRAF homology MTOR mechanistic target of rapamycin NBR1 neighbor of BRCA1 gene 1 NFKB nuclear factor of kappa light polypeptide gene enhancer in B cells NOD nucleotide-binding oligomerization domain containing OPTN optineurin PAMPs pathogen-associated molecular patterns Park2 Parkinson disease (autosomal recessive, juvenile) 2, parkin pdr-1 Parkinson disease related PFTs pore-forming toxins PGRP peptidoglycan-recognition proteins PIK3C3 phosphatidylinositol 3- kinase catalytic subunit type 3 pink-1 PINK (PTEN-I induced kinase) homolog PRKD protein kinase D; PLC, phospholipase C PRKN parkin RBR E3 ubiquitin protein ligase PRRs pattern-recognition receptors PtdIns3P phosphatidylinositol-3-phosphate rab-5 RAB family-5 RB1CC1 RB1-inducible coiled-coil 1 RNAi RNA interference sqst SeQueSTosome related SQSTM1 sequestosome 1 TBK1 TANK-binding kinase 1 TFEB transcription factor EB TGFB/TGF-β transforming growth factor beta TLRs toll-like receptors unc-51 UNCoordinated-51 VPS vacuolar protein sorting; VSV, vesicular stomatitis virus VSV-G VSV surface glycoprotein G Wipi2 WD repeat domain, phosphoinositide interacting 2.
1079	Infusion of leukocytes from HLA haplo-identical familial donors as an adjuvant in the HLH-2004 protocol to treat the virus-associated adult hemophagocytic lymphohistiocytosis: a retrospective study of 26 patients.	29130133	Adult hemophagocytic lymphohistiocytosis (HLH) is a fatal disease with poor survival and a limited role of drug therapies. To help to recognize virus and enhance survival, we infused leukocytes derived from human leukocyte antigen (HLA) haplo-identical familial donors to patients. We retrospectively investigated 26 adult virus-associated hemophagocytic syndrome (VAHS) patients' medical records from 2006-2017. Eleven of the 26 patients accepted relatives' derived leukocytes infusions in addition to drug therapies recommended in the HLH-2004 protocol. The leukocyte doses ranged from 0.75 to 3.30×108 per kilogram of body weight. The other 15 patients accepted immunosuppressive and supportive therapies referred to in the HLH-2004 protocol. We compared the treatment outcomes of the two groups of patients. Patients in the cell infusion group had a lower viral load (P = 0.023) and better laboratory results and prolonged overall survival (60.44 vs. 20.18 weeks, P = 0.047). A factor that might relate to overall survival is platelet count (P = 0.032), except for the leukocyte infusions (P = 0.012). For patients without acceptable donors, infusions of leukocytes from HLA haplo-identical familial donors could be a feasible treatment to prolong overall survival as an adjuvant to drug therapies.
1080	Interdisciplinary care of a neonate with haemophagocytic lymphohistiocytosis.	29115762	Haemophagocytic lymphohistiocytosis (HLH) is a rare condition not commonly observed in neonatal units. It poses a challenge to neonatal staff to source expertise and information when diagnosing and treating patients with this condition. This article uses a case study of a neonate with HLH to analyse interdisciplinary team dynamics in hospital and explore how teams can effectively share knowledge and learn from each other when treating patients with rare and complex conditions.
1081	Macrophage activation syndrome associated with systemic lupus erythematosus treated successfully with the combination of steroid pulse, immunoglobulin and tacrolimus.	29099692	Macrophage activation syndrome (MAS), a variant of secondary hemophagocyticlymphohistiocytosis, is a potentially life-threatening complication of inflammatory and autoimmune diseases. We present a case of MAS as a rare manifestation of systemic lupus erythematosus. Although initial treatment with corticosteroid, with or without cyclosporine A, is justified in patients with MAS, evidence regarding the effectiveness of this treatment protocol remains to be clarified. Our patient was successfully treated with a combination of intravenous immunoglobulin therapy and intravenous methyl predonisolone pulse therapy, which was followed by a course of oral prednisolone and oral tacrolimus. Based on our experience, we propose tacrolimus to provide a more useful adjuvant treatment to corticosteroid therapy than cyclosporine A.
1082	Comparative proteomic analysis: SclR is importantly involved in carbohydrate metabolism in Aspergillus oryzae.	29098410	The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes in organisms from mammals to microbes. We previously identified the bHLH transcription factor SclR in Aspergillus oryzae and found that the loss of SclR function led to significant phenotypic changes, such as rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. The result implied that SclR is potentially important in both traditional fermentative manufacturing and commercial enzyme production in A. oryzae because of its effect on growth. Therefore, this study presents a comparative assessment at the proteome level of the intracellular differences between an sclR-disrupted strain and a control strain using isobaric tandem mass tag (TMT) labeling for quantification. A total of 5447 proteins were identified, and 568 were differentially expressed proteins (DEPs). Of the DEPs, 251 proteins were increased by 1.5-fold, and 317 proteins were decreased by 1.5-fold in an sclR-disrupted strain compared to the control. The comparison of the quantitative TMT results revealed that SclR was mainly involved in carbon metabolism, especially carbohydrate metabolism. In addition, an enzyme profile by a semi-quantitative method (API-ZYM) indicated that three enzymes (β-galactosidase, α-glucosidase, and α-mannosidase) were significantly less active in the ∆sclR strain than in the control. Moreover, quantitative RT-PCR showed that the expression of certain genes was changed similarly to their corresponding proteins. These results suggested that a possible function of SclR during growth of A. oryzae is its important involvement in carbohydrate metabolism.
1083	A First Report of Secondary Hemophagocytic Lymphohistiocytosis Associated With Papillary Thyroid Carcinoma.	29087969	Secondary hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal immune-deficiency disorder that develops in response to diseases that produce strong immunologic activation, such as infection and malignancy. Although secondary HLH is reported to occur in association with various malignancies, there is no report of its association with thyroid cancer. We evaluated a 19-year-old man who presented with HLH. During investigation for an underlying cause of his HLH computed tomographic scan of the neck discovered thyroid nodules that were confirmed with biopsy to be papillary thyroid carcinoma. He was treated with surgery followed by radioactive iodine therapy and remains without any recurrence of malignancy or his secondary HLH. This report documents the first observation of HLH associated with thyroid cancer, and illustrates the need to include imaging of the neck while evaluating patients with secondary HLH for an underlying malignancy.
1084	Macrophage activation syndrome as a complication of juvenile rheumatoid arthritis.	29077164	Juvenile rheumatoid arthritis (JRA), also known as juvenile idiopathic arthritis (JIA), is a rare autoimmune joint disorder of children. The concrete causes for the prevalence of the above pathological state are still unknown. In other words, it is an arthritis affecting mainly children and adolescents. Clinically, it has 3 different clinical subtypes. JRA patients are often noticed with some confirmed symptoms including coagulopathy, disseminated intravascular coagulation (DIC) with hepatosplenomegaly, fall in erythrocyte sedimentation rate and higher levels of liver enzymes leading to a life-threatening outcome. The above complications of JRA are recognized as a macrophage activation syndrome (MAS), which is similar to hemophagocytic lymphohistiocytosis (HLH). Pathogenesis of JRA manly involves deregulation of immunological processes with excessive and persistent activation of antigen presenting cells and T-lymphocytes. Further, abnormalities in the functioning of NK cells are often observed in JIA cases. Also, 40% of patients with these abnormalities are habitually associated with perforin gene mutations. Today, MAS remains a clinical and diagnostic challenge.The diagnosis of MAS is mainly based on clinical grounds. However, laboratory evidence of macrophages in the bone marrow performing phagocytosis of variable hematopoietic cells also help in diagnosis. For confirmation of MAS, there must be present either of two clinical or laboratory criteria. Further, laboratory criteria often appear late and are unable to diagnose the complication right at the beginning stage. Important laboratory findings in macrophage activation syndrome associated with JIA include hypertriglyceridemia, anemia, low erythrocyte sedimentation rate, elevated alanine aminotransferase level, higher than normal bilirubin levels, presence of fibrin degradation products, high lactate dehydrogenase level, low sodium, low albumin, and hyperferritinemia.MAS is a confirmed life threatening complication of patients with JIA. Further, an early diagnosis and treatment of MAS could be a life-saving mode for this syndrome.
1085	Genome-wide discovery of active regulatory elements and transcription factor footprints in Caenorhabditis elegans using DNase-seq.	29074739	Deep sequencing of size-selected DNase I-treated chromatin (DNase-seq) allows high-resolution measurement of chromatin accessibility to DNase I cleavage, permitting identification of de novo active cis-regulatory modules (CRMs) and individual transcription factor (TF) binding sites. We adapted DNase-seq to nuclei isolated from C. elegans embryos and L1 arrest larvae to generate high-resolution maps of TF binding. Over half of embryonic DNase I hypersensitive sites (DHSs) were annotated as noncoding, with 24% in intergenic, 12% in promoters, and 28% in introns, with similar statistics observed in L1 arrest larvae. Noncoding DHSs are highly conserved and enriched in marks of enhancer activity and transcription. We validated noncoding DHSs against known enhancers from myo-2, myo-3, hlh-1, elt-2, and lin-26/lir-1 and recapitulated 15 of 17 known enhancers. We then mined DNase-seq data to identify putative active CRMs and TF footprints. Using DNase-seq data improved predictions of tissue-specific expression compared with motifs alone. In a pilot functional test, 10 of 15 DHSs from pha-4, icl-1, and ceh-13 drove reporter gene expression in transgenic C. elegans Overall, we provide experimental annotation of 26,644 putative CRMs in the embryo containing 55,890 TF footprints, as well as 15,841 putative CRMs in the L1 arrest larvae containing 32,685 TF footprints.
1086	Successful Treatment of Hemophagocytic Lymphohistiocytosis Associated with Lupus Nephritis by Using Mycophenolate Mofetil.	29062579	An estimated 0.9% to 2.4% of patients with systemic lupus erythematosus (SLE) also have hemophagocytic lymphohistiocytosis (HLH). HLH associated with autoimmune diseases is often refractory to corticosteroid treatment; thus, additional immunosuppressive drugs, such as cyclosporine, cyclophosphamide, or tacrolimus, are required. Here, we describe the case of a 44-year-old Japanese woman who developed HLH associated with lupus nephritis. Initially, her HLH was refractory to treatment with a corticosteroid, tacrolimus, and mizoribine. However, alternative treatment with a corticosteroid, mycophenolate mofetil, and tacrolimus improved both her HLH and lupus nephritis. This case suggests the possibility of mycophenolate mofetil as a key drug for treating HLH associated with SLE.
1087	RUNX-mediated growth arrest and senescence are attenuated by diverse mechanisms in cells expressing RUNX1 fusion oncoproteins.	29052866	RUNX gene over-expression inhibits growth of primary cells but transforms cells with tumor suppressor defects, consistent with reported associations with tumor progression. In contrast, chromosomal translocations involving RUNX1 are detectable in utero, suggesting an initiating role in leukemias. How do cells expressing RUNX1 fusion oncoproteins evade RUNX-mediated growth suppression? Previous studies showed that the TEL-RUNX1 fusion from t(12;21) B-ALLs is unable to induce senescence-like growth arrest (SLGA) in primary fibroblasts while potent activity is displayed by the RUNX1-ETO fusion found in t(8;21) AMLs. We now show that SLGA potential is suppressed in TEL-RUNX1 but reactivated by deletion of the TEL HLH domain or mutation of a key residue (K99R). Attenuation of SLGA activity is also a feature of RUNX1-ETO9a, a minor product of t(8;21) translocations with increased leukemogenicity. Finally, while RUNX1-ETO induces SLGA it also drives a potent senescence-associated secretory phenotype (SASP), and promotes the immortalization of rare cells that escape SLGA. Moreover, the RUNX1-ETO SASP is not strictly linked to growth arrest as it is largely suppressed by RUNX1 and partially activated by RUNX1-ETO9a. These findings underline the heterogeneous nature of premature senescence and the multiple mechanisms by which this failsafe process is subverted in cells expressing RUNX1 oncoproteins.
1088	The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans.	29036198	An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.
1089	ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability.	2902606929875365	MDC1 plays a critical role in the DNA damage response (DDR) by interacting directly with several factors including γ-H2AX. However, the mechanism by which MDC1 is recruited to damaged sites remains elusive. Here, we show that MDC1 interacts with a helix-loop-helix (HLH)-containing protein called inhibitor of DNA-binding 3 (ID3). In response to double-strand breaks (DSBs) in the genome, ATM phosphorylates ID3 at serine 65 within the HLH motif, and this modification allows a direct interaction with MDC1. Moreover, depletion of ID3 results in impaired formation of ionizing radiation (IR)-induced MDC1 foci, suppression of γ-H2AX-bound MDC1, impaired DSB repair, cellular hypersensitivity to IR, and genomic instability. Disruption of the MDC1-ID3 interaction prevents accumulation of MDC1 at sites of DSBs and suppresses DSB repair. Thus, our study uncovers an ID3-dependent mechanism of recruitment of MDC1 to DNA damage sites and suggests that the ID3-MDC1 interaction is crucial for DDR.MDC1 is a key component of the DNA damage response and interacts with several factors such as γ-H2AX. Here the authors show that MDC1 interacts with ID3, facilitating MDC1 recruitment to sites of damage and repair of breaks.
1090	Clinical features and outcomes in secondary adult hemophagocytic lymphohistiocytosis.	29025045	Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by an infrequent but immune-mediated life-threatening disease, with confusing clinical manifestations, rapidly deteriorating health, high morbidity and mortality and challenging diagnosis.The purpose of this study was to improve the recognition and understanding of HLH.Retrospective observational cross-sectional study.Data were collected for all cases of adult patients diagnosed with HLH in a large cohort managed at a single medical center from January 2011 to December 2015.The median age was 52 years (range 18-90 years) and 123 (60.0%) were male. Over 95% patients manifested fever, hyperferritinemia and elevated lactate dehydrogenase. Underlying triggers of HLH were as follows: 119 (58.0%) malignancies, 83 (40.5%) infections, 14 (6.8%) unknown triggers and 14 (6.8%) autoimmune disorders. The median overall survival was 55 days. And elderly patients (age ≥60 years) had a markedly worse survival compared with young patients (age <60 years) (median overall survival 24 days vs. 159 days, respectively; P <0.001). In a multivariable analysis, platelet <40 × 109/l (HR = 2.534; 95% CI 1.152-5.573; P = 0.021), PT prolonged >3 s (HR = 1.909; 95% CI 1.127-3.234; P = 0.016) and malignancy (HR = 1.614; 95% CI 1.008-2.582; P = 0.046) were correlated with poor survival.HLH adult patients had very complex clinical manifestations as well as underlying diseases. Patients with PLT <40 ×109/l, PT prolonged >3 s and malignancy had inferior survival. It is of great importance to improve our understanding of this syndrome.
1091	Multivariate analysis of prognosis for patients with natural killer/T cell lymphoma-associated hemophagocytic lymphohistiocytosis.	28982299	A major cause of hemophagocytic lymphohistiocytosis (HLH) is malignant neoplasms of the blood system, among which NK/T cell lymphoma is one of the most common risk factors. Patients with NK/T cell lymphoma hemophagocytic lymphohistiocytosis (NK/T-LAHS) have a worse prognosis and higher mortality. We aimed to explore the factors that affect the prognosis of NK/T-LAHS.Clinical data of 42 patients with NK/T-LAHS diagnosed by Beijing Friendship Hospital from June 2008 to June 2016 were analyzed retrospectively. The survival time was counted until 1 August 2016.For the 42 NK/T-LAHS patients, 1-month survival rate was 48.9%, 2-month survival rate was 36.7%, 3-month survival rate was 28.8%, 6-month survival rate was 23.0%, and 12-month survival rate was 15.4%. NK/T-LAHS patients who underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT) (p = 0.000), exhibited peripheral blood Epstein-Barr virus (EBV)-positivity (p = 0.004), and achieved overall response (OR) remission after initial induction therapy (p = 0.007) had statistical significance.NK/T-LAHS is a disease of poor prognosis and high mortality. NK/T-LAHS patients who achieved OR remission after the initial induction therapy had a better prognosis than non-remission patients and Allo-HSCT was an effective way to prolong the survival of NK/T-LAHS patients. However, EBV positivity in peripheral blood was a poor prognostic factor in NK/T-LAHS patients.
1092	Safe usage of anakinra and dexamethasone to treat refractory hemophagocytic lymphohistiocytosis secondary to acute disseminated histoplasmosis in a patient with HIV/AIDS.	28978596	Hemophagocytic lymphohistiocytosis (HLH) is a serious life-threatening disease if not recognised early. In patients with HIV/AIDS, this association has been reported following acute opportunistic infections, including histoplasmosis. However, optimal treatment is not known. We describe a male aged 46 years with AIDS who developed HLH following acute disseminated histoplasmosis. Presenting symptoms included fever, hepatosplenomegaly and pancytopenia. Bone marrow biopsy confirmed HLH. Initially, he was refractory to the treatment with amphotericin B, antiretroviral therapy and intravenous immunoglobulin (IVIG). Anakinra, an interleukin-1 receptor antagonist, and dexamethasone were initiated. He improved clinically, did not exhibit any harmful effects and ultimately was discharged from the hospital. This, we believe, is the first reported treatment of HLH with anakinra in a patient with AIDS and acute disseminated histoplasmosis.
1093	Combining multiple biomarkers differentiates between active SJIA, SJIA-MAS and EBV-HLH.	2897599928815559	Cytokine storm syndromes are a clinically heterogeneous group of conditions resulting from a maladaptive host response to an inflammatory trigger. These syndromes lead to rapid progression of immune-mediated damage to healthy tissues resulting in life-threatening multi-system organ failure. Prompt recognition of disease and medical intervention to limit damage to healthy tissues is essential to prevent cytokine storm morbidity and mortality. However, the diagnosis of cytokine storm syndromes is challenging, given the clinical heterogeneity in disease presentations. Therefore, expeditious and readily available tests to diagnose disease and differentiate between the various types of cytokine storm syndromes are of clinical utility. The recently published work of Shimizu and colleagues brings us closer to making this a reality.
1094	Hemophagocytic Lymphohistiocytosis Masquerading as Acute Liver Failure: A Single Center Experience.	28970704	Hemophagocytic lymphohistiocytosis (HLH) is a potentially life-threatening disorder of extreme inflammation and unregulated immune response which require prompt recognition and early introduction of definitive therapy. HLH can present with wide range of hepatic dysfunction ranging from mild elevation of transaminases to liver failure. This study is carried out to describe the clinical and laboratory presentation of HLH.Patients who were diagnosed with HLH between January 2013 and December 2015 were retrospectively included in this study.Six patients were diagnosed as secondary HLH with median age of 28.5 years at diagnosis. All patients were presented with history of deep jaundice and high grade fever with pancytopenia and splenomegaly. Underlying diagnosis was viral infections in 4 and probable viral infection in remaining two. Bone marrow hemophagocytosis was present in 3 cases. Three patients were treated with corticosteroids only and one each with corticosteroids with cyclosporine or intravenous immunoglobulin (IVIG) and HLH treatment protocol. One patient died due to acute respiratory distress syndrome (ARDS); another patient died in follow-up due to respiratory failure due to pneumonia.HLH is rare and potentially life-threatening cause of prolonged fever, jaundice and pancytopenia. Early diagnosis and initiation of specific therapy can improve clinical outcome.
1095	Randomized, four-arm, dose-response clinical trial to optimize resistance exercise training for older adults with age-related muscle atrophy.	28964826	The myriad consequences of age-related muscle atrophy include reduced muscular strength, power, and mobility; increased risk of falls, disability, and metabolic disease; and compromised immune function. At its root, aging muscle atrophy results from a loss of myofibers and atrophy of the remaining type II myofibers. The purpose of this trial (NCT02442479) was to titrate the dose of resistance training (RT) in older adults in an effort to maximize muscle regrowth and gains in muscle function.A randomized, four-arm efficacy trial in which four, distinct exercise prescriptions varying in intensity, frequency, and contraction mode/rate were evaluated: (1) high-resistance concentric-eccentric training (H) 3d/week (HHH); (2) H training 2d/week (HH); (3) 3d/week mixed model consisting of H training 2d/week separated by 1 bout of low-resistance, high-velocity, concentric only (L) training (HLH); and (4) 2d/week mixed model consisting of H training 1d/week and L training 1d/week (HL). Sixty-four randomized subjects (65.5±3.6y) completed the trial. All participants completed the same 4weeks of pre-training consisting of 3d/week followed by 30weeks of randomized RT.The HLH prescription maximized gains in thigh muscle mass (TMM, primary outcome) and total body lean mass. HLH also showed the greatest gains in knee extension maximum isometric strength, and reduced cardiorespiratory demand during steady-state walking. HHH was the only prescription that led to increased muscle expression of pro-inflammatory cytokine receptors and this was associated with a lesser gain in TMM and total body lean mass compared to HLH. The HL prescription induced minimal muscle regrowth and generally lesser gains in muscle performance vs. the other prescriptions.The HLH prescription offers distinct advantages over the other doses, while the HL program is subpar. Although limited by a relatively small sample size, we conclude from this randomized dose-response trial that older adults benefit greatly from 2d/week high-intensity RT, and may further benefit from inserting an additional weekly bout of low-load, explosive RT.ClinicalTrials.govNCT02442479.
1096	Comparing hemophagocytic lymphohistiocytosis in pediatric and adult patients.	28957822	Hemophagocytic lymphohistiocytosis (HLH) has long been thought of primarily as a pediatric disease. However, this syndrome may occur secondary to underlying malignancies, infections, and autoimmune diseases, in adult patients. Here, we seek to highlight similarities and differences between pediatric and adult HLH, knowledge gaps, and areas of active research.Malignancy is a more frequent driver of HLH in adults, present in nearly half. Prognosis is poor as compared with nonmalignant HLH. Prognosis in adults is generally worse than pediatric patients, suggesting that age and other comorbid illnesses not surprisingly affect the outcome of HLH. Diagnostic and treatment approaches are more variable in adults, likely contributing to poorer outcomes. The frequency of mutations in HLH-causing genes is higher than had been anticipated in adults, although with a higher frequency of uniallelic and hypomorphic mutations than in children.Optimizing diagnostic criteria for earlier detection may benefit both children and adults. Standardizing treatment approaches in adults will be more difficult because of the variability in triggering illnesses, but a more standardized or algorithmic approach will likely be beneficial. More research into the role of uniallelic and hypomorphic mutations in adults is necessary, to understand treatment and prognostic implications.
1097	Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis presenting with acute sensorineural hearing loss: a case report and review of the literature.	28956548	Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening catastrophic and rarely seen complication of EBV infection especially in adults. While typical presentation of EBV infection is easily diagnosed as mononucleosis syndrome in teenagers and adults, some atypical clinical presentations may be challenged. We did not encounter any patient presenting with sudden sensorineural hearing loss associated with EBV infection in our English medical literature research (1966-2016). In this study, we report an adult patient who was complicated with EBV-HLH under high dose steroid therapy after diagnosis as sensorineural hearing loss. Our aim is to emphasise the atypical presentation of EBV infection and to discuss steroid therapy complication in sensorineural hearing loss that had been simply defined as idiopathic.
1098	A case of leprosy, erythema nodosum leprosum, and hemophagocytic syndrome: A continuum of manifestations of same agent-host interactions.	28944149	A young adult man with 4-years history of lepromatous leprosy (received irregularly multidrug therapy) presented with two and half years history of symptoms suggestive of chronic erythema nodosum leprosum (ENL), initially responded to steroids and thalidomide, but later on failed. During the last 2-months, he developed fever, vomiting, and subsequently altered sensorium. On evaluation, he had hepatosplenomegaly, hyponatremia, hyperferritinemia, hemophagocytosis in bone marrow aspiration, lobular panniculitis in skin biopsy, and multiple parenchymal nodules in chest imaging. Hence diagnosis of ENL with hemophagocytic lymphohistiocytic (HLH) syndrome was established and treatment with dexamethasone (10 mg/m2) started. During hospitalization, he developed sinus bradycardia, QT prolongations, recurrent ventricular tachycardia, and moderate systolic dysfunction. The cardiac complications recovered using a temporary pacemaker and were presumed to be due to micronodular cardiac deposition of ENL. This case iterates that ENL can present with varied presentations like asymptomatic lung nodules and storming cardiac complications. More importantly leprosy, ENL, and HLH are a continuum of manifestations of the same agent-host interactions.
1099	A systematic review of malignancy-associated hemophagocytic lymphohistiocytosis that needs more attentions.	28938698	As an infrequent but potentially life-threatening hyperinflammatory syndrome, hemophagocytic lymphohistiocytosis (HLH) is clinically characterized with prolonged fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hyperferritinemia and hemophagocytosis in bone marrow, liver, spleen or lymph nodes. Malignancy-associated HLH (M-HLH), one type of acquired HLH, usually presents variable overlaps of symptoms with other types of HLH, thus resulting in higher incidence of misdiagnosis and mortality. In recent years, with the increasing awareness to this disease, the diagnosis and management of HLH have gained more and more attention, and improvements have been made accordingly. As a result, the survival of patients is greatly prolonged. However, there is still no consensus on the diagnostic criteria and treatment strategies due to lack of large samples or prospective clinical trials. In order to improve recognition and diagnosis, and provide guidance regarding the treatment of M-HLH, the Study Group in HLH Subtypes of the Histiocyte Society has developed consensus recommendations for the diagnosis and management of M-HLH in 2015. In the present article, we summarized and discussed some updated understandings in M-HLH.
1100	Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis.	28936583	We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.
1101	How Viruses Contribute to the Pathogenesis of Hemophagocytic Lymphohistiocytosis.	28936212	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, hyperinflammatory syndrome, characterized by the uncontrolled activation of macrophages and T cells, eliciting key symptoms such as persistent fever, hepatosplenomegaly, pancytopenia, hemophagocytosis, hyperferritinemia, and coagulopathy. Viral infections are frequently implicated in the onset of active HLH episodes, both in primary, genetic HLH as in the secondary, acquired form. Infections with herpesviruses such as Epstein-Barr virus and cytomegalovirus are the most common. In autoimmune diseases, a link between viral infections and autoreactive immune responses has been recognized for a considerable time. However, the mechanisms by which viruses contribute to HLH pathogenesis remain to be clarified. In this viewpoint, different factors that may come into play are discussed. Viruses, particularly larger DNA viruses such as herpesviruses, are potent modulators of the immune response. By evading immune recognition, interfering with cytokine balances and inhibiting apoptotic pathways, viruses may increase the host's susceptibility to HLH development. In particular cases, a direct connection between the viral infection and inhibition of natural killer cell or T cell cytotoxicity was reported, indicating that viruses may create immunological deficiencies reminiscent of primary HLH.
1102	Confirmed efficacy of etoposide and dexamethasone in HLH treatment: long-term results of the cooperative HLH-2004 study.	2893569529269529	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome comprising familial/genetic HLH (FHL) and secondary HLH. In the HLH-94 study, with an estimated 5-year probability of survival (pSu) of 54% (95% confidence interval, 48%-60%), systemic therapy included etoposide, dexamethasone, and, from week 9, cyclosporine A (CSA). Hematopoietic stem cell transplantation (HSCT) was indicated in patients with familial/genetic, relapsing, or severe/persistent disease. In HLH-2004, CSA was instead administered upfront, aiming to reduce pre-HSCT mortality and morbidity. From 2004 to 2011, 369 children aged <18 years fulfilled HLH-2004 inclusion criteria (5 of 8 diagnostic criteria, affected siblings, and/or molecular diagnosis in FHL-causative genes). At median follow-up of 5.2 years, 230 of 369 patients (62%) were alive (5-year pSu, 61%; 56%-67%). Five-year pSu in children with (n = 168) and without (n = 201) family history/genetically verified FHL was 59% (52%-67%) and 64% (57%-71%), respectively (familial occurrence [n = 47], 58% [45%-75%]). Comparing with historical data (HLH-94), using HLH-94 inclusion criteria, pre-HSCT mortality was nonsignificantly reduced from 27% to 19% (P = .064 adjusted for age and sex). Time from start of therapy to HSCT was shorter compared with HLH-94 (P =020 adjusted for age and sex) and reported neurological alterations at HSCT were 22% in HLH-94 and 17% in HLH-2004 (using HLH-94 inclusion criteria). Five-year pSu post-HSCT overall was 66% (verified FHL, 70% [63%-78%]). Additional analyses provided specific suggestions on potential pre-HSCT treatment improvements. HLH-2004 confirms that a majority of patients may be rescued by the etoposide/dexamethasone combination but intensification with CSA upfront, adding corticosteroids to intrathecal therapy, and reduced time to HSCT did not improve outcome significantly.
1103	Hemophagocytic lymphohistiocytosis (HLH): Elusive diagnosis of disseminated Mycobacterium avium complex infection.	28932715	Hemophagocytic lymphohistiocytosis (HLH) is a clinical syndrome of hyperinflammation leading to an uncontrolled and ineffective immune response, associated with high mortality.A 26-year-old woman with acute lymphoblastic leukemia, 8 months in remission, was found to have HLH. Without any improvement, stem cell transplantation was considered. Then, Mycobacterium avium complex (MAC) infection was identified as cause of her HLH. With appropriate therapy for infection, HLH improved and transplantation was averted.MAC should be included in the list of potential causes of HLH.
1104	Acute HIV infection presenting as hemophagocytic lymphohistiocytosis: case report and review of the literature.	28931369	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon systemic inflammatory condition that can result from infections, autoimmune diseases and malignancies. It is a rarely reported life threatening complication of an acute HIV infection, with only ten documented case reports per our literature search. We present a case of HLH secondary to acute HIV infection with a negative HIV antibody-based assay and high plasma viral load.A 45 year old male with a past medical history of well controlled hypertension presented with fever, dizziness and non-bloody diarrhea. Initial lab work revealed a new thrombocytopenia, marked renal failure and an elevated creatine kinase, ferritin, lactate dehydrogenase and D-dimer. A bone marrow biopsy revealed HLH. As part of the work up for thrombocytopenia, a rapid HIV antibody based assay was done and was negative. The sample was later routinely tested with a fourth generation antigen/antibody assay as per local protocol and was strongly positive. The plasma RNA viral load was >10,000,000 copies /mL confirming the diagnosis of an acute HIV infection. The patient was urgently started on antiretroviral therapy and recovered.This case illustrates a diagnostic approach to HLH which is an uncommon but life threatening multisystem disease, requiring the involvement of a multidisciplinary team of experts. Following any diagnosis of HLH, rapid identification and treatment of the underlying condition is critical. A negative rapid HIV antibody test can be misleading in the context of early HIV infection and the additional use of fourth generation antigen/antibody test or plasma RNA viral load may be required within the right clinical context for diagnosis.
1105	Chimeric antigen receptor T-cell therapy - assessment and management of toxicities.	289259942943433429434335	Immunotherapy using T cells genetically engineered to express a chimeric antigen receptor (CAR) is rapidly emerging as a promising new treatment for haematological and non-haematological malignancies. CAR-T-cell therapy can induce rapid and durable clinical responses, but is associated with unique acute toxicities, which can be severe or even fatal. Cytokine-release syndrome (CRS), the most commonly observed toxicity, can range in severity from low-grade constitutional symptoms to a high-grade syndrome associated with life-threatening multiorgan dysfunction; rarely, severe CRS can evolve into fulminant haemophagocytic lymphohistiocytosis (HLH). Neurotoxicity, termed CAR-T-cell-related encephalopathy syndrome (CRES), is the second most-common adverse event, and can occur concurrently with or after CRS. Intensive monitoring and prompt management of toxicities is essential to minimize the morbidity and mortality associated with this potentially curative therapeutic approach; however, algorithms for accurate and consistent grading and management of the toxicities are lacking. To address this unmet need, we formed a CAR-T-cell-therapy-associated TOXicity (CARTOX) Working Group, comprising investigators from multiple institutions and medical disciplines who have experience in treating patients with various CAR-T-cell therapy products. Herein, we describe the multidisciplinary approach adopted at our institutions, and provide recommendations for monitoring, grading, and managing the acute toxicities that can occur in patients treated with CAR-T-cell therapy.
1106	Successful Treatment of Herpes Simplex Virus (HSV)-1-associated Hemophagocytic Lymphohistiocytosis (HLH) with Acyclovir: A Case Report and Literature Review.	28924117	Hemophagocytic lymphohistiocytosis (HLH) associated with herpes simplex virus (HSV)-1 infection (HSV-1-HLH) is uncommon and is potentially fatal without appropriate treatment. We herein report the case of an adult patient with HSV-1-HLH who was successfully treated with acyclovir. A 69-year-old man developed fever, pancytopenia and liver enzyme elevation after the resolution of pneumonia. These findings and the presence of hemophagocytosis in the patient's bone marrow were consistent with a diagnosis of HLH. The patient was diagnosed with HSV-1-HLH based on the results of a polymerase chain reaction (PCR) for HSV-1. The early administration of acyclovir improved his clinical symptoms and laboratory results within two weeks. In the present case, the rapid and precise diagnosis facilitated the successful treatment of HSV-1-HLH.
1107	The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins.	28919436	Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. We found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets.
1108	T Cell Histiocyte Rich Large B Cell Lymphoma Presenting as Hemophagocytic Lymphohistiocytosis: An Uncommon Presentation of a Rare Disease.	28912991	T cell histiocyte rich large B cell lymphoma (THRLBCL) is a rare subtype of non-Hodgkin's lymphoma characterized by malignant B cells with reactive T lymphocytes. The pathophysiology is thought to involve cytokine-mediated evasion of T cell immune response by malignant B cells. It usually presents at an advanced stage with extranodal involvement. An extremely unusual manifestation of the disease is hemophagocytic lymphohistiocytosis (HLH) which is a hyperinflammatory disorder. We present a case of a 43-year-old male who presented with recurrent fever and recent radiologic imaging showing splenomegaly and right inguinal lymphadenopathy. On presentation, he had a fever of 105°F. Laboratory work-up was consistent with pancytopenia, elevated lactate dehydrogenase, elevated D-dimer, and a ferritin of 24,247 ng/mL. The patient was started on steroid therapy. An excisional biopsy of the right inguinal lymph node was consistent with a diagnosis of THRLBCL and the patient subsequently received six cycles of chemotherapy with R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) after which a PET-CT scan showed no evidence of biologically active disease and ferritin was down to 822 ng/mL. We discuss the clinical manifestations and diagnostic and therapeutic considerations of this rare disease along with a review of reported cases in the literature.
1109	bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.	28899667	Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins.
1110	Hemophagocytosis lymphocytosis presenting as pulmonary-renal syndrome: a case report and literature review.	28878910	Hemophagocytosis Lymphocytosis (HLH) is a rare and life-threatening illness that is more commonly seen in infants; however, its incidence in adults is becoming more common. Recognizing HLH in a complicated clinical scenario is key to early recognition, treatment, as well as improved morbidity and mortality.
1111	Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide.	28878737	GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.
1112	An Intestine-Derived Neuropeptide Controls Avoidance Behavior in Caenorhabditis elegans.	28877481	Adjusting to a continuously changing environment is a key feature of life. For metazoans, environmental changes include alterations in the gut microbiota, which can affect both memory and behavior. The bacteriovorous nematode Caenorhabditis elegans discriminates between pathogenic and non-pathogenic food sources, avoiding the consumption of pathogens. Here, we demonstrate the role of the intestine in regulating C. elegans avoidance to Pseudomonas aeruginosa by an insulin-like neuropeptide encoded by ins-11. The transcriptional expression of ins-11 is controlled through transcription factor hlh-30 and the p38 mitogen-activated protein kinase (MAPK) pathway. ins-11 negatively controls signal pathways in neurons that regulate aversive learning behavior. Attenuation of ins-11 increased avoidance behavior and survival on pathogenic bacteria but decreased opportunities to find a food source as well as lowered energy storage and the number of viable progeny. Our findings support a role for the intestine in avoidance and identify an advantageous role for negative feedback that allows C. elegans to actively balance noxious and favorable environments.
1113	The structure, splicing, synteny and expression of lamprey COE genes and the evolution of the COE gene family in chordates.	28871438	COE genes encode transcription factors that have been found in all metazoans examined to date. They possess a distinctive domain structure that includes a DNA-binding domain (DBD), an IPT/TIG domain and a helix-loop-helix (HLH) domain. An intriguing feature of the COE HLH domain is that in jawed vertebrates it is composed of three helices, compared to two in invertebrates. We report the isolation and expression of two COE genes from the brook lamprey Lampetra planeri and compare these to COE genes from the lampreys Lethenteron japonicum and Petromyzon marinus. Molecular phylogenetic analyses do not resolve the relationship of lamprey COE genes to jawed vertebrate paralogues, though synteny mapping shows that they all derive from duplication of a common ancestral genomic region. All lamprey genes encode conserved DBD, IPT/TIG and HLH domains; however, the HLH domain of lamprey COE-A genes encodes only two helices while COE-B encodes three helices. We also identified COE-B splice variants encoding either two or three helices in the HLH domain, along with other COE-A and COE-B splice variants affecting the DBD and C-terminal transactivation regions. In situ hybridisation revealed expression in the lamprey nervous system including the brain, spinal cord and cranial sensory ganglia. We also detected expression of both genes in mesenchyme in the pharyngeal arches and underlying the notochord. This allows us to establish the primitive vertebrate expression pattern for COE genes and compare this to that of invertebrate chordates and other animals to develop a model for COE gene evolution in chordates.
1114	High concentration of miR-133 is a useful marker for the diagnosis of lymphoma- associated hemophagocytic syndrome.	28869447	Lymphoma associated hemophagocytic syndrome (LAHS) is one of the major adult secondary hemophagocytic lymphohistiocytosis (HLH). Early diagnosis and treatment contribute to improved outcome. No enlarge lymph nodes can often delay the diagnosis of underlying lymphoma.To find out criteria distinguishing LAHS from HLH induced by benign diseases.clinical characteristic and laboratory feature of 31 patients with HLH (10 benign disease-associated HLH and 21 LAHS) were analyzed retrospectively.No significantly differences were observed in the levels of LDH, IL-6, IL-10, TNF-α; however, the level of CRP (C reactive protein) and the mean level of sIL-R (soluble interleukin-2 receptor) were higher in patients with LAHS than those with benign disease associated disease associated HLH while ferritin levels were higher in benign disease associated HLH than in LAHS. Consequently, the serum sIL-2R/ferritin ratio of patients with LAHS was markedly higher than that of patients with benign disease associated HLH (0.33 ± 0.23 vs 5.82 ± 3.26, P= 0.0001). In addition, we found out that the mean level of miR-133 (microRNA-133) was significant higher in LAHS than in benign disease associated HLH (18.83 ± 10.44 vs 5.82 ± 3.26, P⩽ 0.0001).Serum miR-133 is a new very useful marker for diagnosing of LAHS, but it need further confirmation by further clinical studies.
1115	Hemophagocytic syndrome: primary forms and predisposing conditions.	28866302	Hemophagocytic lymphohistiocytosis (HLH, also referred to a hemophagocytic syndrome) is a life-threatening condition in which uncontrolled activation of lymphocytes and macrophages, and thus the secretion of large amounts of inflammatory cytokines, leads to a severe hyperinflammatory state. Over the last few decades, researchers have characterized primary forms of HLH caused by genetic defects that impair lymphocytes' cytotoxic machinery. Other genetic causes of HLH not related to impaired cytotoxicity have also recently been identified. Furthermore, the so-called 'acquired' forms of HLH are encountered in the context of severe infections, autoimmune and autoinflammatory diseases, malignancy, and metabolic disorders, and may also be associated with primary immunodeficiencies. This implies that a variety of disease mechanisms can lead to HLH. Today's research seeks to gain a better understanding of the various pathogenetic and environmental factors that converge to induce HLH.
1116	Significance of Hyperferritinemia in Hospitalized Adults.	28864373	Although high ferritin levels are associated with iron overload, it is known that ferritin is also an acute-phase reactant that may be elevated in conditions associated with acute and chronic inflammation. In addition, an elevated ferritin level is a criterion for the diagnosis of hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS). Therefore, the significance of elevated serum ferritin is often unclear. As HLH/MAS is a medical emergency, prompt diagnosis is important to guide appropriate treatment.To study the spectrum of diagnoses associated with elevated serum ferritin, we did a retrospective review of adult patients admitted to our academic medical center from 2008-2012 with serum ferritin levels greater than 2,000ng/mL. The degree of hyperferritinemia was compared to different diagnoses and selected laboratory values.A total of 333 patients were identified with a serum ferritin level >2,000ng/mL. Hepatocellular injury was the most prevalent diagnosis with n = 126; infection was next with n = 96. Eleven patients were diagnosed with HLH/MAS.Elevated ferritin, as an isolated finding, was not a specific marker for the diagnosis of HLH/MAS. However, as a group, HLH/MAS patients had the highest mean and median ferritin values.
1117	HIF1A is a critical downstream mediator for hemophagocytic lymphohistiocytosis.	28860338	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by overwhelming immune activation. A steroid and chemotherapy-based regimen remains as the first-line of therapy but it has substantial morbidity. Thus, novel, less toxic therapy for HLH is urgently needed. Although differences exist between familial HLH (FHL) and secondary HLH (sHLH), they have many common features. Using bioinformatic analysis with FHL and systemic juvenile idiopathic arthritis, which is associated with sHLH, we identified a common hypoxia-inducible factor 1A (HIF1A) signature. Furthermore, HIF1A protein levels were found to be elevated in the lymphocytic choriomeningitis virus infected Prf1-/- mouse FHL model and the CpG oligodeoxynucleotide-treated mouse sHLH model. To determine the role of HIF1A in HLH, a transgenic mouse with an inducible expression of HIF1A/ARNT proteins in hematopoietic cells was generated, which caused lethal HLH-like phenotypes: severe anemia, thrombocytopenia, splenomegaly, and multi-organ failure upon HIF1A induction. Mechanistically, these mice show type 1 polarized macrophages and dysregulated natural killler cells. The HLH-like phenotypes in this mouse model are independent of both adaptive immunity and interferon-γ, suggesting that HIF1A is downstream of immune activation in HLH. In conclusion, our data reveal that HIF1A signaling is a critical mediator for HLH and could be a novel therapeutic target for this syndrome.
1118	Hemophagocytic Lymphohistiocytosis: A Dangerous Intruder in Pediatric Acute Lymphoblastic Leukemia.	28859036	Hemophagocytic lymphohistiocytosis (HLH) is a condition caused by a pathologic immune activation, which is responsible for its signs and symptoms. It may also appear as a secondary process caused by malignancy. Developing HLH during treatment for acute lymphoblastic leukemia (ALL) is extremely uncommon, but underdiagnosis may be fatal. Two patients with ALL on chemotherapy maintenance treatment who developed HLH triggered by infection are presented here. We emphasize the importance of being aware of this condition when a patient with ALL in complete remission presents with unexplained hepatomegaly, cytopenia, and fever. Early diagnosis and treatment may be lifesaving.
1119	Efficacy of Prompt Initiation of Antiretroviral Therapy in the Treatment of Hemophagocytic Lymphohistiocytosis Triggered by Uncontrolled Human Immunodeficiency Virus.	28856025	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, rapidly progressive hematologic disorder involving uncontrolled immune system activation. HLH has been associated with viral infections, including human immunodeficiency virus (HIV) infections. We report a case of a critically ill 30-year-old female who was hospitalized with HIV-associated HLH, with a CD4 count of 4 cells/mL and HIV viral load of 1,842,730 copies/mL. After ruling out other potential infectious causes of HLH, antiretroviral therapy (ART) was initiated with darunavir, ritonavir, tenofovir, and emtricitabine. Within one week of initiation of ART, the patient began to improve clinically and hematologically and was stable enough for discharge from the hospital three weeks after starting therapy. This case suggests that treatment with ART in patients with HIV-associated HLH should be considered even in critically ill patients with low CD4 counts.
1120	Hemophagocytic Lymphohistiocytosis Secondary to Bone Marrow Only B-Cell Lymphoma: A Very Rare Entity With an Even Rarer Presentation.	32300392	Hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome. It is categorized as familial or acquired, most commonly caused by infections, malignancies, rheumatologic and immunodeficiency disorders. Irrespective of the etiology, the age at the onset is the strongest prognostic factor, hence it is extremely important to have a high suspicion for HLH, diagnose it promptly and initiate treatment without any delay. We encountered a 70-year-old female patient who initially presented with left-sided facial weakness and pancytopenia, secondary to diffuse stage IV B diffuse large B-cell lymphoma with isolated bone marrow involvement with secondary HLH.
1121	Late-Onset Non-HLH Presentations of Growth Arrest, Inflammatory Arachnoiditis, and Severe Infectious Mononucleosis, in Siblings with Hypomorphic Defects in UNC13D.	28848550	Bi-allelic null mutations affecting UNC13D, STXBP2, or STX11 result in defects of lymphocyte cytotoxic degranulation and commonly cause familial hemophagocytic lymphohistiocytosis (FHL) in early life. Patients with partial loss of function are increasingly being diagnosed after presenting with alternative features of this disease, or with HLH later in life. Here, we studied two sisters with lymphocyte degranulation defects secondary to compound heterozygote missense variants in UNC13D. The older sibling presented aged 11 with linear growth arrest and delayed puberty, 2 years prior to developing transient ischemic attacks secondary to neuroinflammation and hypogammaglobulinemia, but no FHL symptoms. Her geno-identical younger sister was initially asymptomatic but then presented at the same age with severe EBV-driven infectious mononucleosis, which was treated aggressively and did not progress to HLH. The sisters had similar natural killer cell degranulation; however, while cytotoxic activity was moderately reduced in the asymptomatic patient, it was completely absent in both siblings during active disease. Following allogeneic bone marrow transplantation at the age of 15, the older child has completely recovered NK cell cytotoxicity, is asymptomatic, and has experienced an exceptional compensatory growth spurt. Her younger sister was also successfully transplanted and is currently disease free. The current study reveals previously unappreciated manifestations of FHL in patients who inherited hypomorphic gene variants and also raises the important question of whether a threshold of minimum NK function can be defined that should protect a patient from serious disease manifestations such as HLH.
1122	Hematopoietic stem cell transplantation in children with Griscelli syndrome: A single-center experience.	28836324	GS2 is a rare autosomal recessive disease characterized by hypopigmentation, variable immunodeficiency with HLH. HSCT is the only curative treatment for GS2. We analyzed the outcome of 10 children with GS2 who underwent HSCT at our center between October 1997 and September 2013. The median age of the patients at transplant was 13.5 months (range, 6-58 months). All of the patients developed HLH before HSCT and received HLH 94 or HLH 2004 protocols. Donors were HLA-identical relatives in 8 patients, HLA-mismatched relatives in 2 patients. Engraftment was achieved in all except one patient. None of the patients developed acute GVHD. Chronic GVHD occurred in one and veno-occlusive disease occurred in four patients. Eight of the patients are under remission without any neurologic sequelae-median time of disease-free survival is 92.4 months. The present study shows successful transplant outcome without long-term neurologic sequelae in patients with GS2 who underwent HSCT from HLA-related donors.
1123	Bone Marrow-Liver-Spleen Type of Large B-Cell Lymphoma Associated with Hemophagocytic Syndrome: A Rare Aggressive Extranodal Lymphoma.	28835859	Recently, an unusual subtype of large B-cell lymphoma (LBCL) with distinctive clinicopathologic features has been recognized; it is characterized by involvement of bone marrow with or without liver and/or spleen, but no lymph node or other extranodal sites, usually associated with fever, anemia, and hemophagocytic lymphohistiocytosis (HLH). Because of this distinctive clinical presentation, it has been designated "bone marrow-liver-spleen" (BLS) type of LBCL. To date there is only one series of 11 cases of BLS type of LBCL with detailed clinical, pathologic, and cytogenetic data. Herein, we describe a case of BLS type LBCL presenting with associated HLH in a 73-year-old female. The bone marrow core biopsy showed cytologically atypical large lymphoma cells present in a scattered interstitial distribution and hemophagocytosis and infrequent large lymphoma cells were seen in the bone marrow aspirate smears. Circulating lymphoma cells were not seen in the peripheral blood smears. The patient underwent treatment with chemotherapy (R-CHOP) but unfortunately passed away 2 months after initial presentation. BLS type of LBCL is a very rare and clinically aggressive lymphoma whose identification may be delayed by clinicians and hematopathologists due to its unusual clinical presentation and pathologic features.
1124	Ruxolitinib for secondary hemophagocytic lymphohistiocytosis: First case report.	28834694	Hemophagocytic lymphohistiocytosis (HLH) is an immune-mediated disorder resulting in hyper-activation of inflammatory cytokines. If left untreated, the uncontrolled inflammatory response can lead to significant tissue injury and potentially life-threatening multi-organ dysfunction. Conventional immunosuppressive agents are available for the management of HLH, including dexamethasone, cyclosporine, and etoposide; however, patients may not respond to these therapies. Clinicians may turn toward alternative pharmacologic agents that likely have less clinical evidence. We describe a case of secondary HLH that did not respond favorably to conventional treatments. Serum inflammatory markers continued to rise significantly with clinical deterioration and worsening pancytopenia. The severe thrombocytopenia and neutropenia were deemed to have contributed to a spontaneous subdural hematoma and candidemia, respectively. Ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, was then utilized as a novel salvage therapy based on available in vivo murine data at the time. Following initiation, there was improvement seen in several disease markers, including serum ferritin, lactate dehydrogenase, fibrinogen, and liver function tests. However, the pancytopenia did not show signs of recovery. The patient ultimately expired after 7days of ruxolitinib treatment. It is unclear if the improvement in disease markers was attributed to JAK inhibition alone. However, this experience combined with the positive in vivo murine data suggests that ruxolitinib may serve as a potential treatment option for HLH, pending the release of more robust data. To our knowledge, this is the first human case report describing the use of ruxolitinib for HLH. Future studies are warranted to determine the role of ruxolitinib in this setting.
1125	Novel use of rituximab in macrophage activation syndrome secondary to systemic lupus erythematosus.	28827301	Macrophage activation syndrome (MAS) is a rare disease characterised by aberrant immune hyperactivation of T lymphocytes and macrophages driven by cytokine dysfunction. The HLH-2004 protocol is commonly used for the treatment of MAS, but significant toxicities are associated. We describe a case of MAS secondary to systemic lupus erythematosus in a young female that responded well to rituximab in lieu of etoposide. She continues to be in remission 1 year following the completion of rituximab infusion and is maintained on hydroxychloroquine. This case highlights the need for further research on the use of rituximab and other available biologics in the setting of MAS in order to help guide further alternative treatment decisions.
1126	Characteristic elevation of soluble TNF receptor II : I ratio in macrophage activation syndrome with systemic juvenile idiopathic arthritis.	2881555928975999	To investigate the clinical significance of soluble tumour necrosis factor receptor (sTNF-R) II/I ratio as an indicator of the diagnosis of macrophage activation syndrome (MAS) complicating systemic juvenile idiopathic arthritis (s-JIA), we measured the serum sTNF-RI and II levels in 117 patients with s-JIA, including 29 patients with MAS, 15 with Epstein-Barr virus-induced haemophagocytic lymphohistiocytosis (EBV-HLH), 15 with Kawasaki disease (KD) and 28 healthy controls (HCs). We determined their correlation with measurements of disease activity and severity. Furthermore, we measured serum interleukin (IL)-18 levels in patients with EBV-HLH and compared these in levels in patients with MAS. The sTNF-RII/I ratio was elevated significantly in MAS and EBV-HLH patients compared with those in the acute phase of s-JIA and KD patients, whereas there were no significant differences between HCs and those in the acute phase of s-JIA. The sTNF-RII/I ratio increased profoundly as MAS developed and correlated positively with disease activity. Serum IL-18 levels were elevated significantly in MAS patients compared with EBV-HLH patients. The monitoring of serum IL-18 and sTNF-RII/I might be useful for the diagnosis of MAS and the differentiation between MAS and EBV-HLH.
1127	Development and Initial Validation of the Macrophage Activation Syndrome/Primary Hemophagocytic Lymphohistiocytosis Score, a Diagnostic Tool that Differentiates Primary Hemophagocytic Lymphohistiocytosis from Macrophage Activation Syndrome.	2880735728838727	To develop and validate a diagnostic score that assists in discriminating primary hemophagocytic lymphohistiocytosis (pHLH) from macrophage activation syndrome (MAS) related to systemic juvenile idiopathic arthritis.The clinical, laboratory, and histopathologic features of 362 patients with MAS and 258 patients with pHLH were collected in a multinational collaborative study. Eighty percent of the population was assessed to develop the score and the remaining 20% constituted the validation sample. Variables that entered the best fitted model of logistic regression were assigned a score, based on their statistical weight. The MAS/HLH (MH) score was made up with the individual scores of selected variables. The cutoff in the MH score that discriminated pHLH from MAS best was calculated by means of receiver operating characteristic curve analysis. Score performance was examined in both developmental and validation samples.Six variables composed the MH score: age at onset, neutrophil count, fibrinogen, splenomegaly, platelet count, and hemoglobin. The MH score ranged from 0 to 123, and its median value was 97 (1st-3rd quartile 75-123) and 12 (1st-3rd quartile 11-34) in pHLH and MAS, respectively. The probability of a diagnosis of pHLH ranged from <1% for a score of <11 to >99% for a score of  ≥123. A cutoff value of ≥60 revealed the best performance in discriminating pHLH from MAS.The MH score is a powerful tool that may aid practitioners to identify patients who are more likely to have pHLH and, thus, could be prioritized for functional and genetic testing.
1128	An Overview of Hemophagocytic Lymphohistiocytosis.	28806468	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by a dysregulated hyperinflammatory response associated with aberrant activation of lymphocytes and macrophages that results in hypercytokinemia. It is classically divided into two types: (1) primary or familial HLH and (2) secondary HLH. Familial HLH is generally an autosomal recessive condition, whereas secondary HLH is usually associated with infectious diseases, autoinflammatory and autoimmune diseases (where it is more commonly known as macrophage activation syndrome), malignancy, immunosuppression, hematopoietic stem cell transplantation, organ transplantation, HIV infection, and metabolic diseases. Although its clinical presentation is often similar to bacterial sepsis or systemic inflammatory response syndrome, HLH can be life-threatening. As such, it is imperative to recognize and diagnose HLH in a timely manner to optimize care. [Pediatr Ann. 2017;46(8):e309-e313.].
1129	Basic helix-loop-helix transcription factors in evolution: Roles in development of mesoderm and neural tissues.	28804953	Basic helix-loop-helix (bHLH) transcription factors have attracted the attention of developmental and evolutionary biologists for decades because of their conserved functions in mesodermal and neural tissue formation in both vertebrates and fruit flies. Their evolutionary history is of special interest because it will likely provide insights into developmental processes and refinement of metazoan-specific traits. This review briefly considers advances in developmental biological studies on bHLHs/HLHs. I also discuss recent genome-wide surveys and molecular phylogenetic analyses of these factors in a wide range of metazoans. I hypothesize that interactions between metazoan-specific Group A, D, and E bHLH/HLH factors enabled a sophisticated transition system from cell proliferation to differentiation in multicellular development. This control mechanism probably emerged initially to organize a multicellular animal body and was subsequently recruited to form evolutionarily novel tissues, which differentiated during a later ontogenetic phase.
1130	Spheroid growth in ovarian cancer alters transcriptome responses for stress pathways and epigenetic responses.	28793334	Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT. Transcripts that included Coiled-Coil Domain Containing 80 (CCDC80), Solute Carrier Family 6 (Neutral Amino Acid Transporter), Member 15 (SLC6A15), Semaphorin 3E (SEMA3E) and PIF1 5'-To-3' DNA Helicase (PIF1) were downregulated more than 10-fold in the 3D cells while Inhibitor Of DNA Binding 2, HLH Protein (ID2), Regulator Of Cell Cycle (RGCC), Protease, Serine 35 (PRSS35), and Aldo-Keto Reductase Family 1, Member C1 (AKR1C1) were increased more than 50-fold. Interestingly, EMT factors, stress responses and epigenetic processes were significantly affected by 3D growth. The heat shock response and the oxidative stress response were also identified as transcriptome responses that showed significant changes upon 3D growth. Subnetwork enrichment analysis revealed that DNA integrity (e.g. DNA damage, genetic instability, nucleotide excision repair, and the DNA damage checkpoint pathway) were altered in the 3D spheroid model. In addition, two epigenetic processes, DNA methylation and histone acetylation, were increased with 3D growth. These findings support the hypothesis that three dimensional ovarian cell culturing is physiologically different from its monolayer counterpart.
1131	Cytomegalovirus induced hemophagocytic lymphocytic histiocytosis in two pediatric patients with acute lymphoblastic leukemia.	28791218	Hemophagocytic lymphohistiocytosis (HLH) is a rare inflammatory condition with tissue destruction due to abnormal immune activation. We present a series of 2 cases of cytomegalovirus-induced HLH in children during maintenance chemotherapy for acute lymphoblastic leukemia. These cases emphasize the importance of considering secondary HLH in this high-risk subset of pediatric patients.
1132	A case of Chediak-Higashi syndrome presented with accelerated phase could be treated effectively by unrelated cord blood transplantation.	28762620	CHS is a rare immunodeficiency syndrome with defects in the functions of cytotoxic cells and neutrophils. Approximately 85% of patients with CHS undergo an AP within the first decade, which is similar to FHLH. Chemotherapy could induce transient remission, but only allogeneic HCT could correct the underlying genetic defect and prevent relapse. We reported a case of CHS diagnosed at 19 months, who had an elder brother who had previously succumbed to the same disease. The little girl presented with severe AP manifestations including recurrent high fever, enlarged superficial lymph nodes, and extraordinary hepatosplenomegaly occupying the whole abdominal and pelvic cavity. Comprehensive therapies including HLH 2004 protocol, supportive care, and antibiotics, especially antituberculous agents, were given to her to induce remission. After remission, the patient received fully HLA-matched UCBT. The transplantation course was uneventful, except for fluctuation of donor chimerism. The patient has been alive for 36 months without infection and neurologic manifestations and is under further follow-up. Our result provides another case that UCBT can be effective for treating patients with CHS and remission before HCT is important for good prognosis.
1133	Hemophagocytic lymphohistiocytosis (HLH) secondary to Ehrlichia chaffeensis with bone marrow involvement.	28762082	NaN
1134	Elevated serum ferritin is not specific for hemophagocytic lymphohistiocytosis.	28762079	Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal, syndrome of excessive and ineffective activation of the immune system. The majority of the reported data on HLH is from pediatric patients and lacks specificity. This makes HLH diagnosis challenging especially in adults where HLH is triggered by many conditions and can resemble many disease entities. Elevated ferritin is one of the diagnostic criteria for HLH. We determined the conditions associated with elevated ferritin at our medical center to assess how specific ferritin is for predicting HLH. We retrospectively reviewed all ferritin results >10,000 μg/L in pediatric and adult patients. The most common condition associated with elevated ferritin was hematologic malignancy in adults (25.7%) and HLH in pediatric patients (48.9%). HLH was diagnosed in 14.2% of adults and 48.9% of children with ferritin >10,000 µg/L. Hyperferritinemia occurs in a variety of conditions and is not specific for adult or pediatric HLH. Common causes of elevated ferritin should be considered before entertaining the possibility of HLH, especially in adult patients.
1135	Diagnostic Challenges of Hemophagocytic Lymphohistiocytosis.	28760295	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by excessive activation of the immune system, resulting in overproduction of inflammatory cytokines. Patients usually present with high fever, cytopenias, hyperferritinemia, and hepatosplenomegaly, and their disease process ranges from mild to fatal multiorgan failure. HLH is a heterogeneous group of disorders that can be triggered by infections, neoplasms, or autoimmune diseases. The HLH diagnosis can be difficult to confidently confirm in critically ill patients while waiting for pathology or reference laboratory results to return, delaying the diagnosis with significantly worsened outcomes. The current HLH-2004 diagnostic guidelines were originally developed for pediatric cases and were not validated to diagnose secondary HLH, whether in children or adults. In addition, some laboratory findings that are common among HLH patients such as hypoalbuminemia and elevated liver enzymes are not represented in the HLH-2004 guidelines. Even more challenging for clinicians is that many of the diagnostic features of this syndrome are nonspecific. For example, the clinical presentation of HLH can meet the diagnostic criteria of systemic inflammatory response syndrome, viral infections, or neoplastic diseases. It is necessary to revisit the diagnostic criteria for HLH by validating the clinical and laboratory findings in large prospective HLH prospective clinical trials or by establishing registries. This will improve our understanding of HLH, help validate and develop newer, more specific, and more rapidly obtainable diagnostic criteria, and, eventually, result in earlier therapy with more consistent monitoring of the response.
1136	Targeting of a Helix-Loop-Helix Transcriptional Regulator by a Short Helical Peptide.	28741867	The Id proteins (Id1-4) are cell-cycle regulators that play a key role during development, in cancer and vascular disorders. They contain a conserved helix-loop-helix (HLH) domain that folds into a parallel four-helix bundle upon self- or hetero-association with basic-HLH transcription factors. By using such protein-protein interactions, the Id proteins inhibit cell differentiation and promote cell-cycle progression. Accordingly, their supporting role in cancer has been convincingly demonstrated, which makes these proteins interesting therapeutic targets. Herein we present a short peptide containing an (i,i+4)-lactam bridge and a hydrophobic (Φ) three-residue motif Φ(i)-Φ(i+3)-Φ(i+6), which adopts a helical conformation in water, shows Id protein binding in the low-micromolar range, penetrates into breast (MCF-7 and T47D) and bladder (T24) cancer cells, accumulates in the nucleus, and decreases cell viability to ∼50 %. Thus, this cyclopeptide is a promising scaffold for the development of Id protein binders that impair cancer cell viability.
1137	EBV-HLH children with reductions in CD4+ T cells and excessive activation of CD8+ T cells.	28738028	BackgroundIn this report, we analyzed the number and activation profiles of T cells in Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) patients and assessed their value for the diagnosis of EBV-HLH.MethodsWe compared the quantity of CD4+ T cells and CD8+ T cells in peripheral blood among HLH patients, a sepsis group, and healthy controls. We then analyzed variations in the activation marker in CD4+ T and CD8+ T cells.ResultsThe percentage of CD4+ T cells decreased in the EBV-HLH patients when compared with healthy controls, which induced significant differences in the ratio of CD4+ T cells/CD8+ T cells. The percentage of CD4+HLA-DR+ T cells, CD8+CD69+ T cells, CD8+HLA-DR+ T cells, and CD8+HLA-DR+ T cells/CD4+CD25+ T cells increased in EBV-HLH patients when compared with that in healthy controls. Compared with EBV-HLH survivors, the median percentage of CD4+CD25+ T cells (7.9 vs. 3.0) were decreased, but the ratios of CD8+HLA-DR+T cells/CD4+CD25+T cells (6.3 vs. 20.3) were increased in the dead patients.ConclusionsThere was a reduction in CD4+ T cells and abnormal activation of CD8+ T cells in the EBV-HLH patients. The percentage of CD4+CD25+ T cells and the ratios of CD8+HLA-DR+ T cells/CD4+CD25+ T cells in EBV-HLH patients had a relation to the prognosis of the disease.
1138	Multi-omics Analyses of Starvation Responses Reveal a Central Role for Lipoprotein Metabolism in Acute Starvation Survival in C. elegans.	28734827	Starvation causes comprehensive metabolic changes, which are still not fully understood. Here, we used quantitative proteomics and RNA sequencing to examine the temporal starvation responses in wild-type Caenorhabditis elegans and animals lacking the transcription factor HLH-30. Our findings show that starvation alters the abundance of hundreds of proteins and mRNAs in a temporal manner, many of which are involved in central metabolic pathways, including lipoprotein metabolism. We demonstrate that premature death of hlh-30 animals under starvation can be prevented by knockdown of either vit-1 or vit-5, encoding two different lipoproteins. We further show that the size and number of intestinal lipid droplets under starvation are altered in hlh-30 animals, which can be rescued by knockdown of vit-1. Taken together, this indicates that survival of hlh-30 animals under starvation is closely linked to regulation of intestinal lipid stores. We provide the most detailed poly-omic analysis of starvation responses to date, which serves as a resource for further mechanistic studies of starvation.
1139	The neutrophil-to-lymphocyte ratio could be a good diagnostic marker and predictor of relapse in patients with adult-onset Still's disease: A STROBE-compliant retrospective observational analysis.	28723775	The neutrophil-to-lymphocyte ratio (NLR) is the proportion of absolute neutrophil count to lymphocytes on routine complete blood count (CBC) tests, and has been studied as a simple marker of the systemic inflammatory response. This study was performed to investigate whether the NLR could be used as a tool to diagnose and predict prognosis in cases of adult-onset Still's disease (AOSD).We retrospectively reviewed 164 patients with suspected AOSD. Among 164 patients with suspected AOSD, 37 patients received another diagnosis (such as viral infection) and were compared with the 127 patients who received a diagnosis of AOSD. Laboratory tests including CBCs, ferritin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and the NLR were evaluated.AOSD patients showed higher neutrophil counts, lower lymphocyte counts, higher NLRs, and higher levels of ferritin, ESR, and CRP than non-AOSD patients (all P < .001). In receiver operating characteristic (ROC) curve analysis of the NLR for diagnosis of AOSD, the area under the curve (AUC) was highest at 0.967 (95% CI = 0.940-0.993) with a cutoff value of 3.08. The cutoff value showed the greatest sensitivity (91.7%), specificity (68.4%), and AUC value (0.967) as a diagnostic tool for AOSD. The NLR and treatment appeared to be significant prognostic factors for relapse, but only age showed a significant relationship with death. Furthermore, the NLR was significantly higher in patients with macrophage activation syndrome than in hemophagocytic lymphohistiocytosis (HLH) patients (P = .007). In ROC analysis, the NLR with a cutoff value of 5.86 showed a sensitivity of 89.4%, specificity of 87.8%, and AUC of 0.794.The NLR can be used as a diagnostic tool and predictor of relapse in AOSD, and for differential diagnosis of HLH.
1140	Diagnostic and prognostic value of low percentage of glycosylated ferritin in acquired hemophagocytic lymphohistiocytosis: A single-center study.	28722256	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome characterized by an excessive immune activation. Glycosylated ferritin (GF) level has been proposed as highly specific of HLH.We have studied 12 subjects with HLH according to the HLH-04 trial criteria and 11 patients with a clinical and laboratoristic suspicion of HLH. The percentage of GF was measured by an in-house assay.The only biomarkers that were significantly different in the two groups were fraction of GF (P<.001) and the presence of hemophagocytosis in bone marrow (P=.006). Subjects with HLH had significantly lower percentage of GF than patients with other inflammatory conditions mimicking HLH. A fraction of GF ≤20% was strongly consistent with a diagnosis of HLH.Fraction of GF is useful to identify subjects at high risk for early death and therefore in need of early treatment.
1141	Novel peptide motifs from lysozyme suppress pro-inflammatory cytokines in macrophages by antagonizing toll-like receptor and LPS-scavenging action.	28711715	Lysozyme is commonly found in spots where bacterial infections are most likely to enter the body. Earlier we found that lysozyme possesses five antimicrobial peptide motifs in its N-terminal region which can be generated by newborn pepsin. In this study, we explore the role of these peptides in the anti-inflammatory activity of lysozyme. The five peptides, helix1 (H1), helix2 (H2), H1 and H2 connected with a loop (HLH), H2 extended with either 2 β-strands (H2-S12) or 3 β-strands (H2-S13), were synthesized and examined for anti-inflammatory action. The five peptides dose-dependently decreased, to different degrees, expression of pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β, in lipopolysaccharide (LPS)- or interferon-gamma (INF-γ)-stimulated mouse macrophage cells (RAW264.7). The HLH peptide and its individual helices (H1 and H2) were markedly the most potent anti-inflammatory. When macrophage cells were stimulated with live bacteria (E. coli), H1 peptide was the most powerful suppressor of TNF-α and IL-6 expression, providing evidence that the peptide is able to antagonize the pathogen-induced inflammatory response. Receptor binding assay and docking simulation provided evidence that H1 peptide bind specifically to the pocket for endotoxin binding of the toll-like receptor 4 (TLR-4) of macrophage. The results demonstrate, for the first time, the molecular basis of anti-inflammatory action of lysozyme that N-terminal helical peptides are the main contributors. This exciting finding offers new classes of therapeutic peptides with potential in the treatment of infection-induced inflammatory diseases.
1142	ThMYC4E, candidate Blue aleurone 1 gene controlling the associated trait in Triticum aestivum.	28704468	Blue aleurone is a useful and interesting trait in common wheat that was derived from related species. Here, transcriptomes of blue and white aleurone were compared for isolating Blue aleurone 1 (Ba1) transferred from Thinopyrum ponticum. In the genes involved in anthocyanin biosynthesis, only a basic helix-loop-helix (bHLH) transcription factor, ThMYC4E, had a higher transcript level in blue aleurone phenotype, and was homologous to the genes on chromosome 4 of Triticum aestivum. ThMYC4E carried the characteristic domains (bHLH-MYC_N, HLH and ACT-like) of a bHLH transcription factor, and clustered with genes regulating anthocyanin biosynthesis upon phylogenetic analysis. The over-expression of ThMYC4E regulated anthocyanin biosynthesis with the coexpression of the MYB transcription factor ZmC1 from maize. ThMYC4E existed in the genomes of the addition, substitution and near isogenic lines with the blue aleurone trait derived from Th. ponticum, and could not be detected in any germplasm of T. urartu, T. monococcum, T. turgidum, Aegilops tauschii or T. aestivum, with white aleurone. These results suggested that ThMYC4E was candidate Ba1 gene controlling the blue aleurone trait in T. aestivum genotypes carrying Th. ponticum introgression. The ThMYC4E isolation aids in better understanding the genetic mechanisms of the blue aleurone trait and in its more effective use during wheat breeding.
1143	Sepsis due to Streptococcus pneumoniae associated with secondary hemophagocytic lymphohistiocytosis in a splenectomized patient for spherocytosis: A case report.	28700505	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome that is characterized by an inappropriate hyperinflammatory immune response - primary, as a consequence of a genetic defect of NK cells and cytotoxic T lymphocytes or - secondary, in the progression of infections, rheumatic or autoimmune diseases, malignancies or metabolic diseases.We present the case of a secondary HLH due to Streptococcus pneumoniae infection in a splenectomised patient for spherocytosis, a 37-year-old patient who was splenectomised in childhood for spherocytosis, without immuneprophylaxis induced by antipneumococcal vaccine.He developed a severe pneumococcal sepsis associated with secondary HLH, with unfavorable outcome and death.To our knowledge, just 2 similar cases had been published in the literature, none in which the secondary HLH was the consequence of an invasive pneumococcal infection in a splenectomized patient for spherocytosis, and the association of splenectomy with HLH is surprizin.
1144	An unusual association between hemophagocytic lymphohistiocytosis, mixed connective tissue disease, and autoimmune hemolytic anemia: A case report.	28700492	In the adult patient, hemophagocytic lymphohistiocytosis (HLH) is uncommon and frequently difficult to diagnose due to its nonspecific presentation and numerous complications.Herein, we present the case of a 25-year-old female who initially presented for evaluation of persistent fevers and fatigue. She was found to have splenomegaly, generalized lymphadenopathy, pancytopenia, and acute hepatic failure.Her course was further complicated by the development of nephrotic syndrome and autoimmune hemolytic anemia (AIHA). Antinuclear antibody and ribonucleoprotein were positive, with concurrent physical examination findings, indicating underlying mixed connective tissue disease (MCTD). Ferritin was greater than 40,000 ng/dL. Viral studies, including hepatitis A, B, and C, cytomegalovirus, and Epstein-Barr virus were negative. On the basis of her clinical presentation, a diagnosis of HLH secondary to MCTD was made. This was later confirmed on liver biopsy. She was started on high-dose prednisone and her symptoms completely resolved. She was then transitioned to azathioprine, hydroxychloroquine, prophylactic antibiotics, and a prednisone taper for long-term management.This case is notable for the association of both AIHA and MCTD with HLH, providing support for a possible relationship between these 3 conditions.
1145	Critically Ill Children With Hemophagocytic Lymphohistiocytosis: A Case Series of 14 Patients.	28697170	Children with hemophagocytic lymphohistiocytosis (HLH) are at an increased risk of critical illness. In this study, we described the clinical characteristics of critically ill children with HLH and identify factors associated with poor clinical outcomes. Children who were diagnosed with HLH with emergent admission to Children's Intensive Care Unit (CICU) between January 1, 2000 and October 31, 2015 were included. The primary outcome was CICU mortality. Over the 15-year period, there were 14 critically ill patients with HLH with 23 CICU admissions. Median age at HLH diagnosis was 8.2 years (interquartile range [IQR], 2.9 to 11.3). Overall CICU mortality was 8 of 23 CICU admissions (34.8%). Factors that were associated with CICU mortality in critically ill children with HLH identified in this study include: a worse median pediatric index of mortality 2 score (4.7% in survivors [IQR, 2.9% to 11.6%] vs. 2.4% [IQR, 1.2% to 4.3%]; P=0.031); higher median peak serum lactate level (mmol/L) within 24 hours of admission (5.6 [IQR, 2.7 to 17.4] vs. 1.6 [IQR, 1.2 to 2.8]; P=0.032); the need for mechanical ventilation (100% vs. 46.7%; P=0.019); inotropic support (87.5% vs. 20.0%; P=0.006); renal replacement therapy (50% vs. 0%; P=0.008); and blood product transfusion episodes (24.5 [IQR, 14.3 to 46.8] vs. 3.0 [IQR, 1.0 to 9.0]; P=0.002). Further studies are required to validate the factors that are associated with poor outcomes in critically ill children with HLH.
1146	Perinatal outcomes of congenital heart disease after emergent neonatal cardiac procedures.	28693354	We compared outcome of neonates with prenatal and post-natal diagnosis of congenital heart disease presenting in our paediatric heart centre between March 2005 and May 2015 who underwent an emergent intervention within 48 h post-partum.In 52/111 (46.8%) with emergent intervention, congenital heart disease was diagnosed prenatally, in 59/111 (53.2%) with no specialized foetal echocardiography, diagnosis was made post-natally. In 98/111 (88.2%), 30-day outcome was known.Regarding the entire cohort, 30-day survival did not differ significantly in prenatal and post-natal diagnosis group (71.2 vs. 72.9%; p > .1). Infants with prenatal diagnosis were more likely to be born by caesarean section (59.6% vs. 33.9%, p = .01). Those with post-natal diagnosis had a higher need for intubation (32.7% vs. 52.5%; p < .05). Subgroup analysis of HLH/HLHC (hypoplastic left heart/hypoplastic left heart complex) patients revealed higher number of deaths within 30 days of life in the post-natal diagnosis group, although the difference did not reach statistical significance (5/7, 71.4% vs. 5/20, 25.0%; p = .075).For newborns who require emergent neonatal cardiac procedures, our results point towards a lower death rate after prenatal diagnosis of HLH/HLHC.
1147	A Case of Familial Hemophagocytic Lymphohistiocytosis Type 4 With Involvement of the Central Nervous System Complicated With Infarct.	28692549	Familial hemophagocytic lymphohistiocytosis (HLH) is a fatal disease affecting infants and very young children. Central nervous system involvement of HLH can cause catastrophic results.We present a case with cranial involvement of familial HLH type 4 who showed diffuse infiltration of white matter complicated with intracranial thrombosis. A 5-year-old girl from a consanguineous couple presented with fever and pancytopenia, and was referred to our hematology unit. Examination revealed fever, lymphadenopathy, and hepatosplenomegaly. Ultrasound examination revealed hepatosplenomegaly and free intra-abdominal fluid. HLH was revealed on bone marrow aspiration biopsy. Defective natural killer and T lymphocyte cytotoxicity using degranulation tests was determined. In the genetic analysis, syntaxin gene mutation was found. On T2-weighted and T2-fluid-attenuated inversion recovery magnetic resonance imaging (MRI), diffuse hyperintense signal changes of cerebral white matter, indicating white matter demyelination, were observed. A second brain MRI showed an acute infarct involving the left temporooccipital region. Immunosuppressive therapy according to the HLH 2004 protocol was started. The infarct resolved but white matter lesions were stable on the brain MRI that was performed 1 month later. Brain MRI taken 4 months after the first examination showed stable cerebral white matter lesions, but hyperintense signal changes appeared in the cerebellar white matter and were regarded as progression. The patient died because of infection despite immunosuppressive therapy.Physicians managing patients with HLH must be vigilant about the possibility of central nervous system involvement including stroke.
1148	N-glycan structures of β-HlH subunit of Helix lucorum hemocyanin.	28672164	The carbohydrate structures of molluscan hemocyanins have recently received particular interest due to their specific monosaccharide composition, as well as their immunostimulatory properties and application in clinical studies. For the first time, we investigated N-glycans of the structural subunit β-HlH of hemocyanin isolated from Helix lucorum. In total, 32 different glycans were enzymatically liberated and characterized by tandem mass spectrometry using a Q-Trap mass spectrometer. Our study revealed a highly heterogeneous mixture of glycans with composition Hex3-7HexNAc2-5MeHex0-4Pent0-1Fuc0-1. The oligosaccharide chains are mostly modified at the inner core by β1-2-linked xylose to β-mannose, by α1-6-fucosylation of the innermost GlcNAc residue (the Asn-bound GlcNAc), and by methylation. The glycans of β-HlH mainly contain a terminal MeHex residue; in some cases even two, three or four of these residues occur. Several carbohydrate chains in β-HlH are core-fucosylated without Xyl and also possess a high degree of methylation. This study shows the presence of mono- and bi-antennary N-glycans as well as hybrid type structures with or without core-fucosylation.
1149	Limited Role of Random Skin Biopsy in the Diagnosis of Intravascular Lymphoma in Adult Patients with Hemophagocytic Lymphohistiocytosis.	28668948	This study examined the role of random normal skin biopsy in the diagnosis of intravascular lymphoma (IVL) in adult Western patients with clinically diagnosed hemophagocytic lymphohistiocytosis (HLH).In a retrospective chart review study, we analyzed a total of 59 skin biopsies that were performed to diagnose IVL in 21 adult patients with HLH seen at Stanford Hospital between 2004 and 2016.Out of the 59 skin biopsies, 42 were taken from clinically normal-appearing skin and 17 from clinically abnormal-appearing skin. None of the 59 biopsies revealed a diagnosis of primary or metastatic malignancy, regardless of the malignancy history, clinical presentation, and biopsy and histopathologic characteristics. A review of 8 positive IVL cases at Stanford Hospital including 1 case associated with HLH showed 1 positive diagnosis by a targeted skin biopsy and other positive diagnoses by bone marrow (n = 4), lung (n = 2), brain (n = 2), muscle (n = 1), and nerve (n = 1).Random skin biopsies have a limited role in diagnosing IVL in adult patients with HLH, in the setting of a single academic institution in the USA. A review of the literature emphasizes the role of a full body skin exam with a selective skin biopsy in these patients.
1150	Eicosapentaenoic acid modulates the synergistic action of CREB1 and ID/E2A family members in the rat pup brain and mouse embryonic stem cells.	28666847	The aim of this study was to investigate the molecular mechanism by which eicosapentaenoic acid (EPA) may exert neuroprotective effects through an "EPA-cyclic AMP response element-binding protein (CREB)" signaling pathway. The current study reveals that EPA modulates the exquisite interplay of interaction of CREB1 with the inhibitor of DNA binding (ID) and E2A family members, thereby delivering mechanistic insights into specific neural differentiation program. In this scenario, our work provides evidence for the capability of CREB1 to sequester ID:E2A family members in brain tissues and neural differentiating mouse embryonic stem cells (mESCs) through formation of a [CREB1]2:ID2:E47 tetrameric complex.In essence, the molecular function of CREB1 is to dynamically regulate the location-specific assembly or disassembly of basic-helix-loop-helix (bHLH):HLH protein complexes to mediate the activation of neural/glial target genes. Together, these findings support the one-to-many binding mechanism of CREB1 and indicate that EPA treatment potentiates the integration of CREB dependent signaling with HLH/bHLH transcriptional network, adding specificity to the CREB1-mediated gene regulation during neural/glial differentiation. Our current research on the EPA-CREB axis could reveal new molecular targets for treating neurogenerative disease.
1151	A Case of Refractory Langerhans Cell Histiocytosis Complicated with Hemophagocytic Lymphohistiocytosis Rescued by Cord Blood Transplantation with Reduced-intensity Conditioning.	28655945	We diagnosed a female infant with Langerhans cell histiocytosis (LCH) who was refractory to conventional chemotherapy. She showed refractory inflammation that was complicated with hemophagocytic lymphohistiocytosis (HLH) during LCH chemotherapy; therefore, we changed the protocol to HLH2004 (dexamethasone, cyclosporine A and VP16). However, there were no signs of hematological recovery. We therefore performed cord blood transplantation with reduced-intensity conditioning, and she achieved complete remission for over 2 years. As salvage therapy for refractory LCH, hematopoietic stem cell transplantation may be a good therapeutic choice, especially when LCH is complicated with HLH.
1152	Lymphoid malignancy-associated hemophagocytic lymphohistiocytosis: Search for the hidden source.	28648938	Secondary hemophagocytic lymphohistiocytosis (HLH) is an uncommon, but life-threatening syndrome of highly stimulated and ineffective immune dysregulation. It is not a disease entity by itself and the current diagnosis of secondary (acquired) HLH is based on constellation of nonspecific clinical and laboratory parameters indicative of overactive immune response. The presenting symptoms are often nonspecific and could potentially be missed, leading to a fatal outcome. Patients with malignancy-associated HLH have a relatively unfavorable overall survival compared with non-malignancy-associated HLH. In this retrospective study, nine adult patients with secondary HLH were identified. Of these four cases were associated with a malignancy and despite a high degree of suspicion, the underlying lymphoid malignancy was not initially evident. Three out of four patients with lymphoid malignancy-associated HLH died over a very short course of time following the diagnosis. The outcome was significantly different for the control group of patients with other underlying cause(s) for HLH. These cases emphasize the importance of a thorough search for a hidden malignant source in patients with secondary HLH for prompt diagnosis and institution of malignancy specific treatment.
1153	Durable Chimerism and Long-Term Survival after Unrelated Umbilical Cord Blood Transplantation for Pediatric Hemophagocytic Lymphohistiocytosis: A Single-Center Experience.	28647558	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune dysregulation characterized by fever, hepatosplenomegaly, cytopenias, central nervous system disease, increased inflammatory markers, and hemophagocytosis. Currently, allogeneic hematopoietic stem cell transplantation is the only curative approach for patients with HLH, with reported survival ranging from 50% to 70% with myeloablative conditioning (MAC) regimens. However, donor availability and transplantation-related mortality associated with conventional MAC are major barriers to success. Unrelated umbilical cord blood transplantation (UCBT) provides a readily available alternative donor source for patients lacking matched related donors. Accordingly, we report the results of UCBT in 14 children treated between 1998 and 2016. All children received standard HLH chemotherapy before UCBT. The median age at diagnosis was 2.7 months (range, .8 to 10.4) and at transplantation was 7.5 months (range, 3.8 to 17). Ten patients received MAC with busulfan/cyclophosphamide/etoposide /antithymocyte globulin (ATG) (n = 5), busulfan/cyclophosphamide /ATG (n = 4), or busulfan /melphalan/ATG (n = 1). Four patients received reduced-toxicity conditioning (RTC) with alemtuzumab/fludarabine/melphalan/hydroxyurea ± thiotepa. Cord blood units were mismatched at either 1 (n = 9) or 2 (n = 5) loci and delivered a median total nucleated cell dose of 11.9 × 107/kg (range, 4.6 to 27.9) and CD34+ dose of 3.1 × 105/kg (range, 1.1 to 6.8). The cumulative incidence of neutrophil engraftment by day 42 was 78.6% (95% confidence interval [CI], 42.9% to 93.4%) with a median of 19 days (range, 13 to 27), and that for platelet (50,000) engraftment by day 100 was 64.3% (95% CI, 28.2% to 85.7%) with a median of 51 days (range, 31 to 94). Six patients developed either grade II (n = 5) or grade IV (n = 1) acute graft-versus-host disease (GVHD); no extensive chronic GVHD was seen. Ten patients (71.4%) are alive and well at a median of 11.2 years after transplantation (range, .85 to 18.25), 9 of whom maintain sustained full donor chimerism after a single UCBT, whereas 1 patient with autologous recovery after first UCBT with RTC has achieved full donor chimerism after a second UCBT with MAC. This series demonstrates that, in combination with standard HLH therapy, UCBT after MAC or RTC conditioning can provide long-term survival with durable complete donor chimerism comparable to that of conventional donors. UCBT should be considered for patients with HLH lacking a fully matched related or unrelated adult donor.
1154	IFNɣ Block, Treosulfan Conditioning and αβ T Cell Deplete PBSCT for XIAP-Deficient HLH.	28639166	NaN
1155	Interleukin 1 Receptor Antagonist Anakinra, Intravenous Immunoglobulin, and Corticosteroids in the Management of Critically Ill Adult Patients With Hemophagocytic Lymphohistiocytosis.	28631531	Hemophagocytic lymphohistiocytosis (HLH) causes multiple organ dysfunction frequently leading to intensive care unit (ICU) referral and/or death. We report on a series of critically ill adult patients treated with a non-etoposide-based regimen including interleukin 1 antagonist anakinra, intravenous immunoglobulin (IVIG), and/or corticosteroids (CS) for HLH.Eight adult (≥18 years) ICU patients having received treatment with anakinra ± IVIG ± CS for HLH between March 2014 and March 2016 at a large tertiary care university hospital (Medical University of Vienna, Vienna, Austria) were retrospectively analyzed.Eight patients (median age: 38 years; range: 20-58 years; 4 males and 4 females) received anakinra together with IVIG (n = 7) and/or high-dose CS (n = 5) for suspected reactive HLH (median H-score: 214; range: 171-288). Seven (88%) patients required vasopressors and invasive mechanical ventilation and 6 (75%) patients required renal replacement therapy (median Sequential Organ Failure Assessment [SOFA] score at HLH diagnosis: 9.5; range: 6-14). Six patients showed a significant decline in the SOFA score at 1 and 2 weeks following treatment initiation (P = .03), and the remainder 2 patients experienced early death. Five patients survived to ICU discharge, 4 of them could further be discharged from hospital (hospital survival rate: 50%). No overt treatment-related toxicity was noted.Anakinra in combination with IVIG and/or CS resulted in a hospital survival rate of 50% in 8 critically ill adult patients with HLH despite a vast degree of organ dysfunction and the need for aggressive ICU treatment. Further research on non-etoposide-based treatment strategies for HLH in critically ill adults is warranted.
1156	Hemophagocytic lymphohistiocytosis associated to Haemophilus parainfluenzae endocarditis- a case report.	28629255	Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe and potentially fatal syndrome that is characterized by increased proliferation and activation of benign macrophages with hemophagocytosis throughout the reticuloendothelial system. This syndrome is classified as primary (genetic) or secondary when acquired in the context of infections (usually viral), malignancies, rheumatologic and metabolic diseases.We report a case of HLH complicating a Haemophilus parainfluenzae mitral valve endocarditis and resolving under antibiotic and surgical treatment alone. We also review other case reports of infective endocarditis associated with HLH.The syndrome is probably underdiagnosed. To our knowledge, this is the first report of a HLH syndrome associated with H. parainfluenzae endocarditis.
1157	Hemophagocytic Lymphohistiocytosis in a Fatal Case of Severe Fever with Thrombocytopenia Syndrome.	28626191	Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease caused by a novel Bunyavirus with a high mortality rate. We herein report a fatal case of an 86-year-old woman with SFTS complaining of a fever, fatigue, and bicytopenia. Her condition deteriorated with rapid progression of bleeding tendency, disturbance of consciousness, and multiple organ failure leading to death on Day 6 of her illness. The histopathological findings in the autopsy revealed marked infiltration of macrophages with hemophagocytosis in the bone marrow, liver, and spleen leading to a diagnosis of hemophagocytic lymphohistiocytosis (HLH). HLH might be a critical pathogenesis in fatal cases of SFTS.
1158	A consensus review on malignancy-associated hemophagocytic lymphohistiocytosis in adults.	28621800	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune activation and dysregulation resulting in extreme and often life-threatening inflammation. HLH has been well recognized in pediatric populations, and most current diagnostic and therapeutic guidelines are based on pediatric HLH. Recently there has been recognition of HLH in adults, especially secondary to immune deregulation by an underlying rheumatologic, infectious, or malignant condition. This review is focused on malignancy-associated HLH (M-HLH), in which possible mechanisms of pathogenesis include severe inflammation, persistent antigen stimulation by the tumor cells, and loss of immune homeostasis because of chemotherapy, hematopoietic stem cell transplantation, or infection. Previously considered rare, M-HLH may occur in up to 1% of patients with hematologic malignancies. M-HLH is often missed or diagnosed late in most published studies, and it has been associated with a poor median survival of less than 2 months. Identification of the clinical and laboratory features specific to M-HLH in adults may allow early detection, consultation with HLH experts, and intervention. Improved management of adult M-HLH with optimal combinations of T-lympholytic and immunosuppressive agents and the incorporation of novel agents based on the pediatric experience hopefully will improve outcomes in adults with M-HLH. Cancer 2017;123:3229-40. © 2017 American Cancer Society.
1159	A bHLH Code for Sexually Dimorphic Form and Function of the C. elegans Somatic Gonad.	28602651	How sexually dimorphic gonads are generated is a fundamental question at the interface of developmental and evolutionary biology [1-3]. In C. elegans, sexual dimorphism in gonad form and function largely originates in different apportionment of roles to three regulatory cells of the somatic gonad primordium in young larvae. Their essential roles include leading gonad arm outgrowth, serving as the germline niche, connecting to epithelial openings, and organizing reproductive organ development. The development and function of the regulatory cells in both sexes requires the basic-helix-loop-helix (bHLH) transcription factor HLH-2, the sole ortholog of the E proteins mammalian E2A and Drosophila Daughterless [4-8], yet how they adopt different fates to execute their different roles has been unknown. Here, we show that each regulatory cell expresses a distinct complement of bHLH-encoding genes-and therefore distinct HLH-2:bHLH dimers-and formulate a "bHLH code" hypothesis for regulatory cell identity. We support this hypothesis by showing that the bHLH gene complement is both necessary and sufficient to confer particular regulatory cell fates. Strikingly, prospective regulatory cells can be directly reprogrammed into other regulatory cell types simply by loss or ectopic expression of bHLH genes, and male-to-female and female-to-male transformations indicate that the code is instructive for sexual dimorphism. The bHLH code appears to be embedded in a bow-tie regulatory architecture [9, 10], wherein sexual, positional, temporal, and lineage inputs connect through bHLH genes to diverse outputs for terminal features and provides a plausible mechanism for the evolutionary plasticity of gonad form seen in nematodes [11-15].
1160	A novel GhBEE1-Like gene of cotton causes anther indehiscence in transgenic Arabidopsis under uncontrolled transcription level.	28600178	Male-sterile lines are very important for selective breeding, and anther dehiscence defect is an effective way to generate male-sterile lines. Although several bHLH-family proteins in Arabidopsis have been characterized, little is known about the role of bHLH-family proteins in cotton. Here, we isolated a novel bHLH protein from cotton (Gossypium hirsutum), named GhBEE1-Like. Protein domain analysis showed that GhBEE1-Like contained a basic domain and an HLH domain. Subcellular localization analysis revealed that GhBEE1-Like was a nuclear-localized protein. Expression pattern analysis showed GhBEE1-Like was highly expressed in floral organs, and its expression was induced by the active brassinosteroid (BR) substance 24-epi-BL. GhBEE1-Like overexpression in Arabidopsis resulted in two types of transgenic lines, one with normal anther dehiscence and the other with defective anther dehiscence. Semi-qRT-PCR and qRT-PCR analyses revealed that GhBEE1-Like transcript levels acted as a check-point determining how anther dehiscence proceeds in these transgenic lines; regulated transcript levels result in normal anther dehiscence, whereas uncontrolled transcript levels lead to anther indehiscence. These results suggest that GhBEE1-Like plays an important role via its accumulation in regulating anther dehiscence. Therefore, controlling the level of GhBEE1-Like expression in cotton could be a convenient tool for generating male-sterile lines to use in selective breeding.
1161	Central nervous system involvement in adults with haemophagocytic lymphohistiocytosis: a single-center study.	28589450	Hemophagocytic lymphohistiocytosis (HLH) is a rare multisystem disorder characterized by proliferation and diffuse infiltration multiple organs with histiocytes, including the central nervous system (CNS). Neurological manifestations of HLH have been recognized in different studies with children, but they remain relatively ill-defined in adults with HLH. From March 2008 to October 2014, 289 adult patients with HLH were admitted to our center. Clinical, radiological, and cerebral spinal fluid (CSF) data of the patients with CNS involvement were reviewed, and a retrospective study in our single-center was carried out. CNS involvement was observed in 29 patients (10%) either in their diagnosis process or during disease course. CNS symptoms included disturbance of consciousness, cranial nerve palsies, seizures, headache, limb paralysis, irritability, meningism, and memory loss. CSF analysis was conducted in 17 patients (59%). Among them, 11 patients (65%) were reported as having abnormal CSF. Neuroradiological studies were performed in 25 patients (86%). Among the 13 cases that underwent CT scan, one patient hemorrhaged. Single or multiple hypodense foci were detected in the other 2 patients. Magnetic resonance imaging (MRI) abnormalities were found in 15 patients, including focal lesions in cortical and adjacent subcortical regions with or without variable nodular or ring contrast-enhancement, multiple lesions in white matter, diffuse white matter signal changes, and meningeal enhancement. Basal ganglia, cerebellum, and brainstem lesions were also observed. CNS involvement could also be found in adult patients with HLH, but not as frequent as it was in children. The clinical manifestations could be diversified. By carrying out rigorous CNS examinations, an early diagnosis could be made and it was of the utmost importance for the prevention of further lesions.
1162	Diffuse Large B-Cell Lymphoma with Secondary Hemophagocytic Lymphohistiocytosis Presenting as Acute Liver Failure.	28584842	Hemophagocytic lymphohistiocytosis (HLH) and newly diagnosed malignant infiltration of liver are rare presentations of acute liver failure associated with poor prognosis. We report a case of a patient with acute liver failure caused by malignant infiltration by diffuse large B-cell lymphoma and secondary HLH.
1163	Hemophagocytic Lymphohistiocytosis Associated with Anaplasmosis.	28584460	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome characterized by unregulated macrophage and T-lymphocyte activation resulting in cytokine overproduction and subsequent histiocytic phagocytosis. Variant infections, particularly viruses have been postulated as the inciting factor for this potentially fatal disease. Herein, we will report a case of HLH associated with anaplasmosis.
1164	Prognostic factors of early death in children with hemophagocytic lymphohistiocytosis.	285826482896581829551450	Hemophagocytic lymphohistiocytosis is a rapidly progressing and fatal disease. Early identification of early death for HLH patients based on the laboratory findings at the time of diagnosis could improve the overall survival. A retrospective study was performed on 95 Chinese pediatric patients with HLH. Patients' data including clinical features and laboratory findings at diagnosis were collected. In a multivariate Cox proportional hazard regression model analysis, albumin≤27.75g/L (hazard ratio (HR)=11.82, 95% confidence interval (CI) 2.58-54.23; P=0.001), LDH≥3707.5 U/L (HR=4.15, 95%CI 1.43-12.01; P=0.009), and IL-10≥456pg/ml (HR=12.39, 95%CI 1.59-96.79; P=0.016) at diagnosis were independent prognostic factors of early death. The risk of early death was 33-fold increase in patients with three risk factors (HR=33.33; 95%CI 8.40-125.00; P<0.001), and 12-fold increase in patients with two risk factors (HR=12.80; 95%CI 2.34-69.80; P=0.002) when compared to it in patients with zero to one risk factor. Our results reveal that HLH patients with the risk of early death can be identified by laboratory findings at diagnosis, which may help guide the treatment decision making for this disease.
1165	Effects of propylene glycol or elevated luteinizing hormone during follicle development on ovulation, fertilization, and early embryo development.	28575154	Seventeen nonlactating Holstein cows were superovulated in a Latin-square designed experiment to determine the effects of increased propylene glycol (PROP) and luteinizing hormone (LH) during antral follicle development on ovarian function, fertilization, and early embryo quality. PROP was orally drenched every 4 h for 7 days to induce hyperinsulinemia and associated metabolic changes. LH concentrations were altered by increasing LH (3-fold) during last 2 days of superovulation. Treatment groups were as follows: (1) control-oral drenching with water plus low-LH preparation; (2) high LH(HLH)-water plus HLH preparation; (3) PROP-drenching with PROP plus low LH; (4) PROP/HLH-PROP plus HLH. PROP increased glucose (P < 0.05) and insulin (P < 0.02) concentrations at all time points analyzed. Neither PROP nor LH affected numbers of follicles > 9 mm at time of gonadotropin-releasing hormone-induced LH surge, although percentage of these follicles that ovulated was decreased by both PROP (P = 0.002) and LH (P = 0.048). In addition, PROP tended (P = 0.056) to decrease total number of ovulations. PROP reduced (P = 0.028) fertilization rate, while LH tended (P = 0.092) to increase fertilization rate. There was no effect of either PROP or LH on any measure of embryo quality including percentage of embryos that were degenerate, quality 1, or quality 1 and 2 of total structures collected or fertilized structures. These results indicate that acute elevation in insulin during the preovulatory follicular wave can decrease percentage of large follicles that ovulate, particularly when combined with increased LH, and reduce fertilization of ovulated oocytes.
1166	Haploidentical hematopoietic stem cell transplantation for adult patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	28573910	To assess the efficacy of haploidentical hematopoietic stem cell transplantation (HSCT) in adult patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH), we retrospectively analyzed 30 cases that presented at our institution. At the time of HSCT, 20 patients (66.7%) had achieved a response after receiving HLH-94 or salvage therapies. All patients underwent myeloablative conditioning followed by peripheral blood HSCT from their related, haploidentical donors. Twenty-six patients (86.7%) achieved donor cell engraftment. Of these, 23 (88.5%) achieved complete chimerism and three (11.5%) demonstrated mixed chimerism. Reactivated EBV infection was found in 25 (96.2%). Acute graft-versus-host disease occurred in 18 (69.2%), with grade I-II in 11 patients and grade III-IV in seven. Chronic graft-versus-host disease occurred in six (23.1%). Nineteen patients survived until the end of follow-up. The three-year overall survival rate was 63.3%. Our results indicate that haploidentical HSCT is an effective treatment for adult patients with EBV-HLH.
1167	Go with the flow: perforin and CD107a in HLH.	2857217428270454	NaN
1168	Mechanistic insights into the activity of Ptf1-p48 (pancreas transcription factor 1a): probing the interactions levels of Ptf1-p48 with E2A-E47 (transcription factor E2-alpha) and ID3 (inhibitor of DNA binding 3).	28571517	Ptf1-p48 (Pancreas specific transcription factor 1a) is transcription regulatory protein known for the activation of exocrine specific genes. Downregulation of its expression formulates early stages of pancreatic adenocarcinoma as deduced by its association with oncogenic bHLH (Basic Helix-Loop-Helix) protein ID3 (Inhibitor of DNA binding 3) protein whose overexpression induces cytoplasmic mislocalization of Ptf1-p48. The precise mechanism and/or functional role of Ptf1-p48in promoting pancreatic cancer is vague. The structural features of the Ptf1-p48 and its dimerization with E47 (Transcription factor E2-alpha) and ID3 mediated by their HLH (Helix-Loop-Helix) domain were perceived through MD (Molecular Dynamics) simulations of 50 ns. The interactions formed by the HLH domain in both Ptf1-E47 and Ptf1-ID3 complexes are favored by the synergistic movement of their domain helices. Accordingly, in the Ptf1-E47 complex α7 of Ptf1-p48 and α1 helix of E47 along with the loop residues of their HLH domain exhibit transitions marked by inward movement toward each other and forms polar and charged interactions. In the Ptf1-ID3 complex, α8 of Ptf1-p48 moves toward the α3 helix of ID3 and forms hydrogen bonds. The interface analysis also reveals better interface in the Ptf1-p48 complex than the Ptf1-ID3 evident by energetics and number of hydrogen bonds. The interactions in each of these complexes, supported by angular displacement and mode vector analyzes, comprehensibly describe the considerable structural changes induced upon dimer formation. It thereby gives an insight into the interfaces that could help in designing of potential inhibitors for ID3 to curb the cancer cell growth.
1169	Hemophagocytic lymphohistiocytosis in a patient with human immunodeficiency virus infection: A case report.	28565867	Hemophagocytic lymphohistiocytosis (HLH), also termed hemophagocytic syndrome, is a severe, life-threatening inflammatory condition that results from an excessive, prolonged and ineffective immune response. The syndrome occurs due to overactive macrophages from the bone marrow or lymph tissue that phagocytose erythrocytes leukocytes and platelets. HLH in a patient with human immunodeficiency virus infection has rarely been studied. The present case study described an uncommon case of this syndrome in combination with human immunodeficiency virus infection in a patient, who eventually succumbed to severe infection and multiple organ failure following the refusal of medical treatment.
1170	Secondary Hemophagocytic Lymphohistocytosis in a Child With Brucellosis.	28562515	Hemophagocytic lymphohistocytosis (HLH) is a potentially fatal hyperinflammatory syndrome that is characterized by proliferation of histiocytes and hemophagocytosis in different organs. The diagnostic criteria include fever, hepatosplenomegaly, bicytopenia, high serum ferritin level, decreased natural killer cell activity, elevated soluble CD25 level, high serum fasting triglyceride level or low fibrinogen level, and hemophagocytosis in the bone marrow, spleen, or lymph nodes. HLH can be classified as primary and secondary. Secondary HLH can be related to infections. Here we report a case of Brucella-related HLH, which has been rarely reported in the literature.
1171	Hemophagocytic Lymphohistiocytosis: A Diagnostic Conundrum.	28553383	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive immune activation causing widespread inflammation and tissue destruction leading to multi-organ dysfunction and failure. Making the diagnosis of HLH could be quite challenging due to the broad range of presenting symptoms and their lack of specificity. After ruling out considerations for differential diagnoses, recognizing the most common presenting signs and symptoms of HLH, including neurologic dysfunction, and having a high clinical suspicion for HLH in the setting of inflammatory/demyelinating diseases are important for prompt diagnosis and treatment.
1172	Isolation, characterization, and expression of proto-oncogene cMyc in large yellow croaker Larimichthys crocea.	28550411	cMyc is a vital transcription factor that involves in the regulation of cell proliferation, growth, differentiation, and apoptosis. In the present study, cMyc in Larimichthys crocea (Lc-cMyc) was cloned and analyzed for investigating its function. The full-length cDNA of Lc-cMyc was 2089 bp encoding a 440-amino-acid protein (Lc-cMyc). Lc-cMyc had the characteristic helix-loop-helix-leucine-zipper (HLH-LZ) DNA-binding domain and highly conservative in evolution. The expression of Lc-cMyc was detected by quantitative real-time PCR (qRT-PCR) and in situ hybridization, respectively. In tissues, the gender differences of Lc-cMyc expression existed only in gonad and Lc-cMyc was extremely significantly expressed in ovary with the highest level in 635-dph ovary, especially in stages II (late) and III (early) oocytes. A certain degree of expression was examined in head kidney of both sexes and testis with high expression in spermatocyte. In embryos, Lc-cMyc was expressed at all embryonic stages. In early embryogenesis (from two-cell stage to mutiple-cell stage), Lc-cMyc was expressed very highly with a peak at two-cell stage. In late embryogenesis (from blastula stage to 1-day-post-hatching stage), the high expression of Lc-cMyc was detected as the following order: 1-day-post-hatching stage > pre-hatching stage > the appearance-of-optic-vesicles stage = mutiple-cell stage > beginning-of-heart-pulsation stage. The distribution of Lc-cMyc concentrated gradually in the back of embryos until in the head. In conclusion, the spatio-temporal expression patterns of Lc-cMyc indicated an essential role in oogenesis and embryogenesis and contributed to insight into the molecular mechanisms of regulating pluripotency in large yellow croaker.
1173	[Hemophagocytic lymphohistiocytosis. Experience in 27 patients].	28548191	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome of excessive immune activation Aim: To describe the clinical characteristics, causes and survival associated with HLH.Review of medical records of patients with HLH attended between 2004 and 2016. They were classified according to their probable cause in: associated with immunosuppression, cancer, post-infectious or idiopathic. Kaplan-Meier survival analysis was performed.Twenty seven patients with HLH aged 18 to 87 years (59% men), were detected. Fourteen (52%) were secondary to immunosuppression, six (22%) were post-infectious, five (18%) were associated with cancer and two (7%) were of unknown cause. There were no significant differences in clinical or laboratory features between these etiologies. Within the immunosuppressed group, 12 (86%) were patients with oncologic or hematologic diseases or bone marrow transplantation. Associated cancers were mostly oncohematologic diseases. Thirty-day mortality was 53.4% (95% confidence intervals (CI) 32.7-70.3%), despite the treatment. Mortality was significantly associated with the presence of renal failure with a hazard ratio (HR) of 3.4 (95% CI of 1.2-9.9, p =0.025). Treatment of the underlying disease proved to be protective against mortality with an HR of 0.3 (95% CI 0.1 to 0.98, p = 0.046).The prognosis of HLH could be related to the treatment of the underlying disease. The study of the pathophysiology of this syndrome will allow a better understanding and treatment.
1174	The effectiveness of cutoff walls to control saltwater intrusion in multi-layered coastal aquifers: Experimental and numerical study.	28527376	The objective of this study was to examine the performance of cutoff walls in controlling saltwater intrusion in stratified heterogeneous coastal aquifers. Numerical and laboratory experiments were completed in laboratory-scale aquifer where the effectiveness of cutoff walls was assessed in three different configurations, including a homogeneous scenario, a stratified aquifer with high K-low K-high K pattern (case HLH) and another stratified aquifer with low K-high K-low K pattern (case LHL). The results show that the cutoff wall was effective in reducing the saltwater wedge in all the investigated cases of layered-aquifers with toe length reduction of up to 43%. The wall exhibited more wedge reduction in shallower than steeper hydraulic gradients. However, the soil stratification appeared to lessen the overall performance of the wall compared to the homogeneous case. The aquifer stratification disrupted the flow dynamics, and thus affected the freshwater velocity at the wall opening to various degrees, depending on the layering pattern. The presence of an interlayer of low k (case HLH) inhibited the downward movement of the freshwater towards the wall opening, and thus decreasing the repulsion ability of the wall. Moreover, the presence of an underlying low permeability layer (case LHL) was found to obstruct the freshwater flow in the lower part of the aquifer, thereby slowing down the velocity through the wall opening. Numerical analysis of other layering patterns of monotonically increasing/decreasing permeability from top to bottom showed that the cutoff wall remained effective in repulsing the seawater wedge.
1175	Hemophagocytic Lymphohistiocytosis in a Newborn Presenting as "Blueberry Muffin Baby".	28523896	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening hyperinflammatory syndrome characterized by uncontrolled activation and proliferation of proinflammatory cytokines. Initial presentation commonly includes fever, hepatosplenomegaly, and pancytopenia; 6 to 65% of cases also have a concurrent cutaneous eruption. We present the case of a 6-day-old premature infant boy with congenital severe thrombocytopenia, anemia, and hepatosplenomegaly who presented with several cutaneous violaceous papules and nodules and was found to have HLH.
1176	Case report: patient with unexplained high fever and pancytopenia.	28521652	We present a case of hemophagocytic lymphohistiocytosis (HLH), giving insight in how to establish diagnosis and start appropiate treatment.A 45-year-old male presented at the emergency ward with high fever and pancytopenia. Repeat bone marrow aspirates showed hemophagocytosis. Extensive work-up with exclusion of other infectious and malignant diseases, eventually lead us to the diagnosis of hemophagocytic lymphohistiocytosis.The patient was treated with Etoposide, Cyclosporine A, and systemic steroids based on the HLH-2004 protocol. Unfortunately the patient responded very poorly to the treatment with prolonged pancytopenia, leading to central hemorrhage and eventually death.Diagnosis of hemopagocytic lymphohistiocytosis is difficult and late because of aspecific signs and low incidence. The HLH-criteria can be of use to exclude or confirm diagnosis in unclear cases. Early diagnosis and treatment is crucial given high morbidity and mortality. Therapy depends on type and etiology of the HLH-syndrome. Besides treating the possible triggering factor, Etoposide, steroids, and Cyclosporine A are the mainstays of treatment, sometimes followed by allogenic hematopoietic stem cell transplantation. More research in adults is needed.
1177	Multifocal Recurrent Osteomyelitis and Hemophagocytic Lymphohistiocytosis in a Boy with Partial Dominant IFN-γR1 Deficiency: Case Report and Review of the Literature.	28516082	Mutations in the genes coding for cytokines, receptors, second messengers, and transcription factors of interferon gamma (IFN-γ) immunity cause Mendelian susceptibility to mycobacterial disease (MSMD). We report the case of a 7-year-old male patient with partial dominant (PD) IFN-γ receptor 1 deficiency who had suffered from multifocal osteomyelitis attributable to bacille Calmette-Guérin vaccination since the age of 18 months. He developed hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory complication, and died with multiorgan dysfunction, despite having been diagnosed and treated relatively early. Patients with PD IFN-γR1 deficiency usually have good prognosis and might respond to human recombinant subcutaneous IFN-γ. Several monogenic congenital defects have been linked to HLH, a catastrophic "cytokine storm" that is usually ascribed to lymphocyte dysfunction and thought to be triggered by interferon gamma. This is the sixth patient with both MSMD and HLH of whom we are aware. The fact that patients with macrophages that cannot respond to IFN-γ still develop HLH, bring these assumptions into question.
1178	Impact of severe hematological abnormalities in the outcome of hospitalized patients with influenza virus infection.	28500507	Although hematological abnormalities have been described among patients with influenza virus infection, little is known about their impact on the outcome of the patients. The aim of this study was to assess the frequency and clinical impact of severe hematological abnormalities in patients with confirmed influenza virus infection. This was an observational retrospective study including all adult patients with diagnosis of influenza virus infection hospitalized from January to May 2016 in our institution. Influenza virus infection was diagnosed by means of rRT-PCR assay performed on respiratory samples. Poor outcome was defined as a composite endpoint in which at least one of the following criteria had to be fulfilled: (a) respiratory failure, (b) SOFA ≥2, or (c) death. Two hundred thirty-nine patients were included. Applying the HLH-04 criteria for the diagnosis of hemophagocytic syndrome, cytopenias (hemoglobin ≤9 g/dl, platelets <100,000/μl or neutrophils <1,000/μl) were present in 51 patients (21%). Patients with hematological abnormalities showed higher SOFA scores, respiratory failure, septic shock and in-hospital mortality than the remaining patients. The composite endpoint was present in 33.3% in the cytopenias group vs. 13.3% in the group without cytopenias (p=0.001). In a multivariate analysis, variables associated with the composite endpoint were: use of steroids prior to present admission (OR: 0.12; 95% CI: 0.015-0.96, p=0.046), presence of any hematological abnormality (OR: 3.54; 95% CI:1.66-7.51, p= 0.001), and LDH>225 U/l (OR:4.45; CI:1-19.71, p=0.049). Hematological abnormalities are not uncommon among hospitalized patients with influenza virus infection, and they are associated with a poorer outcome.
1179	Clinical utility of soluble interleukin-2 receptor in hemophagocytic syndromes: a systematic scoping review.	28497365	The serum-soluble interleukin-2 receptor (sIL-2r) level is considered an important diagnostic test and disease marker in hemophagocytic syndromes/hemophagocytic lymphohistiocytosis (HPS/HLH). However, this cytokine receptor is rarely measured in clinical practice and has been excluded from recent diagnostic/classification criteria such as the HScore and macrophage activation syndrome (MAS) 16. We performed a systematic scoping review of 64 articles (1975-2016) examining the clinical utility of sIL-2r in HPS/HLH. Twenty-two articles describe sIL-2r as a sensitive diagnostic marker for HLH, but only three distinct datasets actually address sensitivity. The original HLH-2004 Guidelines reported sensitivity of 93% and specificity of 100% for sIL-2r ≥ 2400, based on a pediatric dataset (n = 152) which is published for the first time in this review. Two pediatric studies reported sensitivity of 89% for sIL-2r ≥ 2400 in diagnosis of MAS complicating juvenile idiopathic arthritis (JIA) (n = 27) and 88% for secondary HLH in acute liver failure (n = 9). Twenty articles described sIL-2r as a dynamic marker of disease activity that falls with response to treatment, and 15 described high initial sIL-2r levels >10,000 U/mL as a poor prognostic marker. The ability of sIL-2r to distinguish between subtypes of HPS/HLH was inconsistent. This review confirms the importance of soluble IL-2r as a diagnostic and disease marker in HPS/HLH, but also reveals the need for more primary data about its performance characteristics, particularly in adults. More emphasis should be made in including this simple, inexpensive test in clinical practice and studies of HPS/HLH.
1180	Hemophagocytic lymphohistiocytosis in acute African swine fever clinic.	28494931	Hemophagocytic lymphohistiocytosis (HLH) usually has been defined as the combination of a proliferation of cytologically benign, actively phagocytic macrophages in bone marrow, spleen, lymph nodes, etc. in association with fever, cytopenia, splenomegaly, and hypertriglyceridemia. HLH is often triggered by viral infection. The aim of this study was to ascertain the features of HLH involvement in African swine fever virus (ASFV) (genotype II) pathogenesis.The serum levels of macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF), as well as the histological constitution (for hemophagocytic macrophages detection) of various organs of pigs infected with ASFV genotype II were investigated. The diagnosis of HLH was made according to universally accepted human criteria.The association of fever, cytopenias, splenomegaly, and hemophagocytosis was present in 87.5% of the infected pigs (absence of hyperthermia in one of eight pigs). Marked hypertriglyceridemia was observed at 3-4days post infection. Previously it was shown that ASFV induced a significant decrease in the level of fibrinogen from day 5 till the end of experiment. Progression of the HLH coincided with a temporary increase in the serum levels of MCSF levels (early stage of disease) and GMCSF levels (2-3 pays post infection).Hemophagocytic syndrome should be suspected in ASFV (genotypeII) infected pigs.
1181	Similar but not the same: Differential diagnosis of HLH and sepsis.	28477737	Differential diagnosis of hemophagocytic lymphohistiocytosis (HLH; hemophagocytic syndrome) and sepsis is critically important because the life-saving aggressive immunosuppressive treatment, required in the effective HLH therapy, is absent in sepsis guidelines. Moreover, HLH may be complicated by sepsis. Hyperinflammation, present in both states, gives an overlapping clinical picture including fever and performance status deterioration. The aim of this review is to provide aid in this challenging diagnostic process. Analysis of clinical features and laboratory results in multiple groups of patients (both adult and pediatric) with either HLH or sepsis allows to propose criteria differentiating these two conditions. The diagnosis of HLH is supported by hyperferritinemia, splenomegaly, marked cytopenias, hypofibrinogenemia, low CRP, characteristic cytokine profile and, only in adults, hypertriglyceridemia. In the presence of these parameters (especially the most characteristic hyperferritinemia), the other HLH criteria should be assessed. Genetic analyses can reveal familial HLH. Hemophagocytosis is neither specific nor sensitive for HLH.
1182	Hemophagocytic lymphohistiocytosis in patients with metastatic malignant melanoma.	28471834	Hemophagocytic lymphohistiocytosis (HLH) is an autoinflammatory disease that classically occurs because of infections, autoinflammatory, or autoimmune diseases, hematologic cancers, and rarely because of solid tumor. We report a rare case of HLH attributed to metastatic malignant melanoma treated without corticosteroid and with a nonfatal outcome thanks to specific therapies: etoposide for HLH and a selective inhibitor of mutated forms of BRAF kinase associated with a MEK inhibitor for melanoma.
1183	Associations of the Intellectual Disability Gene MYT1L with Helix-Loop-Helix Gene Expression, Hippocampus Volume and Hippocampus Activation During Memory Retrieval.	28470180	The fundamental role of the brain-specific myelin transcription factor 1-like (MYT1L) gene in cases of intellectual disability and in the etiology of neurodevelopmental disorders is increasingly recognized. Yet, its function remains under-investigated. Here, we identify a network of helix-loop-helix (HLH) transcriptional regulators controlled by MYT1L, as indicated by our analyses in human neural stem cells and in the human brain. Using cell-based knockdown approaches and microarray analyses we found that (1) MYT1L is required for neuronal differentiation and identified ID1, a HLH inhibitor of premature neurogenesis, as a target. (2) Although MYT1L prevented expression of ID1, it induced expression of a large number of terminal differentiation genes. (3) Consistently, expression of MYT1L in the human brain coincided with neuronal maturation and inversely correlated with that of ID1 and ID3 throughout the lifespan. (4) Genetic polymorphisms that reduced expression of MYT1L in the hippocampus resulted in increased expression of ID1 and ID3, decreased levels of the proneural basic HLH (bHLH) transcriptional regulators TCF4 and NEUROD6 and decreased expression of genes involved in long-term potentiation and synaptic transmission, cancer and neurodegeneration. Furthermore, our neuroimaging analyses indicated that MYT1L expression associated with hippocampal volume and activation during episodic memory recall, as measured by blood-oxygen-level-dependent (BOLD) signals. Overall, our findings suggest that MYT1L influences memory-related processes by controlling a neuronal proliferation/differentiation switch of ID-bHLH factors.
1184	A case report of novel mutation in PRF1 gene, which causes familial autosomal recessive hemophagocytic lymphohistiocytosis.	28468610	Hemophagocytic Lymphohistiocytosis (HLH) is a life-threatening immunodeficiency and multi-organ disease that affects people of all ages and ethnic groups. Common symptoms and signs of this disease are high fever, hepatosplenomegaly, and cytopenias. Familial form of HLH disease, which is an autosomal recessive hematological disorder is due to disease-causing mutations in several genes essential for NK and T-cell granule-mediated cytotoxic function. For an effective cytotoxic response from cytotoxic T lymphocyte or NK cell encountering an infected cell or tumor cell, different processes are required, including trafficking, docking, priming, membrane fusion, and entry of cytotoxic granules into the target cell leading to apoptosis. Therefore, genes involved in these steps play important roles in the pathogenesis of HLH disease which include PRF1, UNC13D (MUNC13-4), STX11, and STXBP2 (MUNC18-2).Here, we report a novel missense mutation in an 8-year-old boy suffered from hepatosplenomegaly, hepatitis, epilepsy and pancytopenia. The patient was born to a first-cousin parents with no previous documented disease in his parents. To identify mutated gene in the proband, Whole Exome Sequencing (WES) utilizing next generation sequencing was used on an Illumina HiSeq 2000 platform on DNA sample from the patient. Results showed a novel deleterious homozygous missense mutation in PRF1 gene (NM_001083116: exon3: c. 1120 T > G, p.W374G) in the patient and then using Sanger sequencing it was confirmed in the proband and his parents. Since his parents were heterozygous for the identified mutation, autosomal recessive pattern of inheritance was confirmed in the family.Our study identified a rare new pathogenic missense mutation in PRF1 gene in patient with HLH disease and it is the first report of mutation in PRF1 in Iranian patients with this disease.
1185	Pyogenic brain abscesses treated with antibiotics in a patient with hemophagocytic lymphohistiocytosis on HLH-94 protocol.	28466263	NaN
1186	Visceral leishmaniasis-associated hemophagocytosis: A tale of two unexpected diagnoses from a nonendemic region.	28459018	A case of visceral leishmaniasis (VL)-associated hemophagocytic lymphohistiocytosis (HLH) in an immunocompetent native from a nonendemic area was reported. The patient belonged to Ravi river valley area (altitude 996 meters above the mean sea level) of Chamba, Himachal Pradesh, India. VL and HLH were not a differential diagnosis. Identification of the Leishman-Donovan bodies and hemophagocytosis in bone marrow aspirate and biopsy provided the diagnosis. The patient recovered to the treatment with amphotericin B.
1187	Differences in Granule Morphology yet Equally Impaired Exocytosis among Cytotoxic T Cells and NK Cells from Chediak-Higashi Syndrome Patients.	28458669	Chediak-Higashi syndrome (CHS) is caused by autosomal recessive mutations in LYST, resulting in enlarged lysosomal compartments in multiple cell types. CHS patients display oculocutaneous albinism and may develop life-threatening hemophagocytic lymphohistiocytosis (HLH). While NK cell-mediated cytotoxicity has been reported to be uniformly defective, variable defects in T cell-mediated cytotoxicity has been observed. The latter has been linked to the degree of HLH susceptibility. Since the discrepancies in NK cell- and T cell-mediated cellular cytotoxicity might result from differences in regulation of cytotoxic granule release, we here evaluated perforin-containing secretory lysosome size and number in freshly isolated lymphocytes from CHS patients and furthermore compared their exocytic capacities. Whereas NK cells from CHS patients generally contained a single, gigantic perforin-containing granule, cytotoxic T cells predominantly contained several smaller granules. Nonetheless, in a cohort of 21 CHS patients, cytotoxic T cell and NK cell granule exocytosis were similarly impaired upon activating receptor stimulation. Mechanistically, polarization of cytotoxic granules was defective in cytotoxic lymphocytes from CHS patients, with EEA1, a marker of early endosomes, mislocalizing to lysosomal structures. The results leads to the conclusion that lysosome enlargement corresponds to loss of distinct organelle identity in the endocytic pathway, which on a subcellular level more adversely affects NK cells than T cells. Hence, vesicular size or numbers do not per se dictate the impairment of lysosomal exocytosis in the two cell types studied.
1188	A Case of Hemophagocytic Lymphohistiocytosis and Kawasaki Disease: Concurrent or Overlapping Diagnoses?	28452857	Hemophagocytic lymphohistiocytosis (HLH) is not always suspected at the time of presentation. It is often mistaken for other diagnoses; complicated by the fact that it is often associated with an inciting event that has significant overlap. Kawasaki disease, along with other disorders, such as Ebstein Barr Virus infection, are conditions that may lead HLH. Our patient had a presentation that was consistent with Kawasaki disease on initial presentation, however subsequently met the diagnostic criteria of HLH. It provided an interesting discussion about diagnoses with clinical criteria and how the overlap can sometimes delay or complicate initial diagnosis.
1189	Local injection of Lenti-Olig2 at lesion site promotes functional recovery of spinal cord injury in rats.	28452182	Olig2 is one of the most critical factors during CNS development, which belongs to b-HLH transcription factor family. Previous reports have shown that Olig2 regulates the remyelination processes in CNS demyelination diseases models. However, the role of Olig2 in contusion spinal cord injury (SCI) and the possible therapeutic effects remain obscure. This study aims to investigate the effects of overexpression Olig2 by lentivirus on adult spinal cord injury rats.Lenti-Olig2 expression and control Lenti-eGFP vectors were prepared, and virus in a total of 5 μL (108 TU/mL) was locally injected into the injured spinal cord 1.5 mm rostral and caudal near the epicenter. Immunostaining, Western blot, electron microscopy, and CatWalk analyzes were employed to investigate the effects of Olig2 on spinal cord tissue repair and functional recovery.Injection of Lenti-Olig2 significantly increased the number of oligodendrocytes lineage cells and enhanced myelination after SCI. More importantly, the introduction of Olig2 greatly improved hindlimb locomotor performances. Other oligodendrocyte-related transcription factors, which were downregulated or upregulated after injury, were reversed by Olig2 induction.Our findings provided the evidence that overexpression Olig2 promotes myelination and locomotor recovery of contusion SCI, which gives us more understanding of Olig2 on spinal cord injury treatment.
1190	Eculizumab in a child with atypical haemolytic uraemic syndrome and haemophagocytic lymphohistiocytosis triggered by cytomegalovirus infection.	28446488	We present the case of a 21-month-old girl with two rare and life-threatening conditions, atypical haemolytic uraemic syndrome (aHUS) and haemophagocytic lymphohistiocytosis (HLH), triggered by a cytomegalovirus (CMV) infection. Soon after admission, the girl became anuric and required continuous venovenous haemodiafiltration.Initial treatments included methylprednisolone, fibrinogen and plasma infusion (for HLH), plasmapheresis (for thrombotic microangiopathy), immunoglobulins (for inflammation), ganciclovir (for CMV infection) and the antibiotic cefotaxime. On day 5, eculizumab (600 mg) was given for aHUS, with rapid improvement in haematological and nephrological parameters. Despite a subsequent isolated episode of right heart thrombosis that resolved with heparin treatment, the patient showed a favourable response to eculizumab (300 mg/15 days), with improved renal function, normal haematological values, and no treatment complications. In conclusion, eculizumab effectively treated aHUS in this case despite a comorbid immunological disease.
1191	Haemophagocytic lymphohistiocytosis as a consequence of untreated B-cell chronic lymphocytic leukaemia.	28446487	Haemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening syndrome characterised by defective cytotoxic function and hypercytokinaemia leading to macrophage expansion and haemophagocytosis. Patients often present with unexplained fevers, hepatosplenomegaly and pancytopenia, with elevation in serum ferritin and triglyceride. Acquired forms are triggered by infection, malignancy or rheumatological disorders. HLH in the setting of chronic lymphocytic leukaemia is rarely reported, however, and is usually associated with infection or as a consequence of chemotherapy. We present a case of HLH in a 64-year-old Caucasian woman with the only identified trigger being her hitherto untreated CLL.
1192	Haemophagocytic lymphohistiocytosis presenting as HELLP syndrome: a diagnostic and therapeutic challenge.	28433984	Haemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal, haematological disorder, which can be clinically challenging to diagnose and manage. We report a case of HLH in a previously healthy 33-year-old primigravida. The patient presented at 22 weeks gestation with dyspnoea, abdominal pain, anaemia, thrombocytopenia and elevated liver enzymes suggestive of HELLP syndrome.HELLP, a syndrome characterised by haemolysis, elevated liver enzymes and low platelets is considered a severe form of pre-eclampsia. Despite delivery of the fetus, her condition deteriorated over 3-4 days with high-grade fever, worsening thrombocytopenia and anaemia requiring transfusion support. A bone marrow biopsy showed haemophagocytosis and a diagnosis of HLH was made. Partial remission was achieved with etoposide-based chemotherapy and complete remission following bone marrow transplantation. Eleven months post-transplant, the disease aggressively recurred, and the patient died within 3 weeks of relapse.
1193	Recent advances in diagnostic and therapeutic guidelines for primary and secondary hemophagocytic lymphohistiocytosis.	28433836	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome characterized by excessive activation of macrophages and T cells resulting from defective cytotoxicity. It is potentially life threatening due to the large amounts of cytokines released by the activated macrophages and lymphocytes secondary to a hyperinflammatory response. It has a high fatality in children with an incidence of approximately 1.2cases/million per year.The literature was extensively searched in PubMed, MEDLINE and Google scholar.A variable and nonspecific symptomatology can delay the diagnosis and hence requires a high index of suspicion in both primary and secondary HLH.
1194	Metronomic regimen as an effective treatment for aggressive T-LGL leukemia with central nervous system infiltration: clinical experience and review of literature.	28427176	A 71-year-old man was diagnosed with T-Large granular lymphocytic (LGL) leukemia, which usually represents a relatively indolent clinical course. While the clinical manifestation of this patient we report herein was aggressive with lasting fever, splenomegaly and hemophagocytic lymphohistiocytosis (HLH). T-cell immunophenotype was CD3+CD4-CD8-CD5-CD7-TCRαβ+. After comprehensive evaluation, an adjusted chemotherapy regimen CEOP (cyclophosphamide, vincristine, etoposide, prednisone) with etoposide, a potential effective regimen for HLH was administrated to the patient. Although he received intensive regimen, the patient showed drug resistance and disease progression with central nervous system (CNS) involvement during treatment and showed only transiently response to intrathecal methotrexate, cytarabine and dexamethasone. Therefore, considering the refractory elderly patient with fragile physical condition, metronomic regimen T-PEPC (oral administration of thalidomide, prednisone, cyclophosphamide, etoposide and methyhydrazine) was recommended, which refers to the frequent even daily administration of cytotoxic drugs at comparatively low doses with minimal or prolonged drug-free breaks. The patient responded well to this treatment and remained symptom-free for 8-month follow-up. To our knowledge, this is the first case of reporting this unique immunophenotype of dual CD4-/CD8- with aggressive clinical course and CNS involvement that successfully treated with metronomic regimen, suggesting that low dose metronomic regimen could be a better option for elderly patient with aggressive T-LGL leukemia.
1195	Tuberculosis-associated hemophagocytic lymphohistiocytosis with initial presentation of fever of unknown origin in a general hospital: An analysis of 8 clinical cases.	28422850	The study aimed to investigate the clinical features and prognoses of patients with tuberculosis (TB) who had secondary hemophagocytic lymphohistiocytosis (HLH).Patients first presenting with fever of unknown origin, who were ultimately diagnosed with TB-associated secondary HLH, were assessed retrospectively. We summarized and analyzed clinical manifestations, laboratory examinations, diagnoses, treatments, and prognoses of patients using clinical data, outpatient follow-up, and telephone follow-up in combination with literature review.Among patients admitted to the hospital with fever of unknown origin in the past 10 years, 371 patients were diagnosed with TB. Among them, 8 cases were diagnosed as tuberculosis-associated HLH (TB-HLH). The proportion of females among TB-HLH patients was higher than the proportion of females among TB patients. Within the same time period, 227 cases met the diagnostic criteria for HLH, among which TB-HLH patients accounted for 3.52% of the cases. None of the 8 TB-HLH patients had underlying diseases, and a majority of them had short symptom durations, rapid progression, along with multisystem and multiorgan dysfunctions. Their clinical manifestations were inconsistent with the typical clinical manifestations and imaging results characteristic of TB. Compared with patients with TB in our hospital during the same period, the 8 TB-HLH patients had a higher proportion of blood-disseminated TB and tuberculous meningitis. Apart from this, the hematological damage in these patients was higher than the common clinical manifestations of TB, and they also had a high proportion of respiratory failure. All 8 TB-HLH patients received antitubercular therapy, and 6 of them were also treated for HLH. However, their morbidity and mortality were significantly higher than that for reported cases of TB-HLH cases, both domestically and abroad, which may be attributed to the fever of unknown origin.Patients with TB-HLH had poor prognoses and no specific clinical manifestations. Therefore, cases of atypical TB and severe TB should be carefully monitored to achieve early diagnosis and early intervention.
1196	Elevation of CD16+CD56+ NK-cells and down-regulation of serum interleukin-21 (IL-21) and IL-1α after splenectomy in relapsed hemophagocytic lymphohistiocytosis of unknown cause.	28413901	Encouraging progress has been made in application of splenectomy in the treatment of relapsed hemophagocytic lymphohistiocytosis (HLH) of unknown cause. The aim was to determine the roles of lymphocyte subpopulations and inflammatory cytokines in splenectomy.We retrospectively analyzed changes in lymphocyte subpopulations and levels of inflammatory cytokines at different time-points before and after splenectomy in the patients with relapsed HLH of unknown cause, as well as the correlations between these changes and the disease prognosis.During the period from June 2006 to June 2016, we enrolled 107 patients with relapsed HLH of unknown cause, of whom 29 were treated with splenectomy. Among the 29 patients, 7 cases were non-Hodgkin lymphomas based on spleen pathology, 1 case withdrew and the remaining 21 non-lymphoma cases were available for analysis. Results showed a significant increase in both percentage of CD16+CD56+ NK cells (P = 0.003) and NK cell activity (P = 0.028) at 24 wk after splenectomy compared to their baseline pre-surgery levels. We also examined seven patients for the changes in cytokine levels before and after splenectomy and found that IL-21 and IL-1α decreased at 4 wk after splenectomy (P < 0.05). Seven non-lymphoma patients determined as no response to treatment (NR) prior to splenectomy had significantly longer survival (P = 0.001) compared to the 24 patients with relapsed HLH of unknown cause who were also determined as NR but not treated by splenectomy.Splenectomy can improve clinical symptoms and survival of patients with relapsed HLH of unknown cause. The mechanism is likely related to the changes in percent NK cells and cytokines (IL-21 and IL-1α) after surgery.
1197	Severe dengue due to secondary hemophagocytic lymphohistiocytosis: A case study.	28409119	Dengue, transmitted by the mosquito Aedes aegypti affects millions of people worldwide every year. Dengue induced hemophagocytic lymphohistiocytosis (HLH) is a serious condition and may prove fatal if not detected early and treated appropriately. Diagnosis of HLH is challenging and usually missed as clinical and laboratory findings are nonspecific. Moreover, the pathophysiology of the systemic inflammatory response syndrome and/or sepsis is remarkably similar to HLH. Secondary HLH following infection by the dengue virus is now being increasingly recognized as a cause of severe form of the disease. We report a case of dengue associated HLH in an otherwise healthy person who deteriorated during the course of hospitalization. A disproportionately high ferritin level and persistent bicytopenia prompted investigations for HLH. Diagnosis of dengue fever with virus-associated hemophagocytic syndrome was established according to the diagnostic criteria laid down by the Histiocyte Society. We discuss the diagnosis and management of this complex case and try to generate awareness about dengue induced HLH as one of the possible causes for severe manifestations of this infection.
1198	Acute Cytomegalovirus (CMV) Infection Associated with Hemophagocytic Lymphohistiocytosis (HLH) in an Immunocompetent Host Meeting All Eight HLH 2004 Diagnostic Criteria.	28409071	Hemophagocytic lymphohistiocytosis (HLH) is a rare and often deadly syndrome characterized by severe inflammation and cytokine dysregulation. The disease is defined by the HLH-2004 criteria, requiring five of eight findings, and is further differentiated into either primary or secondary causes. Primary HLH tends to be of genetic etiology, while secondary HLH results from other insults such as infection. Secondary HLH is most commonly associated with viral infections in immunocompromised patients. Acute cytomegalovirus (CMV) associated HLH in the immunocompetent host is exceedingly rare and only documented in four case reports to date. We describe the fifth documented case of CMV-associated HLH in an immunocompetent patient, and furthermore, we demonstrate that this patient is the first published case of its type to satisfy all eight of HLH-2004 criteria.
1199	Novel NLRC4 Mutation Causes a Syndrome of Perinatal Autoinflammation With Hemophagocytic Lymphohistiocytosis, Hepatosplenomegaly, Fetal Thrombotic Vasculopathy, and Congenital Anemia and Ascites.	28403691	Autoinflammatory diseases are caused by pathologic activation of the innate immune system. Primary hemophagocytic lymphohistiocytosis (HLH) is an aggressive syndrome of excessive immune activation caused by monogenic mutations resulting in cytotoxic cell defects and subsequent failure to eliminate activated macrophages. Secondary HLH is often diagnosed in cases without a known Mendelian inheritance. However, some cases of "secondary" HLH have been shown to harbor mutations with partial dysfunction of the cytotoxic system. Recently, macrophage intrinsic abnormalities caused by NLRC4 inflammasome mutations have been linked to autoinflammation and recurrent macrophage activation syndromes resembling a primary HLH. We report a case of a former 28-week preterm infant with congenital anemia, ascites, and a heavy edematous placenta with fetal thrombotic vasculopathy, who developed hepatosplenomegaly and unexplained systemic inflammation with laboratory features of HLH in the early postnatal course and died at 2 months of age. Postmortem examination confirmed the hepatosplenomegaly with marked sinusoidal hemophagocytosis, along with striking hemophagocytosis in the bone marrow and lymph nodes. There was extensive acute and chronic ischemic bowel disease with matted bowel loops, fibrous adhesions, and patchy necrotizing enterocolitis features. Whole exome sequencing analysis demonstrated a novel mosaic heterozygous NLRC4 512 C> T (p.Ser171Phe) de novo mutation predicated to cause a dominant, gain-of-function mutation resulting in a constitutively active protein. The assembly of NLRC4-containing inflammasomes via an induced self-propagation mechanism likely enables a perpetuating process of systemic macrophage activation, presumed to be initiated in utero in this patient.
1200	The effect of transport on the physiologic stability of neonates with ductal-dependent single-ventricle lesions.	28393581	To compare the status of infants with hypoplastic left heart syndrome (HLHS) or pulmonary atresia-hypoplastic right heart (PA-HRH) before and following transport using the validated Transport Risk Index of Physiologic Stability (TRIPS) score.In this retrospective review of infants with HLHS or PA-HRH transported to a Children's Hospital by a pediatric transport team, an increase in TRIPS score (temperature, blood pressure, respiratory status, and response to stimuli) following transport was defined as deterioration. Statistical analyses included t-test (paired and independent), χ2, and McNemar's tests for comparisons between groups with and without deterioration and before and after transport.Our cohort [n = 64; 39 (61%) HLHS and 25 (39%) PA-HRH] was predominantly female (61%), black (56%), and diagnosed antenatally (78%). Median transport time was 20 (10-30) min and age was <12 h in 48 (75%) infants. TRIPS scores worsened after transport in 24 (37.5%) infants, due to temperature (n = 10) or respiratory (n = 7) dysregulation. Infants who deteriorated during transport had HLH more often (83 versus 48%) and lower pH [7.27 (0.12) versus 7.33 (0.07)]. HLH was significantly predictive of deterioration during transport [OR 5.60 (95% C.I. 1.18-26.62)].The physiologic deterioration in a third of infants with single ventricle following short transports is intriguing and may have implications on their optimal place of birth.
1201	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis during pregnancy.	28381688	An 11-week pregnant, 32-year-old Japanese woman who had recovered from infectious mononucleosis visited our center due to fever, anorexia, and bilateral hypochondrial pain. Blood tests revealed leukopenia, thrombocytopenia and elevated ferritin. She was diagnosed with hemophagocytic lymphohistiocytosis (HLH). A high viral load of the Epstein-Barr virus (EBV) was recognized, indicating EBV-HLH. She was treated with a single dose of dexamethasone to protect the fetus. However, the disease was uncontrollable, necessitating etoposide and cyclosporine administration. Remission was obtained with these medications, and she has remained in remission for the 10 months since completion of chemotherapy. Although the occurrence of EBV-HLH during pregnancy is rare, it is possible that a change in cellular immunity associated with the pregnancy may contribute to EBV-HLH development.
1202	Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans.	28369379	Twist transcription factors, members of the basic helix-loop-helix family, play crucial roles in mesoderm development in all animals. Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Here, we describe a new clinical entity, Sweeney-Cox syndrome, associated with distinct de novo amino acid substitutions (p.Glu117Val and p.Glu117Gly) at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1, in two subjects with frontonasal dysplasia and additional malformations. Although about one hundred different TWIST1 mutations have been reported in patients with the dominant haploinsufficiency Saethre-Chotzen syndrome (typically associated with craniosynostosis), substitutions uniquely affecting the Glu117 codon were not observed previously. Recently, subjects with Barber-Say and Ablepharon-Macrostomia syndromes were found to harbor heterozygous missense substitutions in the paralogous glutamic acid residue in TWIST2 (p.Glu75Ala, p.Glu75Gln and p.Glu75Lys). To study systematically the effects of these substitutions in individual cells of the developing mesoderm, we engineered all five disease-associated alleles into the equivalent Glu29 residue encoded by hlh-8, the single Twist homolog present in Caenorhabditis elegans. This allelic series revealed that different substitutions exhibit graded severity, in terms of both gene expression and cellular phenotype, which we incorporate into a model explaining the various human disease phenotypes. The genetic analysis favors a predominantly dominant-negative mechanism for the action of amino acid substitutions at this highly conserved glutamic acid residue and illustrates the value of systematic mutagenesis of C. elegans for focused investigation of human disease processes.
1203	Griscelli syndrome subtype 2 with hemophagocytic lympho-histiocytosis: A case report and review of literature.	28357189	Griscelli syndrome (GS) is a rare autosomal recessive disorder resulting in pigmentary dilution of the skin and hair with variable phenotypes depending upon subtypes. Mutations in 3 distinct genes MYO5A, RAB27A, MLPH are responsible for 3 subtypes (GS1, GS2, and GS3) of GS respectively. GS subtype 2 commonly develops hemophagocytic lymphohistiocytosis (HLH) and recurrent infections due to immunodeficiency. We hereby report a 20 month old male child presenting with silvery gray hair, hypomelanosis and features of hemophagocytosis. The diagnosis of a type 2 GS was made in response to a set of clinical features: hypopigmentation of skin and the silvered reflection of the hair, absence of psychomotor retardation, the occurrence of an accelerated phase (hemophagocytosis) and, above all, a pathognomonic appearance by microscopic examination of a hair. The absence of giant granules in the nucleated cells made it possible to eliminate Chediak-Higashi syndrome, which shares a close clinical spectrum with GS. This case promotes awareness about this rare case of GS as a high indicator of suspicion about this potentially fatal condition and aids in prompt diagnosis and foresees complications. Early bone marrow transplant is the only curative treatment for GS-2.
1204	Hemophagocytic lymphohistiocytosis secondary to hemodialysis catheter-related blood stream infection.	28356667	A 57-year-old man on dialysis presented with fever due to Pseudomonas septicemia. Workup revealed very high triglycerides and serum ferritin levels. A bone marrow examination showed hemophagocytosis. A diagnosis of hemophagocytic lymphohistiocytosis (HLH) was made and steroids were started. He was put on automated peritoneal dialysis. Patients' condition continued to deteriorate and he succumbed to his illness. This case illustrates the development of HLH secondary to infections which are increasingly being recognized in the literature. Often this diagnosis is missed as it becomes difficult to differentiate between sepsis and HLH. The presence of high ferritin, hypertriglyceridemia, and hemophagocytosis in the bone marrow confirms the diagnosis.
1205	[The clinical characteristics of adult hemophagocytic lymphohistiocytosis treated with haploidentical donor hematopoietic stem cell transplantation].	28355720	Objective: To analyze the clinical characteristics of adult patients with hemophagocytic lymphohistiocytosis (HLH) receiving haploidentical donor hematopoietic stem cell transplantation (HID HSCT). Method: We retrospectively reviewed 20 adult patients with HLH from August 2009 to August 2014.The clinical features and outcome were analyzed. Results: Conditioning regimens consisted of total body irradiation/etoposide/cyclophosphamide (TBI/VP-16/CTX) and busulfan (Bu)/VP-16/CTX in HLH with anti-thymocyte globulin (ATG) 8 mg/kg.The stem cells were mobilized from donors' peripheral blood.Median time to white blood cell engraftment was 13 (9-27) days.Median time to platelet engraftment was 14 (10-28) days.Mixed chimerism after transplantation developed in 4 patients and no patient presented graft failure.Eight patients developed grade Ⅱ to Ⅲ acute graft-versus-host disease (GVHD), while as chronic GVHD occurred in 9 patients.Among 12 patients with EB virus(EBV) reactivation, 2 patients developed post-transplant lymphoproliferative disorder (PTLD), 7 were suspected as PTLD and 3 were considered as relapse of primary disease.With a median follow-up of 20 months (range: 0.5-108 months) after transplantation, the estimated 2-year overall survival (OS) rate was (60.0±11.0)% in all patients.During the follow-up, 12 patients survived, 8 died including 5 within 100 days after HSCT.Among 5 non-remission patients before HSCT, 4 patients died within 100 days after HCT. Conclusions: HID HSCT is an effective treatment for adult patients with HLH to achieve remission and long-term survival. High proportion of mixed chimerism has been seen at early stage after transplantation.EBV reactivation and early transplant-related mortality are common.目的： 探讨亲缘单倍体造血干细胞移植(HID HSCT)治疗成人噬血细胞性淋巴组织细胞增生症(HLH)的临床特征。 方法： 回顾性分析首都医科大学附属北京友谊医院2009年8月至2014年8月HID HSCT治疗的20例成人HLH的疗效和预后。 结果： 预处理方案包括全身照射/依托泊苷/环磷酰胺和马利兰/依托泊苷/环磷酰胺，抗人胸腺细胞球蛋白剂量为8 mg/kg。干细胞来源均为外周血。中位白细胞植活时间13 (9～27) d，中位血小板植活时间14 (10～28) d。移植后出现混合嵌合状态4例，无病例植入失败。发生Ⅱ～Ⅳ度急性移植物抗宿主病8例，慢性移植物抗宿主病9例。EB病毒(EBV)复燃12例，复燃中位时间33(15～80) d，其中确诊移植后淋巴组织增殖性疾病(PTLD) 2例，疑似PTLD 7例，原发病复发3例。移植后中位随访20 (0.5～108)个月，2年总生存率为(60.0±11.0)%。截至随访结束，存活12例，死亡8例，死于移植后100 d内5例。移植前未获得缓解者5例，4例死亡，均死于移植后100 d内。 结论： HID HSCT治疗成人HLH在移植早期容易发生混合嵌合，EBV复燃发生显著，移植早期相关死亡表现突出。.
1206	A novel frameshift mutation of Chediak-Higashi syndrome and treatment in the accelerated phase.	28355352	Chediak-Higashi syndrome (CHS) is a rare autosomal recessive immunodeficiency disease characterized by frequent infections, hypopigmentation, progressive neurologic deterioration and hemophagocytic lymphohistiocytosis (HLH), known as the accelerated phase. There is little experience in the accelerated phase of CHS treatment worldwide. Here, we present a case of a 9-month-old boy with continuous high fever, hypopigmentation of the skin, enlarged lymph nodes, hepatosplenomegaly and lung infection. He was diagnosed with CHS by gene sequencing, and had entered the accelerated phase. After 8 weeks of therapy, the boy had remission and was prepared for allogenic stem cell transplantation.
1207	Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.	28355222	Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.
1208	Exome sequencing for simultaneous mutation screening in children with hemophagocytic lymphohistiocytosis.	28353193	In the present study, we used exome sequencing to analyze PRF1, UNC13D, STX11, and STXBP2, as well as genes associated with primary immunodeficiency disease (RAB27A, LYST, AP3B1, SH2D1A, ITK, CD27, XIAP, and MAGT1) in Thai children with hemophagocytic lymphohistiocytosis (HLH). We performed mutation analysis of HLH-associated genes in 25 Thai children using an exome sequencing method. Genetic variations found within these target genes were compared to exome sequencing data from 133 healthy individuals. Variants identified with minor allele frequencies <5% and novel mutations were confirmed using Sanger sequencing. Exome sequencing data revealed 101 non-synonymous single nucleotide polymorphisms (SNPs) in all subjects. These SNPs were classified as pathogenic (n = 1), likely pathogenic (n = 16), variant of unknown significance (n = 12), or benign variant (n = 72). Homozygous, compound heterozygous, and double-gene heterozygous variants, involving mutations in PRF1 (n = 3), UNC13D (n = 2), STXBP2 (n = 3), LYST (n = 3), XIAP (n = 2), AP3B1 (n = 1), RAB27A (n = 1), and MAGT1 (n = 1), were demonstrated in 12 patients. Novel mutations were found in most patients in this study. In conclusion, exome sequencing demonstrated the ability to identify rare genetic variants in HLH patients. This method is useful in the detection of mutations in multi-gene associated diseases.
1209	Biophysical characterization of the b-HLH-LZ of ΔMax, an alternatively spliced isoform of Max found in tumor cells: Towards the validation of a tumor suppressor role for the Max homodimers.	28350847	It is classically recognized that the physiological and oncogenic functions of Myc proteins depend on specific DNA binding enabled by the dimerization of its C-terminal basic-region-Helix-Loop-Helix-Leucine Zipper (b-HLH-LZ) domain with that of Max. However, a new paradigm is emerging, where the binding of the c-Myc/Max heterodimer to non-specific sequences in enhancers and promoters drives the transcription of genes involved in diverse oncogenic programs. Importantly, Max can form a stable homodimer even in the presence of c-Myc and bind DNA (specific and non-specific) with comparable affinity to the c-Myc/Max heterodimer. Intriguingly, alterations in the Max gene by germline and somatic mutations or changes in the gene product by alternative splicing (e.g. ΔMax) were recently associated with pheochromocytoma and glioblastoma, respectively. This has led to the proposition that Max is, by itself, a tumor suppressor. However, the actual mechanism through which it exerts such an activity remains to be elucidated. Here, we show that contrary to the WT motif, the b-HLH-LZ of ΔMax does not homodimerize in the absence of DNA. In addition, although ΔMax can still bind the E-box sequence as a homodimer, it cannot bind non-specific DNA in that form, while it can heterodimerize with c-Myc and bind E-box and non-specific DNA as a heterodimer with high affinity. Taken together, our results suggest that the WT Max homodimer is important for attenuating the binding of c-Myc to specific and non-specific DNA, whereas ΔMax is unable to do so. Conversely, the splicing of Max into ΔMax could provoke an increase in overall chromatin bound c-Myc. According to the new emerging paradigm, the splicing event and the stark reduction in homodimer stability and DNA binding should promote tumorigenesis impairing the tumor suppressor activity of the WT homodimer of Max.
1210	Epstein barr virus hemophagocytic lymphohistiocytosis related to rituximab use and immunopathogenetic insights.	28340951	Anti-CD20-based chemo-immunotherapeutic regimens have been suggested to assist in the management of Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis (HLH) and EBV-associated post-transplant lymphoproliferative disorders (EBV-PTLD), by reducing EBV viral load and EBV-induced inflammation. Herein we report a fatal EBV-related HLH in the context of Hodgkin lymphoma (HL)-like Richter's transformation of B chronic lymphocytic leukemia (B-CLL), two months after rituximab treatment. The complex balance between EBV driven T-cell stimulation and immunosuppressive therapy in the context of multiple immune deficits is discussed.
1211	Hormetic heat shock and HSF-1 overexpression improve C. elegans survival and proteostasis by inducing autophagy.	28333578	The cellular recycling process of macroautophagy/autophagy is an essential homeostatic system induced by various stresses, but it remains unclear how autophagy contributes to organismal stress resistance. In a recent study, we report that a mild and physiologically beneficial ("hormetic") heat shock as well as overexpression of the heat-shock responsive transcription factor HSF-1 systemically increases autophagy in C. elegans. Accordingly, we found HSF-1- and heat stress-inducible autophagy to be required for C. elegans thermoresistance and longevity. Moreover, a hormetic heat shock or HSF-1 overexpression alleviated PolyQ protein aggregation in an autophagy-dependent manner. Collectively, we demonstrate a critical role for autophagy in C. elegans stress resistance and hormesis, and reveal a requirement for autophagy in HSF-1 regulated functions in the heat-shock response, proteostasis, and aging.
1212	Interferon gamma and interleukin 10 polymorphisms in Chinese children with hemophagocytic lymphohistiocytosis.	28332776	The aim of the study is to investigate the association of interferon gamma (IFN-γ) and interleukin-10 (IL-10) gene single nucleotide polymorphisms with the susceptibility of hemophagocytic lymphohistiocytosis (HLH) in Chinese children without known family history of HLH.Forty children with HLH and 160 age- and gender-matched healthy controls from Xuzhou Children's Hospital were enrolled in the study. Serum IFN-γ and IL-10 levels were measured by enzyme linked-immunosorbent assay. Polymorphisms of the IFN-γ gene at position +874 and +2109, and IL-10 at position -1082 were analyzed by allele-specific PCR.Median serum concentrations of IFN -γ and IL-10 were significantly higher in children with HLH compared to healthy controls. The frequencies of IFN-γ +874 T/A and T/T genotypes, as well as T allele, were significantly higher in the HLH group compared with those in the control group. The frequencies of IL-10 -1082 G/A genotype and G allele were significantly increased in HLH patients compared with healthy controls. No significant difference was found in the distribution of IFN-γ +2109G/A genotypes between children with HLH and controls.This study presents preliminary evidence for the association between IFN +874 T/A, T/T, IL-10 -1082 A/G genotypes, and HLH susceptibility in Chinese children with HLH.
1213	A Newborn With Familial Hemophagocytic Lymphohistiocytosis Complicated With Transfusion Associated Graft Versus Host Disease.	28328614	Hemophagocytic lymphohistiocytosis (HLH) is characterized by activation of cytotoxic T and natural killer (NK) cells, and macrophages related to a spectrum of hyperinflammatory disorders. The clinical findings mainly include high fever, cytopenia, splenomegaly, phagocytosis, and proliferation of histiocytes in lymphoreticular tissue. To the best of our knowledge, transfusion-associated graft versus host disease (TA-GVHD) in a 13-day old male newborn with HLH is being reported first time in the literature. The aim of this report was to emphasize the importance of blood products irradiation in the prevention of the development of graft versus host disease especially among high-risk subjects such as newborns with HLH.
1214	Hemophagocytic Lymphohistiocytosis, an Unclear Nosologic Entity: Case Report of an Adult Man with Rising of Amylase and Lipase and Spinal Cord Infiltration.	28286628	Here we present the case of a 57-years old patient affected by hemophagocytic lymphohistiocytosis (HLH), a rare disease characterized by an uncontrolled immune activation, resulting in clinical and biochemical manifestations of extreme inflammation. In a previous hospitalization, the patient showed fever, hepato-splenomegaly, pancytopenia, hyperferrtitinemia, lymphadenopathy and cholestasis. No diagnosis was done, however, he totally recovered after splenectomy. Eight months later, he relapsed, showing also hypofibrinogenemia, hypertriglyceridemia, hemophagocytic signs in bone marrow, cholestatic jaundice, high LDH and high PT-INR. Interestingly, he presented increased levels of amylase and lipase in absence of radiologic signs of pancreatitis. He was treated with Dexamethasone and Cyclosporine according to HLH-2004 guidelines. The clinical and biochemical manifestations disappeared in a few weeks, but he was newly hospitalized for lower limbs hypotonia caused by a hemophagocytic lesion of the cauda equina and lumbar cord. The death occurred in a few days, despite the immunosuppressive treatment.
1215	TFE3 regulates whole-body energy metabolism in cooperation with TFEB.	28283651	TFE3 and TFEB are members of the MiT family of HLH-leucine zipper transcription factors. Recent studies demonstrated that they bind overlapping sets of promoters and are post-transcriptionally regulated through a similar mechanism. However, while Tcfeb knockout (KO) mice die during early embryonic development, no apparent phenotype was reported in Tfe3 KO mice. Thus raising the need to characterize the physiological role of TFE3 and elucidate its relationship with TFEB TFE3 deficiency resulted in altered mitochondrial morphology and function both in vitro and in vivo due to compromised mitochondrial dynamics. In addition, Tfe3 KO mice showed significant abnormalities in energy balance and alterations in systemic glucose and lipid metabolism, resulting in enhanced diet-induced obesity and diabetes. Conversely, viral-mediated TFE3 overexpression improved the metabolic abnormalities induced by high-fat diet (HFD). Both TFEB overexpression in Tfe3 KO mice and TFE3 overexpression in Tcfeb liver-specific KO mice (Tcfeb LiKO) rescued HFD-induced obesity, indicating that TFEB can compensate for TFE3 deficiency and vice versa Analysis of Tcfeb LiKO/Tfe3 double KO mice demonstrated that depletion of both TFE3 and TFEB results in additive effects with an exacerbation of the hepatic phenotype. These data indicate that TFE3 and TFEB play a cooperative, rather than redundant, role in the control of the adaptive response of whole-body metabolism to environmental cues such as diet and physical exercise.
1216	Influence of hydrolysis behaviour and microfluidisation on the functionality and structural properties of collagen hydrolysates.	28274424	The functionality and structural properties of pig skin hydrolysates with different degrees of hydrolysis (DH, 10% and 20%) and microfluidisation (120MPa), prepared by pepsin and Alcalase® have been investigated in this study. Extensive hydrolysis can significantly improve the absolute value of the zeta potential and surface hydrophobicity. The particle distribution of hydrolysates decreased with increasing DH. The numbers of free sulfhydryl (SH) and disulfide bonds (SS) were significantly increased with increasing DH (p<0.05). Hydrolysates with a lower DH showed a better emulsifying property than those with a higher DH. Microfluidisation led to the transformation of structural and interfacial properties of the hydrolysates and increased the value of the zeta potential, S0, and gel strength. Microfluidisation results in limited breakage of chemical bonds, the number of SS and SH bonds unchanged in the treatment. These results reflect the functionality and structural properties of collagen-rich pig skin hydrolysates.
1217	Perforin and CD107a testing is superior to NK cell function testing for screening patients for genetic HLH.	2827045428572174	Primary hemophagocytic lymphohistiocytosis (HLH) can be caused by biallelic mutations in PRF1, encoding perforin, or UNC13D, STXBP2, STX11, RAB27A, LYST, and AP3B1, encoding proteins involved in cytotoxic lymphocyte degranulation. Natural killer (NK)-cell cytotoxicity assays can quickly screen for all of these genetic diseases, facilitating treatment, but combining NK-cell perforin expression and CD107a upregulation tests can as well. To determine the relative diagnostic accuracies for each approach, we retrospectively reviewed screening test performance in 1614 patients referred for HLH evaluation. For each test, we generated a receiver operating characteristic (ROC) curve, and calculated area under the curve (AUC) and diagnostic parameters at optimal threshold. We generated an AUC for combining perforin and CD107a tests by creating a logistic regression model and applying model-generated coefficients to patient values. Sensitivities of NK-cell function, perforin mean channel fluorescence (MCF), and CD107a MCF to detect biallelic mutations were 59.5%, 96.6%, and 93.8%, with specificities of 72.0%, 99.5%, and 73%. AUCs for NK-cell cytotoxicity, perforin MCF, CD107a MCF, and combined perforin and CD107a MCFs were 0.690, 0.971, 0.860, and 0.838. Perforin and CD107a tests are more sensitive and no less specific compared with NK cytotoxicity testing for screening for genetic HLH and should be considered for addition to current HLH criteria.
1218	Rescue of Cytokine Storm Due to HLH by Hemoadsorption in a CTLA4-Deficient Patient.	28265964	NaN
1219	Infection associated haemophagocytic syndrome in severe dengue infection - a case series in a district hospital.	28255145	Haemophagocytic lymphohistiocytosis (HLH) is a potentially fatal disorder resulting from uncontrolled hyperinflammatory response. There had been increase in cases of one of the secondary form of HLH, i.e., infectionassociated haemophagocytic syndrome (IAHS) in severe dengue in recent years. However, the condition remains under diagnosed due to lack of awareness compounded by the lack of validated diagnostic criteria. Severe hepatitis with prolonged cytopenias, severe hyperferritinemia, hypofibrinogenemia and persistent fever were evident in all four cases reported. All the subjects survived with supportive care and adjuvant steroid therapy. Prospective controlled studies are needed to develop diagnostic criteria and management protocol for IAHS in severe dengue.
1220	Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52.	28252832	Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant-negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52-kDa FK506-binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52-dependent AR transcriptional activity was observed in L(-)ID4 cells. Moreover, pharmacological inhibition of FKBP52-AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(-)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling.
1221	Dynamic changes of laboratory parameters and peripheral blood lymphocyte subsets in severe fever with thrombocytopenia syndrome patients.	28249810	The aim of this study was to dynamically investigate laboratory parameters and peripheral blood lymphocyte subsets in severe fever with thrombocytopenia syndrome (SFTS) patients at different stages, to evaluate the significance of these changes in the infection process and its influence on prognosis.Case-control study was used in the research. Sixty-nine confirmed thrombocytopenia syndrome virus(SFTSV) infected patients were enrolled. They were divided into two groups, recovery group and poor prognosis group, according to the clinical prognosis of the diseases. The laboratory parameters were measured by matched fully-automatic detector. The dynamic lymphocyte subsets of each group were tested by flow cytometry. Independent-group Student's t-test, Bonferroni test and Nemenyi test were used to compare the mean value of every group.The clinical manifestations typically became worse on about the 7th day. Most of them had multi organ dysfunction, and part of them had hemophagocytic lymphohistiocytosis histiocytosis (HLH). The characteristic laboratory findings in the early stage were the drop of platelets (PLT), while the increase of alanine aminotransferase (ALT), aspartate amino transferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH). SFTSV viral loads reached the highest on Days 7-10 after onset of fever in SFTS patients. CD3+, CD3+CD4+ T cell counts were significantly reduced in poor prognosis group, more so on Days 7-10 after onset of fever. CD3-CD19+ (B cell) counts in SFTS patients were significantly higher than that of healthy controls. 11 days after illness onset, symptoms were improved, accompanied by resolution of laboratory abnormalities.These results indicated that SFTS had an acute onset and self-limited course. It was a systemic infection. The host immune response caused tissues and organs injury. The improvement of symptoms and laboratory tests was consistent with the elimination of the virus and recover of immune response. Further investigation should be done in order to reveal the mechanisms of SFTSV pathogenesis and guide the clinical treatment.
1222	The CDK8 Complex and Proneural Proteins Together Drive Neurogenesis from a Mesodermal Lineage.	28238659	At least some animal species can generate neurons from mesoderm or endoderm, but the underlying mechanisms remain unknown. We screened for C. elegans mutants in which the presumptive mesoderm-derived I4 neuron adopts a muscle-like cell fate. From this screen, we identified HLH-3, the C. elegans homolog of a mammalian proneural protein (Ascl1) used for in vitro neuronal reprogramming, as required for efficient I4 neurogenesis. We discovered that the CDK-8 Mediator kinase module acts together with a second proneural protein, HLH-2, and in parallel to HLH-3 to promote I4 neurogenesis. Genetic analysis revealed that CDK-8 most likely promotes I4 neurogenesis by inhibiting the CDK-7/CYH-1 (CDK7/cyclin H) kinase module of the transcription initiation factor TFIIH. Ectopic expression of HLH-2 and HLH-3 together promoted expression of neuronal features in non-neuronal cells. These findings reveal that the Mediator CDK8 kinase module can promote non-ectodermal neurogenesis and suggest that inhibiting CDK7/cyclin H might similarly promote neurogenesis.
1223	Crohn's disease complicated by Epstein-Barr virus-driven haemophagocytic lymphohistiocytosis successfully treated with rituximab.	28228435	We report a case of Epstein-Barr virus (EBV)-driven haemophagocytic lymphohistiocytosis (HLH) in a man with Crohn's disease treated with 6-mercaptopurine and adalimumab therapy who was successfully treated with rituximab therapy alone. This is the first published case in an adult patient with EBV-driven HLH in the setting of thiopurine use and inflammatory bowel disease to be successfully treated with rituximab therapy alone. Here, we will discuss putative immunological mechanisms which may contribute to this potentially life-threatening complication.
1224	Dengue-associated hemophagocytic lymphohistiocytosis in an adult: A case report and literature review.	2822549831305642	Infection-associated hemophagocytic syndrome (IAHS) is potentially a fatal disease caused by systemic infection complicated by hemophagocyticlymphohistiocytosis (HLH). Here, we report a case of HLH associated with dengue hemorrhagic fever (DHF) after a trip to Thailand.A 33-year-old healthy female patient presented with 3 days of fever, myalgia, and skin rash. Serotype 3 dengue virus was isolated. Clinical and laboratory findings fulfilled the criteria of HLH. After the initiation of corticosteroid therapy, the patient recovered and laboratory findings were normalized.It would be important to differentially diagnose dengue-associated HLH from severe DHF. Early recognition and initiation of steroid treatment would be crucial for the successful treatment of dengue fever complicated by HLH.
1225	Is coronary arteritis a feature in secondary haemophagocytic lymphohistiocytosis?	28222654	Haemophagocytic lymphohistiocytosis (HLH) is rare. Although Kawasaki disease (KD) has been reported as a precursor to HLH, coronary arteritis occurring at the onset of secondary HLH, not in association with KD, has not been reported. An 8-year-old girl presented with virus-induced secondary HLH associated with a giant aneurysm and ectasia of the coronary arteries which was detected incidentally at onset of the disease. She did not fulfill the criteria for diagnosis of KD. The coronary lesions improved after 6 months of treatment with dexamethasone and etoposide. Echocardiography early in the course of HLH is a useful tool to detect the unusual finding of coronary arteritis which may carry significant clinical sequelae.
1226	Cytokine profiles as novel diagnostic markers of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children.	28222313	The aim of this study was to identify specific laboratory indices to distinguish Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) in children.In this prospective study, Th1/Th2 cytokines, including IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ, were analyzed in patients with EBV-HLH or sepsis at the onset of disease by flow cytometry.IL-10, IFN-γ, IL-10/IL-6, and IFN-γ/IL-6 were higher and IL-6 was lower in EBV-HLH patients compared to sepsis patient levels. When using the criteria of IL-10 >20.9 pg/mL, IFN-γ >17.9 pg/mL, IL-10/IL-6 >0.5 and IFN-γ/IL-6 >0.7, the sensitivity was 89.8%, 93.2%, 93.2%, and 91.5%, while the specificity was 89.8%, 100%, 94.9%, and 100%, respectively. After treatment of EBV-HLH patients, IL-6, IL-10, TNF-α, and IFN-γ were significantly reduced (IL-6: P<.001; IL-10: P<.001; TNF-α: P=.011; IFN-γ: P<.001).This study showed that IFN-γ, IL-10/IL-6, and IFN-γ/IL-6 are novel specific indicators for differential diagnosis of EBV-HLH. Additionally, IL-6, IL-10, TNF-α, and IFN-γ are useful indices for monitoring the effects of treatment on EBV-HLH.
1227	Incidence and Outcomes of Central Nervous System Hemophagocytic Lymphohistiocytosis Relapse after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation.	28219834	Hemophagocytic lymphohistiocytosis (HLH) is an immune regulatory disorder that commonly presents with central nervous system (CNS) involvement. The only cure for genetic HLH is hematopoietic stem cell transplantation (HSCT), typically treated with reduced-intensity conditioning (RIC) regimens. We sought to estimate the incidence of CNS relapse after RIC HSCT, determine risk factors, and evaluate outcomes. We performed a retrospective chart review of 94 consecutive children and young adults with primary HLH who received RIC HSCT. CNS relapse within 1 year after transplantation was diagnosed by review of clinical symptoms, cerebral spinal fluid (CSF), and radiologic findings. Four (4.25%) patients developed symptoms of possible CNS HLH after HSCT and 3 patients were diagnosed. Eight patients underwent screening lumbar puncture because of history of active CNS disease at the onset of the conditioning regimen and 4 had evidence of continued disease. The overall incidence of CNS relapse and continued CNS disease after RIC HSCT was 8%. All patients with CNS disease after HSCT responded to CNS-directed therapy. Whole blood donor chimerism at the time of CNS relapse was low at 1% to 34%, but it remained high at 88% to 100% for patients with continued CNS disease. Overall survival for patients with CNS relapse was 50%, compared with 75% for patients without CNS disease (P = .079). Our data suggest that a low level of donor chimerism or active CNS disease at the time of transplantation increase the risk of CNS HLH after HSCT. Surveillance CSF evaluation after allogeneic RIC HSCT should be considered in patients with risk factors and CNS-directed treatment should be initiated if appropriate.
1228	Living-Donor Lung Transplantation After Bone Marrow Transplantation for Chediak-Higashi Syndrome.	28219570	An 8-year-old girl with Chediak-Higashi syndrome (CHS) had pulmonary complications after hematopoietic stem cell transplantation (HSCT) for hemophagocytic lymphohistiocytosis (HLH) and eventually underwent single living-donor lobar lung transplantation (LDLLT). Electron micrographic findings showed vagus nerve tissue in extracted lung having granular inclusions, which are pathognomonic for CHS. Because her mother was the donor for both hematopoietic stem cell and lung transplantations, she was weaned from immunosuppression and is doing well 3 years after lung transplantation. Furthermore, an induced pluripotent stem (iPS) cell line was established from her skin fibroblasts for investigation and potential future treatment for CHS.
1229	Hemophagocytic Lymphohistiocytosis in a Patient With Hodgkin lymphoma and Concurrent EBV, CMV, and Candida Infections.	28210636	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by immune activation and subsequent widespread organ damage. Patients affected by HLH commonly develop fever, cytopenias, liver damage, neurologic manifestations, and hypercytokinemia. In this case, we describe a 60-year-old male who presented with HLH and concurrent Epstein-Barr virus, cytomegalovirus, and Candida infections and was subsequently diagnosed with a Hodgkin lymphoma. This case highlights the importance of considering a cancer diagnosis in the differential diagnosis of patients presenting with HLH.
1230	Hematologic manifestations of babesiosis.	28202022	Babesiosis, a zoonotic parasitic infection transmitted by the Ixodes tick, has become an emerging health problem in humans that is attracting attention worldwide. Most cases of human babesiosis are reported in the United States and Europe. The disease is caused by the protozoa of the genus Babesia, which invade human erythrocytes and lyse them causing a febrile hemolytic anemia. The infection is usually asymptomatic or self-limited in the immunocompetent host, or follows a persistent, relapsing, and/or life threatening course with multi-organ failure, mainly in the splenectomized or immunosuppressed patients. Hematologic manifestations of the disease are common. They can range from mild anemia, to severe pancytopenia, splenic rupture, disseminated intravascular coagulopathy (DIC), or even hemophagocytic lymphohistiocytosis (HLH).A 70 year old immunocompetent female patient living in New York City presented with a persistent fever, night sweats, and fatigue of 5 days duration. Full evaluation showed a febrile hemolytic anemia along with neutropenia and thrombocytopenia. Blood smear revealed intraerythrocytic Babesia, which was confirmed by PCR. Bone marrow biopsy was remarkable for dyserythropoiesis, suggesting possible HLH, supported by other blood workup meeting HLH-2004 trial criteria.Human babesiosis is an increasing healthcare problem in the United States that is being diagnosed more often nowadays. We presented a case of HLH triggered by Babesia microti that was treated successfully. Also, we presented the hematologic manifestations of this disease along with their pathophysiologies.
1231	Characterization of a novel disease-causing mutation in exon 1 of SH2D1A gene through amplicon sequencing: a case report on HLH.	28196537	Hemophagocytic lymphohistocytosis (HLH) is a rare but fatal hyperinflammatory syndrome caused by uncontrolled proliferation of activated macrophages and T lymphocytes secreting high amounts of inflammatory cytokines. Genetic defect is a common cause of HLH. HLH is complicated to be diagnosed as there are many common symptoms with other disorders.Here we report on an HLH case caused by 1 bp deletion in gene SH2D1A. Patient was a 3-years-old boy and had fever for more than 8 days. Splenomegaly and hemophagocytosis in bone marrow were observed in examination. The results of the blood analysis suggested the diagnosis of HLH. Genetic test based on high throughput amplicon sequencing was then conducted by targeting all six known HLH-causing genes simultaneously. It took only one single day to accomplish the amplicon sequencing library preparation, sequencing and data analysis. Finally, a novel 1 bp deletion in gene SH2D1A was discovered. The result was also confirmed by Sanger sequencing. The result of the genetic test served as a good basis for further diagnosis of HLH.This is the first case that the disease-causing genetic defect of HLH was quickly determined by high throughput amplicon sequencing. This diagnosis was also confirmed by Sanger sequencing and cross-validated by blood analysis and other clinical criteria. This case suggests that genetic test based on amplicon sequencing is a powerful tool for diagnosis of HLH and other diseases caused by genetic defect.
1232	Infliximab Is Not Associated With Increased Risk of Malignancy or Hemophagocytic Lymphohistiocytosis in Pediatric Patients With Inflammatory Bowel Disease.	281935152888118528881189	Immunosuppressive therapy for inflammatory bowel disease (IBD) in pediatric patients is thought to increase the risk of malignancy and lymphoproliferative disorders, including hemophagocytic lymphohistiocytosis (HLH). We compared unadjusted incidence rates of malignancy and HLH in pediatric patients with IBD exposed to infliximab (IFX) with patients not exposed to biologics and calculated standardized incidence ratios (SIRs).We collected and analyzed data from 5766 participants in a prospective study of long-term outcomes of pediatric patients with IBD (NCT00606346), from May 31, 2007 through June 30, 2016. Patients were 17 years old or younger and had Crohn's disease, ulcerative colitis, or IBD-unclassified with 24,543.0 patient-years of follow-up. We estimated incidence rates for malignancy and HLH as events/1000 patient-years of follow-up. We calculated age-, sex-, and race-adjusted SIRs, with 95% confidence intervals (CIs), using the Surveillance, Epidemiology, and End Results Program (SEER) database.Thirteen of the 15 patients who developed a malignancy and all 5 of the patients who developed HLH had been exposed to thiopurines; 10 patients with malignancy had also been exposed to a biologic agent. Unadjusted incidence rates showed no increased risk of malignancy (0.46/1000 patient-years) or HLH (0.0/1000 patient-years) in patients exposed to IFX as the only biologic vs those unexposed to biologics (malignancy: 1.12/1000 patient-years; HLH: 0.56/1000 patient-years). SIRs did not demonstrate an increased risk of malignancy among patients exposed to IFX (SIR, 1.69; 95% CI, 0.46-4.32) vs patients not exposed to a biologic agent (SIR, 2.17; 95% CI, 0.59-5.56), even when patients were stratified by thiopurine exposure.In determination of age-, sex-, and race-adjusted SIRs using data from a large clinical study and the SEER database, we found that IFX exposure did not associate with increased risk of malignancy or HLH in pediatric patients with IBD. Thiopurine exposure is an important precedent event for the development of malignancy or HLH in pediatric patients with IBD.
1233	Leishmania Infantum and Epstein-Barr Virus Co-Infection in a Patient with Hemophagocytosis.	28191297	The authors describe a rare case of a 27- year old previously healthy male presenting with high grade fever, pancytopenia, hepatosplenomegaly, high levels of ferritin and triglyceride, suggesting a diagnosis of hemophagocytic lymphohistiocytosis (HLH) syndrome. Other investigations showed a positive Leishmania infantum serology and high Epstein-Barr virus (EBV) viremia. The diagnosis of a visceral leishmaniasis was confirmed by bone morrow biopsy, which showed Leishman-Donovan bodies and evidence of HLH. The patient received liposomal amphotericin B and he had a complete resolution of his symptoms and clearance of EBV viremia. This case of HLH associated with visceral leishmaniasis and EBV co-infection raises the question about the significance of EBV in patients with HLH. The treatment of actual etiological agent can lead to complete cure while using current recommend chemotherapy for HLH-related EBV in a patient with hidden infection may have deleterious effects.
1234	Hemophagocytic Lymphohistiocytosis in a Neonate: Case Report.	28188950	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of pathologic immune activation, occurring as either a familial disorder or a sporadic condition, in association with a variety of triggers. This article will introduce a neonate with HLH in Iran. We report a case of HLH presenting with respiratory distress and fever, hepatosplenomegaly, jaundice and pancytopenia on the second day of life. Typical clinical and laboratory findings were detected in the neonate. HLH was diagnosed according to HLH-2004 guidelines. In spite of initiating the treatment, the disease did not cure. Post-mortem, extensive hemophagocytosis was found in multiple organs. No specific genetic defect was identified. Since HLH is a potentially lethal childhood illness, early diagnosis of this disorder and commences the therapy is important for pediatricians.
1235	Diagnostic dilemmas in HLH: Can T-cell phenotyping help?	2818525227925643	The diagnosis of hemophagocytic lymphohistiocytosis (HLH) can be a difficult one, and the distinction between primary versus secondary HLH can be particularly challenging during the early stages of diagnosis. This distinction is important to make as primary HLH requires allogeneic hematopoietic cell transplantation for a definitive cure. Flow cytometric screening tests for many of the genetic forms of HLH are available. However, not all patients with primary HLH are captured by these screening tests, due to the fact that no screening test is 100% sensitive, and additionally, some patients with "primary" forms of HLH may have mutations in genes which are yet to be discovered. In this issue of the European Journal of Immunology, Ammann et al. [Eur. J. Immunol. 2017. 47: 364-373] compare T-cell phenotype patterns among patients with primary and secondary HLH, and find that assessment of T-cell activation and differentiation may assist with the diagnosis of HLH. Furthermore, this phenotypic analysis has the potential to help make the important distinction between primary and secondary HLH.
1236	Application of an improved flow cytometry-based NK cell activity assay in adult hemophagocytic lymphohistiocytosis.	28185204	Low or absent natural killer (NK) cell activity is included as one of the HLH-2004 diagnostic criteria. To improve the diagnosis of HLH, we aimed to establish a rapid and reliable NK cell activity assay that avoids the use of radioactivity. The K562 cell line, as standard NK target cells, was engineered to stably express enhanced green fluorescent protein (EGFP), which can be quantified by flow cytometry. The EGFP-flow cytometry method for measuring NK cell activity was improved by double staining of early and late apoptotic target cells. Whole-blood samples from healthy volunteers were assessed with this method, which demonstrated that optimal conditions were effector-target ratio of 10:1 and incubation time of 4 h. This method was further evaluated for samples from 113 HLH patients and 64 healthy volunteers. Mean NK cell activity in either primary or secondary HLH patients was significantly lower (P < 0.001) than in healthy individuals (20.23 ± 4.12%). Furthermore, primary HLH patients (10.76 ± 2.54%) exhibited even lower (P < 0.001) NK cell activity compared with secondary HLH patients (15.01 ± 3.62%). We have optimized and implemented this method in clinically relevant samples.
1237	"Hemophagocytic Lymphohistiocytosis after EBV reactivation and ibrutinib treatment in relapsed/refractory Chronic Lymphocytic Leukemia".	28180067	Hemophagocytic Lymphohistiocytosis (HLH) is a rare syndrome characterized by ineffective T-cell and NK response. We report the clinical course of a patient with relapsed CLL who developed acute symptoms soon after starting ibrutinib. Hyperpyrexia, splenomegaly, hyperferritinemia, hypertriglyceridemia, cytopenias, and a typical cytokine pattern, i.e. high interleukin (IL)-6, IL10 and IL18, were consistent with a diagnosis of HLH. Coexistent Epstein Barr virus reactivation was documented. Ibrutinib-induced impairment of NK degranulation, associated with EBV reactivation and CLL-related immunodeficiency may have contributed to the development of HLH in our patient.
1238	Role of Skin Biopsies in the Diagnosis of Hemophagocytic Lymphohistiocytosis.	28178007	This is a report of a 15-year-old female who presented with a skin eruption and clinical features consistent with severe sepsis and septic shock. A diagnosis of acquired hemophagocytic lymphohistiocytosis (HLH) was made and confirmed by bone marrow analysis. Skin biopsy showed hemophagocytosis and CD163-positive staining of macrophages. We briefly review the clinical, histologic, and laboratory findings of hemophagocytic lymphohistiocytosis in addition to the potential role of skin biopsies in this condition.
1239	HAX1 mutation positive children presenting with haemophagocytic lymphohistiocytosis.	28169428	The genetic basis of haemophagocytic lymphohistiocytosis (HLH) has not been elucidated in 10% of affected patients. In this study, we report four HLH episodes in three patients with HAX1 gene mutations. We screened the mutations associated with congenital neutropenia (CN) because the neutropenia persisted following HLH treatment. There were homozygous HAX1 mutations detected in all patients. This is the first case series of patients with CN caused by HAX1 mutation who presented with HLH. We hypothesize that severe neutropenia persists after an HLH episode in children without HLH mutations (especially infants) because these patients have CN caused by HAX1 mutations.
1240	[Correctly adDRESS the cause of hemophagocytic lymphohistiocytosis].	28161232	Hemophagocytic lymphohistiocytosis (HLH) is a rare and severe syndrome usually associated with a cytotoxicity deficiency, which leads to an excess of immune response driven by activated macrophages and cytotoxic T cells. In children, HLH can be genetic, as part of a familial lymphohistiocytosis, or secondary: the most frequent causes are systemic-onset juvenile idiopathic arthritis, hematological malignancies, and severe infections, especially with Ebstein-Barr virus or leishmaniosis. We report on the case of a 3-year-old girl with no past medical history, who presented inaugural Pseudomonas aeruginosa maxillary osteitis, with secondary HLH. The rarity of this osteitis, the characteristics of the pathogen, and the onset of HLH oriented the diagnosis toward primary immunodeficiencies, malignancies, or systemic diseases. Steroids were initiated at 2mg/kg/day and were very effective in improving the systemic symptoms. Antibiotic therapy was continued unchanged. A few days after discontinuation of steroids, while the patient was still under antibiotics, she presented with erythroderma. Skin biopsy revealed eosinophil infiltrate in line with the diagnosis of a drug reaction with eosinophilia and systemic symptoms (DRESS), even though we only observed very transient eosinophilia, up to 0.98G/L, during HLH. Stopping antibiotics normalized the symptoms without using systemic corticosteroids. Patch tests confirmed an allergy to piperacillin. These atypical manifestations of DRESS underline that causative diagnosis of HLH is challenging, and DRESS syndrome should be considered.
1241	How to Treat Involvement of the Central Nervous System in Hemophagocytic Lymphohistiocytosis?	28155064	Central nervous system (CNS)-hemophagocytic lymphohistiocytosis (HLH) is not a disease in itself, but it is part of a systemic immune response. The vast majority of patients with CNS-HLH also have systemic HLH and a large number of patients with primary and secondary HLH have CNS involvement. Reactivations within the CNS are frequent during the course of HLH treatment and may occur concomitant with or independent of systemic relapses. It is also important to consider primary HLH as an underlying cause of "unknown CNS inflammation" as these patients may present with only CNS disease. To initiate proper treatment, a correct diagnosis must be made. A careful review of the patient's history and a thorough neurological examination are essential. In addition to the blood tests required to make a diagnosis of HLH, a lumbar puncture with cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI) should always be done in all cases regardless of the presence or absence of neurological signs or symptom. Treatment options for CNS-HLH include, but are not limited to, those commonly used in systemic HLH, including corticosteroids, etoposide, cyclosporine A, alemtuzumab, and ATG. In addition, intrathecal treatment with methotrexate and corticosteroids has become a standard care and is likely to be beneficial. Therapy must be initiated without inappropriate delay to prevent late effects in HLH. An interesting novel approach is an anti-IFN-gamma antibody (NI-0501), which is currently being tested. Hematopoietic stem cell transplantation (HSCT) also represents an important CNS-HLH treatment; patients with primary HLH may benefit from immediate HSCT even if there is active disease at time of transplantation, though care should be taken to monitor CNS inflammation through HSCT and treat if needed. Since CNS-HLH is a condition leading to the most severe late effects of HLH, early expert consultation is recommended.
1242	Extremely High Ferritinemia Associated with Haemophagocytic Lympho Histiocytosis (HLH).	28149024	Hyperferritinemia (>10,000 ng/ml) is an important hallmark used as an indicator of infection triggered macrophage activation syndrome leading to hemophagocytic lympho histiocytosis (HLH). Measurement of serum ferritin can be used in diagnosis as well as disease monitoring indicator and prognosis related to HLH, cAPS, sepsis, neoplasm and inflammatory conditions. It is a major contributor to manage critically ill patients as predicting and monitoring indicator. It can be used as acute phase response in conditions of MAS, AOSD, cAPS etc. A case study in our hospital showed extremely high ferritin values along with low hemoglobin, elevated LDH and triglycerides with positive MRSA in sputum culture and macrophage proliferation and hemophagocytosis in the bone marrow examination. Patient showed definite inverse relation with steroid therapy and serum ferritin levels.
1243	Pho4 Is Essential for Dissemination of Cryptococcus neoformans to the Host Brain by Promoting Phosphate Uptake and Growth at Alkaline pH.	28144629	Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signaling pathway. Cryptococcus neoformans disseminates from the lung to the brain and is the commonest cause of fungal meningitis worldwide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we characterized a transcription factor knockout strain (hlh3Δ/pho4Δ) defective in phosphate acquisition. Despite little similarity with other fungal Pho4 proteins, Hlh3/Pho4 functioned like a typical phosphate-responsive transcription factor in phosphate-deprived cryptococci, accumulating in nuclei and triggering expression of genes involved in phosphate acquisition. The pho4Δ mutant strain was susceptible to a number of stresses, the effect of which, except for alkaline pH, was alleviated by phosphate supplementation. Even in the presence of phosphate, the PHO pathway was activated in wild-type cryptococci at or above physiological pH, and under these conditions, the pho4Δ mutant had a growth defect and compromised phosphate uptake. The pho4Δ mutant was hypovirulent in a mouse inhalation model, where dissemination to the brain was reduced dramatically, and markedly hypovirulent in an intravenous dissemination model. The pho4Δ mutant was not detected in blood, nor did it proliferate significantly when cultured with peripheral blood monocytes. In conclusion, dissemination of infection and the pathogenesis of meningitis are dependent on cryptococcal phosphate uptake and stress tolerance at alkaline pH, both of which are Pho4 dependent. IMPORTANCE Cryptococcal meningitis is fatal without treatment and responsible for more than 500,000 deaths annually. To be a successful pathogen, C. neoformans must obtain an adequate supply of essential nutrients, including phosphate, from various host niches. Phosphate acquisition in fungi is regulated by the PHO signaling cascade, which is activated when intracellular phosphate decreases below a critical level. Induction of phosphate acquisition genes leads to the uptake of free phosphate via transporters. By blocking the PHO pathway using a Pho4 transcription factor mutant (pho4Δ mutant), we demonstrate the importance of the pathway for cryptococcal dissemination and the establishment of brain infection in murine models. Specifically, we show that reduced dissemination of the pho4Δ mutant to the brain is due to an alkaline pH tolerance defect, as alkaline pH mimics the conditions of phosphate deprivation. The end result is inhibited proliferation in host tissues, particularly in blood.
1244	Efficacy and safety of follitropin alfa/lutropin alfa in ART: a randomized controlled trial in poor ovarian responders.	2813775428541398	How does the efficacy and safety of a fixed-ratio combination of recombinant human FSH plus recombinant human LH (follitropin alfa plus lutropin alfa; r-hFSH/r-hLH) compare with that of r-hFSH monotherapy for controlled ovarian stimulation (COS) in patients with poor ovarian response (POR)?The primary and secondary efficacy endpoints were comparable between treatment groups and the safety profile of both treatment regimens was favourable.Although meta-analyses of clinical trials have suggested some beneficial effect on reproductive outcomes with r-hLH supplementation in patients with POR, the definitions of POR were heterogeneous and limit the comparability across studies.Phase III, single-blind, active-comparator, randomized, parallel-group clinical trial. Patients were followed for a single ART cycle. A total of 939 women were randomized (1:1) to receive either r-hFSH/r-hLH or r-hFSH. Randomization, stratified by study site and participant age, was conducted via an interactive voice response system.Women classified as having POR, based on criteria incorporating the ESHRE Bologna criteria, were down-regulated with a long GnRH agonist protocol and following successful down-regulation were randomized (1:1) to COS with r-hFSH/r-hLH or r-hFSH alone. The primary efficacy endpoint was the number of oocytes retrieved following COS. Safety endpoints included the incidence of adverse events, including ovarian hyperstimulation syndrome (OHSS). Post hoc analyses investigated safety outcomes and correlations between live birth and baseline characteristics (age and number of oocytes retrieved in previous ART treatment cycles or serum anti-Müllerian hormone (AMH)). The significance of the treatment effect was tested by generalized linear models (Poisson regression for counts and logistic regression for binary endpoints) adjusting for age and country.Of 949 subjects achieving down-regulation, 939 were randomized to r-hFSH/r-hLH (n = 477) or r-hFSH (n = 462) and received treatment. Efficacy assessment: In the intention-to-treat (ITT) population, the mean (SD) number of oocytes retrieved (primary endpoint) was 3.3 (2.71) in the r-hFSH/r-hLH group compared with 3.6 (2.82) in the r-hFSH group (between-group difference not statistically significant). The observed difference between treatment groups (r-hFSH/r-hLH and r-hFSH, respectively) for efficacy outcomes decreased over the course of pregnancy (biochemical pregnancy rate: 17.3% versus 23.9%; clinical pregnancy rate: 14.1% versus 16.8%; ongoing pregnancy rate: 11.0% versus 12.4%; and live birth rate: 10.6% versus 11.7%). An interaction (identified post hoc) between baseline characteristics related to POR and treatment effect was noted for live birth, with r-hFSH/r-hLH associated with a higher live birth rate for patients with moderate or severe POR, whereas r-hFSH was associated with a higher live birth rate for those with mild POR. A post hoc logistic regression analysis indicated that the incidence of total pregnancy outcome failure was lower in the r-hFSH/r-hLH group (6.7%) compared with the r-hFSH group (12.4%) with an odds ratio of 0.52 (95% CI 0.33, 0.82; P = 0.005). Safety assessment: The overall proportion of patients with treatment-emergent adverse events (TEAEs) occurring during or after r-hFSH/r-hLH or r-hFSH use (stimulation or post-stimulation phase) was 19.9% and 26.8%, respectively. There was no consistent pattern of TEAEs associated with either treatment.Despite using inclusion criteria for POR incorporating the ESHRE Bologna criteria, further investigation is needed to determine the impact of the heterogeneity of POR in the Bologna patient population. The observed correlation between baseline clinical characteristics related to POR and live birth rate, as well as the observed differences between groups regarding total pregnancy outcome failure were from post hoc analyses, and the study was not powered for these endpoints. In addition, the attrition rate for pregnancy outcomes in this trial may not reflect general medical practice. Furthermore, as the patient population was predominantly White these results might not be applicable to other ethnicities.In the population of women with POR investigated in this study, although the number of oocytes retrieved was similar following stimulation with either a fixed-ratio combination of r-hFSH/r-hLH or r-hFSH monotherapy, post hoc analyses showed that there was a lower rate of total pregnancy outcome failure in patients receiving r-hFSH/r-hLH, in addition to a higher live birth rate in patients with moderate and severe POR. These findings are clinically relevant and require additional investigation. The benefit:risk balance of treatment with either r-hFSH/r-hLH or r-hFSH remains positive.This study was funded by Merck KGaA, Darmstadt, Germany. P.H. has received honoraria for lectures and unrestricted research grants from Ferring, Merck KGaA and MSD. D.R. is a former employee of EMD Serono, a business of Merck KGaA, Darmstadt, Germany. J.S., J.H. and W.C. are employees of EMD Serono Research and Development Institute, a business of Merck KGaA, Darmstadt, Germany. T.D.'H. and S.L. are employees of Merck KGaA, Darmstadt, Germany.ClinicalTrials.gov identifier: NCT02047227; EudraCT Number: 2013-003817-16.ClinicalTrials.gov: 24 January 2014; EudraCT: 19 December 2013.30 January 2014.
1245	Carbazole-based N4-donor Schiff base macrocycles: obtained metal free and as Cu(ii) and Ni(ii) complexes.	28127594	The very different multi-step routes to the closely related pair of diformyl-carbazole head units, 1,8-diformyl-3,6-di-tert-butyl-9H-carbazole (1tBu) and 1,8-diformyl-9H-carbazole (1H), are detailed and compared. The first examples of Schiff base macrocycles derived from diformyl-carbazole head units are reported. Specifically, the direct cyclisation of 1tBu or 1H with diethylenetriamine gives the two metal-free [1 + 1] Schiff base macrocycles HLH and HLtBu in high yields. Four carbazole-based macrocyclic complexes, [CuIIL(OH2)]OAc and [NiIIL]OAc, where L = LH or LtBu, were accessed either by metallation of these macrocycles, or by metal templated reaction of the macrocycle components. [CuIILtBu(OH2)]OAc·0.5(Ether) and [NiLH]OAc·EtOH, were structurally characterised, confirming the nickel(ii) complexes are square planar (both show diamagnetic NMR spectra) and that the copper(ii) complexes are square pyramidal with a water molecule bound in the axial site. Like porphyrins, both of these N4-donor macrocycles, which differ only in the R group present at the 3 and 6 positions (H or tBu), impose a strong ligand field.
1246	The Id-protein family in developmental and cancer-associated pathways.	28122577	Inhibitors of DNA binding and cell differentiation (Id) proteins are members of the large family of the helix-loop-helix (HLH) transcription factors, but they lack any DNA-binding motif. During development, the Id proteins play a key role in the regulation of cell-cycle progression and cell differentiation by modulating different cell-cycle regulators both by direct and indirect mechanisms. Several Id-protein interacting partners have been identified thus far, which belong to structurally and functionally unrelated families, including, among others, the class I and II bHLH transcription factors, the retinoblastoma protein and related pocket proteins, the paired-box transcription factors, and the S5a subunit of the 26 S proteasome. Although the HLH domain of the Id proteins is involved in most of their protein-protein interaction events, additional motifs located in their N-terminal and C-terminal regions are required for the recognition of diverse protein partners. The ability of the Id proteins to interact with structurally different proteins is likely to arise from their conformational flexibility: indeed, these proteins contain intrinsically disordered regions that, in the case of the HLH region, undergo folding upon self- or heteroassociation. Besides their crucial role for cell-fate determination and cell-cycle progression during development, other important cellular events have been related to the Id-protein expression in a number of pathologies. Dysregulated Id-protein expression has been associated with tumor growth, vascularization, invasiveness, metastasis, chemoresistance and stemness, as well as with various developmental defects and diseases. Herein we provide an overview on the structural properties, mode of action, biological function and therapeutic potential of these regulatory proteins.
1247	Hemophagocytic Lymphohistiocytosis mimics many common conditions: case series and review of literature.	28120605	Introduction. Hemophagocytic lymphohistiocytosis (HLH), a rare but potentially fatal disease, is characterized by excessive immune activation and cytokine release which stimulates bone marrow macrophages to engulf hematopoietic cells. HLH could be secondary to infections: viral, fungal, and bacterial; malignancies and autoimmune diseases. The diagnosis of HLH is usually delayed due to the presence of non-specific symptoms at presentation. This delay contributes to increased mortality. Cases and review. We present the case of 4 patients who presented with subjective fevers and extreme fatigue. Patients all had systemic inflammatory response syndrome (SIRS). All patients were initially managed as in sepsis from an underlying infection. All unfortunately progressed to multiple organs dysfunction and died. The underlying causes for HLH in the patients were considered to be: HIV/AIDS, T cell lymphoma, histoplasmosis and juvenile rheumatoid arthritis. We have also included a brief review of the literature on HLH highlighting the treatment and outcomes of patients in case series; and the many conditions which can trigger HLH. Conclusion. Patients with HLH usually share various non-specific symptoms, such as fever and malaise, with patients across a wide spectrum of conditions: from bacterial sepsis to malignancies. Since early suspicion and diagnosis is critical to prompt therapy and improved mortality, including HLH as a possible cause of fever particularly in patients with prolonged fever of unknown origin and cytopenias will be crucial.
1248	Clinical significance of lymphocyte subset changes in hemophagocytic lymphohistiocytosis of children.	28105088	In order to examine the role of peripheral blood lymphocyte subsets on the diagnosis, treatment and prognosis of hemophagocytic lymphohistiocytosis (HLH), 30 affected children during the acute period of the disease and 30 healthy children within the same age range were selected to test their peripheral blood lymphocyte subsets using flow cytometry and compare these subsets. At the same time, the peripheral blood lymphocyte subsets of 20 children with complete remission from HLH were compared to those of 10 cases who succumbed to the disease. The proportion of CD3+ and CD8+ T cells were increased in children during the acute period. Additionally, the proportion of CD4+ T and CD3-CDl6+CD56+ natural killer (NK) cells and the ratio of CD4+/CD8+ cells were decreased in the same group of children, with the differences being statistical significance (P<0.05). The proportion of CD19+ B cells showed no differences in the affected and healthy groups. HLH children during the remission period had a higher proportion of CD3+ and CD8+ T cells than that in the control group, but the ratio of CD4+ T and CD4+/CD8+ were lower than that in the control group, with the differences being statistically significant (P<0.05). The proportion of CD19+ B cells and CD3-CD16+CD56+ NK cells revealed no significant difference between the two groups. In addition, regarding the proportion of CD3+, CD4+, CD8+ T, CD19+ B cells, CD3-CDl6+CD56+ NK cells and the ratio of CD4+/CD8+, and there were no significant differences. The results showed that HLH modifies the peripheral blood lymphocyte subsets and causes cellular immunity disorders. Thus, monitoring these dynamic changes can be useful in the diagnosis of HLH and evaluate the response to therapy.
1249	Macrophage Activation Syndrome: different mechanisms leading to a one clinical syndrome.	28095869	Macrophage activation syndrome (MAS) is a severe complication of rheumatic disease in childhood, particularly in systemic Juvenile Idiopathic Arthritis (sJIA). It is characterize by an uncontrolled activation and proliferation of T lymphocytes and macrophages.MAS is currently classified among the secondary or acquired forms of haemophagocytic lymphohistiocytosis (sHLH). The reason is that MAS shares clinical and laboratory features with primary genetic HLH (pHLH). In this context is conceivable that some of the pathogenic mechanisms of pHLH may be involved in other forms of HLH. Heterozygosity for mutations of genes involved in pHLH may lead to a cytotoxic defect and to a development of clinical overt disease. But other different contributors might be involved to the development of MAS such as infections or underlying inflammation. In MAS, the inflammatory status of the patient is a major contributor of the disease. Indeed, the majority of the MAS episodes occurs during active disease phases or at disease onset. In addition, recent evidence in animals and humans suggest that genetics may also play a major role in contributing to hyperinflammation and particularly to macrophages hyper-responses.We hypothesize that HLH may be one unique clinical syndrome, to whose generation different mechanisms may contribute, and maintained by one final effector mechanism.
1250	Secondary haemophagocytic lymphohistiocytosis triggered by postnatally acquired cytomegalovirus infection in a late preterm infant.	28093696	Haemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory condition with impairment of cytotoxic T-cells and natural killer cells. Causes in infants are mostly hereditary immune defects as well as various infectious triggering factors, amongst these cytomegalovirus (CMV). Vertical CMV transmission may occur in utero, during birth, and by breast feeding. Usually, a CMV infection transmitted via breast milk is symptomatic only in very immature preterm infants. We report on a late preterm infant born after 35 + 5 weeks of gestation with a birth weight of 1840 g, being admitted to our intensive care unit at the age of 9 weeks with acute enteritis and severe dehydration. After a prolonged recovery, the infant developed a sepsis-like condition with hyperpyrexia, hepatosplenomegaly, and pancytopenia. Combination with high ferritin levels (2809 μg/l), hypertriglyceridaemia (481 mg/dl), elevated soluble IL-2 receptor (sCD25, 9120 U/ml), and reduced perforin expression allowed diagnosis of HLH, caused by an acute CMV infection. Since connatal CMV infection had been ruled out earlier, we report the rare case of secondary HLH triggered by a postnatally acquired symptomatic CMV infection in an immunocompetent infant, most likely transmitted via breast milk. The infant was successfully treated with ganciclovir without need for immunosuppressive therapy.
1251	Human Granulocytic Anaplasmosis as a Cause of Febrile Illness in Korea Since at Least 2006.	28093540	AbstractHuman granulocytic anaplasmosis (HGA) is a tick-borne rickettsial zoonosis with fever, thrombocytopenia, and leukopenia. HGA has been reported in Korea in 2013 but it is uncertain how long it has existed. A retrospective study was conducted on patients who underwent bone marrow examination due to fever and cytopenia, with no clear hematologic or microbiologic causes, from 2003 through 2012. Laboratory diagnosis was made by detecting 16S rRNA genes of Anaplasma phagocytophilum from the stored blood samples. Among the 70 patients, five (7.1%) HGA cases were found, and the earliest case dated back to 2006. Two cases met the diagnostic criteria of hemophagocytic lymphohistiocytosis (HLH) and were fatal. Although HGA has been prevalent in Korea since at least 2006, it is not always diagnosed and has posed a possible lethal health risk to the people in Korea. HGA should be considered as a cause of fever with cytopenia or HLH.
1252	A case of pulmonary arterial hypertension associated with adult hemophagocytic lymphohistiocytosis.	28090306	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive, life-threatening syndrome of excessive immune activation. Presentation is most common among the pediatric population, and cases in adults are rare. The number of nonhematologic presentations described in relation to HLH has been growing. We present a case involving a woman who developed HLH after autologous stem cell transplantation for mantle cell lymphoma. Months later, she received a diagnosis of pulmonary arterial hypertension (PAH) while undergoing treatment for her HLH. To our knowledge, PAH associated with adult HLH has only been described in the literature once before. PAH may now be a potential differential diagnosis for patients with HLH who present with respiratory symptoms.
1253	Hemophagocytic lymphohistiocytosis in adults: An under recognized entity.	28070498	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune activation with macrophage and T-cell infiltration resulting in, multi organ damage. HLH may be primary or secondary in etiology. A high index of suspicion is essential for early diagnosis and treatment. Diagnostic criteria need to be refined and newer treatment options to be explored in order to improve survival especially in adult HLH and malignancy-associated HLH (M-HLH). We report a case of malignancy associated HLH (M-HLH) in adult treated on one of the only FDA-approved protocols for adult HLH to highlight the diagnostic and therapeutic challenges of this disease entity.
1254	Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.	28069895	Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway.
1255	Secondary Hemophagocytic Lymphohistiocytosis: Do We Really Need Chemotherapeutics for All Patients?	2806013328926358	Because of the acute and life-threatening course of the hemophagocytic lymphohistiocytosis (HLH) syndrome, International Histiocyte Society guidelines recommend chemoimmune therapy for the treatment of both primary and secondary HLH (sHLH). To manage children with sHLH, instead of HLH-2004 protocol we considered less immunosuppressive/cytotoxic approach. We assessed 12 children who fulfilled the diagnostic criteria for sHLH between January 2009 and March 2015. Multivariate Cox regression analysis showed that ferritin levels (hazard ratio=1.02, P=0.006), pediatric logistic organ dysfunction scores (hazard ratio=1.01, P=0.001) were the predictors of the survival. The hospital survival was 83% for patients with sHLH who were treated with less immunosuppressive therapy. In conclusion initiation of HLH-specific therapy for the patients with hyperferritinemia-associated sHLH should be delayed while awaiting resolution of systemic inflammation with less immunosuppressive therapy.
1256	The Maze of Diagnosing Hemophagocytic Lymphohistiocytosis: Single-Center Experience of a Series of 6 Clinical Cases.	28052298	Clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) are based on an excessive inflammatory response. Not only the diversity of the putative underlying etiologies of this rare syndrome but also the subsequent large variety of symptoms complicate the diagnosis of HLH in adult patients. However, early diagnosis and immediate treatment initiation are imperative for clinical outcome. In this article, we will review the diagnostic criteria of HLH and, in this context, discuss 6 cases, each of whom presented with a different clinical appearance causally associated with distinct malignant and nonmalignant diseases, exemplifying the spectrum of associations and manifestations of HLH.
1257	Tuberculosis-associated hemophagocytic lymphohistiocytosis in an umbilical cord blood transplant recipient.	28040556	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal condition that can be primary or secondary. Secondary HLH due to Mycobacterium tuberculosis (TB) is uncommon. We report a case of tuberculosis-associated HLH in an umbilical cord blood transplant (UCBT) recipient and discuss its clinical characteristics and challenges.Hematologic investigations, bone marrow aspirates, Xpert MTB/RIF test of TB with peripheral blood were performed. Immune modulation with anti-TB therapy was initiated.Subsequent treatment with anti-TB treatment resulted rapid clinical response and disease remission.It is important to consider TB as one of the underlying cause of HLH in high-risk patients, particularly those in immunodeficient states. Early diagnosis and treatment can improve the survival rates of patients with tuberculosis-associated HLH.
1258	A Differentiation Transcription Factor Establishes Muscle-Specific Proteostasis in Caenorhabditis elegans.	28036392	Safeguarding the proteome is central to the health of the cell. In multi-cellular organisms, the composition of the proteome, and by extension, protein-folding requirements, varies between cells. In agreement, chaperone network composition differs between tissues. Here, we ask how chaperone expression is regulated in a cell type-specific manner and whether cellular differentiation affects chaperone expression. Our bioinformatics analyses show that the myogenic transcription factor HLH-1 (MyoD) can bind to the promoters of chaperone genes expressed or required for the folding of muscle proteins. To test this experimentally, we employed HLH-1 myogenic potential to genetically modulate cellular differentiation of Caenorhabditis elegans embryonic cells by ectopically expressing HLH-1 in all cells of the embryo and monitoring chaperone expression. We found that HLH-1-dependent myogenic conversion specifically induced the expression of putative HLH-1-regulated chaperones in differentiating muscle cells. Moreover, disrupting the putative HLH-1-binding sites on ubiquitously expressed daf-21(Hsp90) and muscle-enriched hsp-12.2(sHsp) promoters abolished their myogenic-dependent expression. Disrupting HLH-1 function in muscle cells reduced the expression of putative HLH-1-regulated chaperones and compromised muscle proteostasis during and after embryogenesis. In turn, we found that modulating the expression of muscle chaperones disrupted the folding and assembly of muscle proteins and thus, myogenesis. Moreover, muscle-specific over-expression of the DNAJB6 homolog DNJ-24, a limb-girdle muscular dystrophy-associated chaperone, disrupted the muscle chaperone network and exposed synthetic motility defects. We propose that cellular differentiation could establish a proteostasis network dedicated to the folding and maintenance of the muscle proteome. Such cell-specific proteostasis networks can explain the selective vulnerability that many diseases of protein misfolding exhibit even when the misfolded protein is ubiquitously expressed.
1259	A case of pseudorheumatism with submasseteric abscess and HLH in a patient with visceral leishmaniasis: A diagnostic dilemma.	28035119	NaN
1260	Acquired hemophagocytic syndrome in a patient with synovial sarcoma: a case report.	28031902	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by severe hyperinflammation due to an overwhelming ineffective immune response to different triggers. Most important symptoms are fever, hepatosplenomegaly and cytopenias. Biochemical signs include elevated ferritin, hypertriglyceridemia and low fibrinogen. Hemophagocytosis in the bone marrow is a hallmark of this syndrome. Based on the pathogenetic mechanism, it can be classified into primary (inherited) or secondary (acquired) HLH. We report, to our knowledge, the first case of acquired hemophagocytic syndrome that arose in a 20-year-old man affected by synovial sarcoma as a complication during chemotherapy.
1261	A novel trimeric complex in plant cells that contributes to the lamina inclination of rice.	28029278	The architecture determining grain production in rice is mainly affected by factors such as lamina angle, tillering, plant height and panicle morphology. In particular, leaf angle, the degree of bending between the leaf blade and leaf sheath directly affects crop architecture and grain yields. Balancing activities between the 2 antagonistic groups of proteins, atypical helix-loop-helix (HLH) and basic HLH (bHLH) proteins have been regarded as one of the major molecular machineries regulating lamina angles through the control of cell elongation in the lamina joint of rice. Recently, formation of a complex consisting of atypical HLH protein, OsBUL1, a KxDL motif-containing protein, LO9-177 and a bHLH protein, OsBC1 has been reported to play a positive role in lamina inclination of rice unraveling a novel layer of cell elongation control in rice lamina joint. Here, we demonstrate a trimeric complex formation in rice cells by a combination of BiFC and FRET-FLIM assays.
1262	[Children's NK/T Cell Lymphoma-Associated Hemophagocytic Syndrome: Clinical Analysis of 6 Cases].	28024491	To study the clinical features, treatment and prognosis of patients with NK/T cell lymphoma-associated hemophagocytic syndrome(NK/T-LAHPS).Retrospective analysis was used to explore the clinical data of 6 children with NK/T-LAHPS who were admitted in Department of Pediatric Hematology of Fujian Medical University Union Hospital from July 2012 to June 2016. The 6 patients included 4 boys and 2 girls, with a median age of 4 years(range 1.75 to 11). In 4 patients the hemophagocytic syndrome(HPS) occurred as the main primary manifestations of underlying lymphoma, in the other 2 patients HPS occurred during lymphoma progression. The clinical manifestations included persistent fever(6/6), hepatomegaly(6/6), splenomegaly(6/6) and pancytopenia(6/6). Laboratory data indicated that the level of ferritin(2179-15000 ng/ml) , LDH(608-3899 IU/L) and EBV-DNA(>105 copies/ml ) was elevated obviously. The other common clinical features of NK/T-LAHPS were hypoproteinemia(6/6), hepatic dysfunction(5/6), hypofibrinogenimia(5/6), hypertriglyceridemia(3) and hemophagocytosis in bone marrow(5/6).After being treated according to the HLH-2004 protocol combined with supported therapy for 1 or 2 weeks, all the patients achieved a clinical response, and the laboratory indicators of HPS were improved. The combined chemotherapy of SMILE was given to 4 patients timely, among them 2 patients achieved complete remission(CR) and long term survival, 1 patient achieved partial remission(PR) and died of relapse after drug withdrawal and 1 patient died of aggravated lymphoma. The other 2 patients did not receive chemotherapy in time, HPS relapsed quickly, because of the progression of lymphoma, and all died of severe hepatic dysfunction and coagulopathy.The NK/T-LAHPS is an invariably fatal disease with poor prognosis, and typically occurrs at the advanced stage or the terminal phase of the disease. HLH-2004- based protocol in combination with comprehensive therapy is hopeful for the patients with NK/T-LAHPS, which may delay the disease progression and provide opportunities for the treatment of primary disease. Once the laboratory indicators of HPS are improved, it is important to treat lymphoma timely with the combined chemotherapy of SMILE, which is significant for improving the prognosis.
1263	A Rare Case of Hemophagocytic Lymphohistiocytosis Associated with Parvovirus B19 Infection.	28018767	Hemophagocytic lymphohistiocytosis (HLH) is a rare but life-threatening syndrome resulting from excessive immune activation. Secondarily, HLH is often associated with autoimmune disease, infection, and malignancy. The most common infectious trigger is Epstein-Barr virus (EBV) infection. HLH is rarely triggered by parvovirus B19. We discuss a case of a 62-year-old male who presented with multi-organ failure with presumed septic shock who eventually was diagnosed with HLH, with positive parvovirus B19 deoxyribonucleic acid (DNA) polymerase chain reaction (PCR). Prompt treatment with dexamethasone resulted in significant clinical resolution.
1264	Combination Immunosuppressive Therapy Including Rituximab for Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis in Adult-Onset Still's Disease.	28018698	Hemophagocytic lymphopcytosis (HLH) is a life-threatening condition. It can occur either as primary form with genetic defects or secondary to other conditions, such as hematological or autoimmune diseases. Certain triggering factors can predispose individuals to the development of HLH. We report the case of a 25-year-old male patient who was diagnosed with HLH in the context of adult-onset Still's disease (AOSD) during a primary infection with Epstein-Barr virus (EBV). During therapy with anakinra and dexamethasone, he was still symptomatic with high-spiking fevers, arthralgia, and sore throat. His laboratory values showed high levels of ferritin and C-reactive protein. His condition improved after the addition of rituximab and cyclosporine to his immunosuppressive regimen with prednisolone and anakinra. This combination therapy led to a sustained clinical and serological remission of his condition. While rituximab has been used successfully for HLH in the context of EBV-associated lymphoma, its use in autoimmune diseases is uncommon. We hypothesize that the development of HLH was triggered by a primary EBV infection and that rituximab led to elimination of EBV-infected B-cells, while cyclosporine ameliorated the cytokine excess. We therefore propose that this combination immunosuppressive therapy might be successfully used in HLH occurring in the context of autoimmune diseases.
1265	The Conundrum in the Diagnosis and Management of Atypical Fulminant Measles in a Leukemia Survivor on Maintenance Chemotherapy.	27992394	We report the diagnostic and therapeutic challenges in an unusually fulminant presentation of measles, presenting as severe necrotizing bronchiolitis with secondary hemophagocytic lymphohistiocytosis (HLH) in the absence of classical clinical features in an immunocompromised host on maintenance chemotherapy. Our patient had presented with features of a viral pneumonitis without the classical exanthem, in combination with HLH. Although rhinovirus-induced HLH was highly unusual, the positive rhinovirus swab result had distracted us from the eventual diagnosis. This calls for greater impetus to increase awareness and public education to improve vaccination rates and herd immunity to curb outbreaks and protect the immunocompromised patients.
1266	Comprehensive genetic testing for primary immunodeficiency disorders in a tertiary hospital: 10-year experience in Auckland, New Zealand.	27980540	New Zealand is a developed geographically isolated country in the South Pacific with a population of 4.4 million. Genetic diagnosis is the standard of care for most patients with primary immunodeficiency disorders (PIDs).Since 2005, we have offered a comprehensive genetic testing service for PIDs and other immune-related disorders with a published sequence. Here we present results for this program, over the first decade, between 2005 and 2014.We undertook testing in 228 index cases and 32 carriers during this time. The three most common test requests were for X-linked lymphoproliferative (XLP), tumour necrosis factor receptor associated periodic syndrome (TRAPS) and haemophagocytic lymphohistiocytosis (HLH). Of the 32 suspected XLP cases, positive diagnoses were established in only 2 patients. In contrast, genetic defects in 8 of 11 patients with suspected X-linked agammaglobulinemia (XLA) were confirmed. Most XLA patients were initially identified from absence of B cells. Overall, positive diagnoses were made in about 23% of all tests requested. The diagnostic rate was lowest for several conditions with locus heterogeneity.Thorough clinical characterisation of patients can assist in prioritising which genes should be tested. The clinician-driven customised comprehensive genetic service has worked effectively for New Zealand. Next generation sequencing will play an increasing role in disorders with locus heterogeneity.
1267	Cord Blood Transplantation Following Reduced-Intensity Conditioning for Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis during Systemic Lupus Erythematosus Treatment.	27980302	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a serious disorder in which monoclonal growth of T cells infected with EBV and macrophage activation cause pancytopenia, high fever and acute liver failure. Patients with chemotherapy- or immunosuppression-resistant EBV-HLH require allogeneic hematopoietic stem cell transplantation (allo-HSCT), but patients who have no sibling donors may not have time to wait for an unrelated donor. In pediatric patients, there are some reports on allogeneic cord blood transplantation (allo-CBT) for the treatment of EBV-HLH; however, in adult patients, reports of allo-CBT for EBV-HLH are quite limited. The present case of a 20-year-old woman with chemotherapy-resistant EBV-HLH and systemic lupus erythematosus (SLE) who underwent allo-CBT following reduced-intensity conditioning (RIC-CBT). She achieved and maintained a complete donor type and the EBV-DNA load, and the titers of anti-double stranded DNA and antinuclear antibodies became negative. It is therefore considered that RIC-CBT is an effective treatment option for adult onset HLH. However, because the effectiveness of allo-HSCT for SLE remains unclear and transplant-related mortality is high, it is recommended that allo-HSCT for SLE is restricted to patients concomitant with oncohematological disease as with our present case.
1268	Donor cell leukemia with bone marrow necrosis.	27941282	A 60-year-old man with myelodysplastic syndrome underwent allogeneic transplantation of female umbilical cord blood in 2010 and sustained a complete remission. He experienced severe pain in his left hip joint and was admitted to the orthopedic surgery division of our institution in February 2015. After admission, he was suspected to have hemophagocytic syndrome (HPS) and was thus transferred to the hematology division. Bone marrow aspiration revealed hyper-cellular marrow filled with abnormal collapsed cells, consistent with bone marrow necrosis (BMN). As there was no evidence of infection, collagen disease, or occult cancer, he was diagnosed with HPS of unknown origin and treated with dexamethasone, cyclosporine A, and etoposide according to the HLH-2004 protocol. Although his general condition and laboratory findings showed amelioration, morphologically abnormal cells appeared in peripheral blood two weeks after treatment. Bone marrow aspiration showed BMN with increased abnormal cells, positive for CD117 and MPO. Sex chromosome FISH analysis revealed donor chimerism and cytogenetic analysis showed 46XX, +1, der (1;7) (q10;q10). He was diagnosed with donor cell leukemia (DCL) and received salvage chemotherapy. However, he died because of severe pneumonia and sepsis without neutrophil recovery at day 68. We herein report this rare case of DCL with BMN.
1269	Combining an antiviral with rituximab in EBV-related haemophagocytic lymphohistiocytosis led to rapid viral clearance; and a comprehensive review.	27941111	Epstein-Barr virus (EBV)-related haemophagocytic lymphohistiocytosis (EBVr-HLH) has a better prognosis when the virus is rapidly cleared, but the best antiviral approach is controversial. We present a patient to whom the therapeutic standard rituximab was co-administered with valacyclovir and an HLH-specific treatment with favourable viral and clinical responses. We conducted an extensive literature review and contacted several world reference centres and experts to inquire about their approaches and experience. We conclude that antivirals are infrequently used for EBVr-HLH, despite their laboratory-proven and likely clinical beneficial effect on some EBV-related diseases. However, the role of antivirals remains obscure. Concerns about their lack of efficacy are based on observational data and reports of the cellular tropism of EBV. Therefore, the adjunct use of antivirals may be considered when myelotoxicity is not the primary concern, and related outcomes should be systematically recorded to produce higher quality evidence.
1270	Rapidly Fatal Hemophagocytic Lymphohistiocytosis Developing Within Six Days Following Deceased-Donor Renal Transplantation: Case Report.	27932162	Hemophagocytic lymphohistiocytosis (HLH) is an often fatal hyperinflammatory syndrome that may complicate malignancy, infection, rheumatic disease, or immunosuppression. HLH after kidney transplantation is most often triggered by infection, usually Herpes viruses such as cytomegalovirus and Epstein-Barr virus (EBV). It usually occurs early after transplantation. We present a case of HLH triggered by reactivation of EBV that pursued a rapidly fatal course within 6 days of receiving a deceased-donor kidney transplant. This case serves to remind transplant clinicians to consider HLH when cytopenias and hyperinflammation are atypical for the usual post-transplantation course. We discuss pitfalls in diagnosis and suggestions for treatment in this setting.
1271	Primary and secondary hemophagocytic lymphohistiocytosis have different patterns of T-cell activation, differentiation and repertoire.	2792564328185252	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening inflammatory syndrome characterized by hyperactivation of lymphocytes and histiocytes. T cells play a key role in HLH pathogenesis, but their differentiation pattern is not well characterized in patients with active HLH. We compared T-cell activation patterns between patients with familial HLH (1°HLH), 2°HLH without apparent infectious trigger (2°HLH) and 2°HLH induced by a viral infection (2°V-HLH). Polyclonal CD8+ T cells are highly activated in 1°HLH and 2°V-HLH, but less in 2°HLH as assessed by HLA-DR expression and marker combination with CD45RA, CCR7, CD127, PD-1 and CD57. Absence of increased HLA-DR expression on T cells excluded active 1° HLH with high sensitivity and specificity. A high proportion of polyclonal CD127- CD4+ T cells expressing HLA-DR, CD57, and perforin is a signature of infants with 1°HLH, much less prominent in virus-associated 2°HLH. The similar pattern and extent of CD8+ T-cell activation compared to 2° V-HLH is compatible with a viral trigger of 1°HLH. However, in most 1°HLH patients no triggering infection was documented and the unique activation of cytotoxic CD4+ T cells indicates that the overall T-cell response in 1°HLH is different. This may reflect different pathways of pathogenesis of these two HLH variants.
1272	Hemophagocytic lymphohistiocytosis triggered by Gaucher disease in a preterm neonate.	27922757	To present the diagnostic workup in an extremely low birth weight infant patient with signs of both sepsis and hemophagocytosis.A preterm infant presented with clinical and laboratory signs of early-onset sepsis including hepatosplenomegaly, thrombocytopenia, direct hyperbilirubinemia, and elevated liver enzymes.Despite extensive septic workup, no underlying infection was detected. Additional hyperferritinemia and other elevated inflammatory parameters raised the suspicion of a primary or secondary hemophagocytic lymphohistiocytosis (HLH).However, further metabolic analysis yielded a positive result for Gaucher disease (GD) type 2, a rare, but possible trigger of HLH.Our case shows that GD may lead to the picture of a secondary HLH and that a metabolic workup should always be performed in patients in whom primary HLH has been excluded.
1273	Acute Liver Failure Secondary to Hemophagocytic Lymphohistiocytosis during Pregnancy.	27921061	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive immune activation that mimics and occurs with other systemic diseases. A 35-year-old female presented with signs of viral illness at 13 weeks of pregnancy and progressed to acute liver failure (ALF). We discuss the diagnosis of HLH and Kikuchi-Fujimoto (KF) lymphadenitis in the context of pregnancy and ALF. HLH may respond to comorbid disease-specific therapy, and more toxic treatment can be avoided.
1274	Usual and Unusual Manifestations of Familial Hemophagocytic Lymphohistiocytosis and Langerhans Cell Histiocytosis.	27894453	Familial hemophagocytic lymphohistiocytosis (FHL) and Langerhans cell histiocytosis (LCH) are histiocytic diseases that occur most commonly in young children. Improvements in recognition and treatment have been substantial for both diseases in the past decade, although early and late morbidity continue to be major concerns. These two diagnoses behave differently, although the clinical spectra for both diseases are diverse and can lead to confusion and delays in diagnosis and treatment. This article focuses on the clinical and genetic spectrum of FHL as well as the clinical and treatment variations of LCH.
1275	Acute liver failure caused by hemophagocytic lymphohistiocytosis in adults: A case report and review of the literature.	27893685	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition that can be caused by a primary or acquired disorder of uncontrolled immune response. Liver injury is a common complication of HLH; however, HLH presenting as acute liver failure (ALF) has rarely been reported in adults.A 34-year-old man was admitted to our hospital with nausea and fatigue persisting for 2 weeks and jaundice for 1 week. He had hyperthermia at the onset of disease. At admission, he had severe liver injury with unknown etiology. The laboratory data showed that he had hyperferritinemia, thrombocytopenia, anemia, hypertriglyceridemia, and hypofibrinogenemia. Finally, a bone marrow biopsy revealed hemophagocytic cells, and he was diagnosed with HLH. The patient was treated with prednisone and plasma exchange. However, the liver function of the patient deteriorated, and he finally died of multiorgan failure.Reports of adult patients with ALF caused by HLH have increased, and HLH should be suspected in patients with ALF of indeterminate cause. Although the efficacy of the treatment strategy recommended by the HLH 2004 remains to be confirmed in adult patients with ALF caused by HLH, early diagnosis and prompt combined treatment with steroids and cyclosporin A or etoposide should be emphasized.
1276	Ectopic fat deposition contributes to age-associated pathology in Caenorhabditis elegans.	27884963	Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat deposition in nonadipose tissues. Ectopic fat contributes to lipotoxicity and has been implicated in the development of a metabolic syndrome that increases risk of age-associated diseases. However, the molecular mechanisms coupling ectopic fat accumulation with aging remain obscure. Here, we use nonlinear imaging modalities to visualize and quantify age-dependent ectopic lipid accumulation in Caenorhabditis elegans We find that aging is accompanied by pronounced deposition of lipids in nonadipose tissues, including the nervous system. Importantly, interventions that promote longevity such as low insulin signaling, germ-line loss, and dietary restriction, which effectively delay aging in evolutionary divergent organisms, diminish the rate of ectopic fat accumulation and the size of lipid droplets. Suppression of lipotoxic accumulation of fat in heterologous tissues is dependent on helix-loop-helix (HLH)-30/transcription factor EB (TFEB) and autophagy. Our findings in their totality highlight the pivotal role of HLH-30/TFEB and autophagic processes in the maintenance of lipid homeostasis during aging, in addition to establishing nonlinear imaging as a powerful tool for monitoring ectopic lipid droplet deposition in vivo.
1277	Severe Fever with Thrombocytopenia Syndrome Presenting with Hemophagocytic Lymphohistiocytosis.	27883371	Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the newly discovered SFTS Bunyavirus, and there have been no case reports of SFTS patients presenting with hemophagocytic lymphohistiocytosis (HLH) in the English literature. We report a case of SFTS presenting with HLH in a 73-year-old immunocompetent male farmer. Although the patient had poor prognostic factors for SFTS, such as old age and central nervous system symptoms, he recovered fully with supportive care.
1278	Clinically Mild Encephalitis/Encephalopathy With a Reversible Splenial Lesion Accompanied by Epstein-Barr Virus Hemophagocytic Lymphohistiocytosis: A Case Report and Review of the Literature.	27879539	Central nervous system involvement in hemophagocytic lymphohistiocytosis (HLH) is associated with a poor outcome. For such patients, it is unknown whether more aggressive therapies, such as intrathecal methotrexate or hydrocortisone, are inevitably required. We present a very rare case of 3-year-old Japanese girl who developed mild encephalitis/encephalopathy with a reversible splenial lesion, accompanied by Epstein-Barr virus-associated HLH, and review previous similar reports. Our case and previous reports suggest that mild encephalitis/encephalopathy with a reversible splenial lesion accompanied by Epstein-Barr virus-associated HLH has a relatively good prognosis, even in the absence of intrathecal treatments.
1279	Rice Leaf Angle and Grain Size Are Affected by the OsBUL1 Transcriptional Activator Complex.	27879391	Rice atypical HLH protein Oryza sativa BRASSINOSTEROID UPREGULATED 1-LIKE1 (OsBUL1) is preferentially expressed in the lamina joint where it controls cell elongation and positively affects leaf angles. OsBUL1 knockout mutant (osbul1) and transgenic rice for double-stranded RNA interference (dsRNAi) of OsBUL1 produced erect leaves with smaller grains, whereas OsBUL1 overexpressors and an activation tagging line of OsBUL1 exhibited increased lamina inclination and grain size. Moreover, OsBUL1 expression was induced by brassinolide (BL) and osbul1 did not respond to BL treatment. To understand the molecular network of OsBUL1 function in rice, we isolated a novel OsBUL1-interacting protein, LO9-177, an uncharacterized protein containing a KxDL motif, and functionally studied it with respect to the lamina inclination and grain size of rice. OsBUL1 COMPLEX1 (OsBC1) is a basic helix-loop-helix (bHLH) transcriptional activator that interacts with OsBUL1 only in the presence of LO9-177 forming a possible trimeric complex for cell elongation in the lamina joint of rice. Expression of OsBC1 is also upregulated by BL and has a similar pattern to that of OsBUL1 Transgenic rice plants expressing OsBC1 under the control of OsBUL1 promoter showed increased grain size as well as leaf bending, while transgenic lines for dsRNAi and/or expressing a dominant repressor form of OsBC1 displayed reduced plant height and grain size. Together, these results demonstrated that a novel protein complex consisting of OsBUL1, LO9-177, and OsBC1 is associated with the HLH-bHLH system, providing new insight into the molecular functional network based on HLH-bHLH proteins for cell elongation.
1280	HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans.	27875098	Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we reveal that bacterial membrane pore-forming toxin (PFT) induces autophagy in an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.
1281	Pediatric acute liver failure of undetermined cause: A research workshop.	27862115	Pediatric acute liver failure (PALF) is a potentially devastating condition that occurs in previously healthy children of all ages and frequently leads to a rapid clinical deterioration. An identified cause for liver injury is lacking in approximately 30% of cases. Children with undetermined diagnosis have lower spontaneous survival and higher rates of transplantation and death than other diagnostic groups. A single-day workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases brought together clinicians and basic scientists to integrate aligned research findings and develop a foundation for new mechanistic studies and future treatment trials. The clinical phenotype of indeterminate PALF shares important similarities to the hyperinflammatory state characteristic of hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS). A failure of cytotoxic T cells to limit or contract inflammatory responses may propagate injury and lead to a local and systemic milieu that does not support normal hepatic regeneration. Evidence was presented that bone marrow (BM)-derived Sinusoidal endothelial cell PROgenitor Cells (sprocs) play a vital role in hepatic regeneration. Overwhelming systemic inflammatory responses may suppress mobilization of BM sprocs and dampen hepatic recovery.Experience gained through treatment trials of HLH and MAS in childhood may inform study design for therapy of PALF. Successful approaches to limiting neuroinflammation through reduction of systemic inflammation and standardized neuroprotection protocols that limit glial injury could significantly improve intact survival. Finally, given that PALF is a rare disease, investigative efforts must include broad multicenter collaboration and careful stewardship of biorepository specimens. (Hepatology 2017;65:1026-1037).
1282	Miz-1 and Max compete to engage c-Myc: implication for the mechanism of inhibition of c-Myc transcriptional activity by Miz-1.	27859590	c-Myc is a basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factor deregulated in the majority of human cancers. As a heterodimer with Max, another b-HLH-LZ transcription factor, deregulated and persistent c-Myc accumulates at transcriptionally active promoters and enhancers and amplifies transcription. This leads to the so-called transcriptional addiction of tumor cells. Recent studies have showed that c-Myc transcriptional activities can be reversed by its association with Miz-1, a POZ transcription factor containing 13 classical zinc fingers. Although evidences have led to suggest that c-Myc interacts with both Miz-1 and Max to form a ternary repressive complex, earlier evidences also suggest that Miz-1 and Max may compete to engage c-Myc. In such a scenario, the Miz-1/c-Myc complex would be the entity responsible for the inhibition of c-Myc transcriptional amplification. Considering the implications of the Miz-1/c-Myc interaction, it is highly important to solve this duality. While two potential c-Myc interacting domains (hereafter termed MID) have been identified in Miz-1 by yeast two-hybrid, with the b-HLH-LZ as a bait, the biophysical characterization of these interactions has not been reported so far. Here, we report that the MID located between the 12th and 13th zinc finger of Miz-1 and the b-HLH-LZ of Max compete to form a complex with the b-HLH-LZ of c-Myc. Our results support the notion that the repressive action of Miz-1 on c-Myc does not rely on the formation of a ternary complex. The implications of these observations for the mechanism of inhibition of c-Myc transcriptional activity by Miz-1 are discussed. Proteins 2017; 85:199-206. © 2016 Wiley Periodicals, Inc.
1283	Hemophagocytic lymphohistiocytosis in a patient with Sjögren's syndrome: case report and review.	27853859	Hemophagocytic lymphohistiocytosis (HLH) is a very rare syndrome with a mortality up to 95% of cases if not treated. It is characterised by an excessive activation of the immune system that leads to a disproportionate and destructive inflammatory response. The high mortality rates are in part due to a delay in the diagnosis, and therefore clinicians must maintain a high index of suspicion. When the treatment is started early, the survival rate reaches around 55% of cases. HLH usually presents with persistent fever, pancytopenia, and organomegaly and is associated with very high levels of serum ferritin. In this manuscript, we present the case of a patient with primary Sjögren's syndrome who developed HLH after an acute infection by Cytomegalovirus. We will describe and discuss the pathogenesis, differential diagnosis and a pragmatic approach to the treatment for this critically important and, when diagnosed early, potentially curable syndrome.
1284	Adult Onset Still's Disease Associated with Mycoplasma pneumoniae Infection and Hemophagocytic Lymphohistiocytosis.	27847518	Adult Onset Still's Disease (AOSD) is a systemic inflammatory disorder that can be associated with hemophagocytic lymphohistiocytosis (HLH), a rare but potentially fatal disease of overactive histiocytes and lymphocytes. We present a unique case of AOSD complicated by Mycoplasma pneumonia infection and HLH. A 28-year-old female developed joint pains followed by a diffuse, erythematous, pruritic skin rash that quickly spread throughout the body. The patient deteriorated and developed fever, chills, cough, and dyspnea and had to be intubated. She had hypoalbuminemia, elevated liver enzymes, a very high serum ferritin level, positive anti-Mycoplasma pneumonia IgG and IgM antibodies, and normal rheumatoid factor and anti-nuclear antibodies. The chest X-ray showed diffuse bilateral infiltrates. Bone marrow biopsy revealed hemophagocytosis. The patient was treated with azithromycin, methylprednisolone, and anakinra and was discharged home on cyclosporine and prednisone. This case highlights that patients can develop features of both AOSD and HLH at the beginning of the disease and early diagnosis and treatment increase the likelihood of recovery.
1285	The Non-Langerhans Cell Histiocytoses (Rare Histiocytoses) - Clinical Aspects and Therapeutic Approaches.	27846659	Rare histiocytoses, also called non-Langerhans cell histiocytoses, include all proliferative disorders of histiocytes, macrophages and dendritic cells that are not classified as Langerhans cell histiocytosis (LCH) and do not belong to the hemophagocytic lymphohistiocytosis (HLH) group of diseases. Thus, the term includes numerous benign or malignant, localized or systemic, adult or pediatric diseases. The classification of the histiocytic disorders has been revised several times. Here, we follow the classification recently published by Jean Francois Emile and an international expert panel, defining subgroups of histiocytoses described as L-Group, C-Group, M-Group, R-Group, and H-Group, which stands for LCH-like, cutaneous or mucocutaneous, malignant, Rosai-Dorfman-Disease like and HLH like. Some of the diseases have an excellent prognosis after resection or even disappear spontanously, others progress rapidly, requiring intensive systemic therapies. The malignant non-Langerhans cell histiocytoses in general have a poor prognosis, here, complex chemotherapy protocols are usually applied, with inconsistant results. An interesting perspective in non-malignant rare histiocytoses might be small molecular inhibitors, in particular BRAF inhibitors, since BRAF mutations have been found in some subtypes of non-Langerhans cell histiocytoses. By prospective and retrospective collection of experiences in a new registry (the "International Rare Histiocytic Disorders Registry", IRHDR), knowledge about these rare diseases might hopefully be improved.
1286	OL-EDA-ID Syndrome: a Novel Hypomorphic NEMO Mutation Associated with a Severe Clinical Presentation and Transient HLH.	27838798	NaN
1287	Diagnosing haemophagocytic syndrome.	27831908	Haemophagocytic syndrome, or haemophagocytic lymphohistiocytosis (HLH), is a hyperinflammatory disorder characterised by uncontrolled activation of the immune system. It can result from mutations in multiple genes involved in cytotoxicity or occur secondary to a range of infections, malignancies or autoimmune rheumatic diseases. In the latter case, it is also known as macrophage activation syndrome (MAS). Characteristic features are persistent fever, hepatosplenomegaly, petechial/purpuric rash, progressive cytopenias, coagulopathy, transaminitis, raised C reactive protein, falling erythrocyte sedimentation rate, hypertriglyceridaemia, hypofibrinogenaemia and extreme hyperferritinaemia often associated with multi-organ impairment. Distinguishing HLH from systemic sepsis can present a major challenge. Criteria for diagnosis and classification of HLH and MAS are available and a serum ferritin >10 000 µg/L is strongly supportive of HLH. Without early recognition and appropriate treatment, HLH is almost universally fatal. However, with prompt referral and advancements in treatment over the past two decades, outcomes have greatly improved.
1288	Histoplasmosis-Induced Hemophagocytic Lymphohistiocytosis in an Adult Patient: A Case Report and Review of the Literature.	27830097	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threating immune dysregulation syndrome characterized by persistent activation of the mononuclear phagocytic system leading to uncontrolled systemic hyperinflammatory response. The proliferation and activation of histiocytes and lymphocytes lead to production of large amounts of cytokines, also called cytokine storm. Hematopoietic and lymphoid tissues are directly involved while other organs are damaged by circulating cytokines. Primary HLH is attributed to genetic defects of the immune system and secondary HLH is usually seen in adults secondary to malignancy, infection, or autoimmune diseases. Zoonotic diseases including fungal infections are an important cause of HLH. Secondary HLH can delay the recognition of the underlying zoonoses. We report the case of a 61-year-old female with history of rheumatoid arthritis with histoplasmosis associated hemophagocytic lymphohistiocytosis.
1289	Recurrent macrophage activation syndrome since toddler age in an adolescent boy with HLA B27 positive juvenile ankylosing spondylitis.	27826329	Recurrent macrophage activation syndrome (MAS) is very rare. We present the case of an adolescent boy with human leukocyte antigen (HLA) B27-positive ankylosing spondylitis (AS), who experienced episodes of recurrent MAS since he was a toddler. A 16-year-old boy was admitted because of remittent fever with pancytopenia and splenomegaly after surgical intervention for an intractable perianal abscess. He had been diagnosed with hemophagocytic lymphohistiocytosis (HLH) 4 different times, which was well controlled with intravenous immunoglobulin and steroids since the age of 3. We were unable to identify the cause for the HLH. He remained symptom-free until the development of back pain and right ankle joint pain with swelling at 15 years of age. He was diagnosed with HLA B27-positive AS with bilateral active sacroiliitis. He showed symptom aggravation despite taking naproxen and methotrexate, and the symptoms improved with etanercept. On admission, his laboratory data showed leukopenia with high ferritin and triglyceride levels. Bone marrow biopsy examination showed histiocytic hyperplasia with hemophagocytosis. There was no evidence of infection. He received naproxen alone, and his symptoms and laboratory data improved without any other immunomodulatory medications. Genetic study revealed no primary HLH or inflammasome abnormalities. In this case, underlying autoimmune disease should have been considered as the cause of recurrent MAS in the young patient once primary HLH was excluded.
1290	Hematopoietic Stem Cell Transplantation for XIAP Deficiency in Japan.	27815752	X-linked inhibitor of apoptosis protein (XIAP) deficiency is a rare immunodeficiency that is characterized by recurrent hemophagocytic lymphohistiocytosis (HLH) and splenomegaly and sometimes associated with refractory inflammatory bowel disease (IBD). Although hematopoietic stem cell transplantation (HSCT) is the only curative therapy, the outcomes of HSCT for XIAP deficiency remain unsatisfactory compared with those for SLAM-associated protein deficiency and familial HLH.To investigate the outcomes and adverse events of HSCT for patients with XIAP deficiency, a national survey was conducted.A spreadsheet questionnaire was sent to physicians who had provided HSCT treatment for patients with XIAP deficiency in Japan.Up to the end of September 2016, 10 patients with XIAP deficiency had undergone HSCT in Japan, 9 of whom (90%) had survived. All surviving patients had received a fludarabine-based reduced intensity conditioning (RIC) regimen. Although 5 patients developed post-HSCT HLH, 4 of them survived after etoposide administration. In addition, the IBD associated with XIAP deficiency improved remarkably after HSCT in all affected cases.The RIC regimen and HLH control might be important factors for successful HSCT outcomes, with improved IBD, in patients with XIAP deficiency.
1291	Establishment and characterization of a novel Hodgkin lymphoma cell line, AM-HLH, carrying the Epstein-Barr virus genome integrated into the host chromosome.	27813134	We describe the establishment and characterization of a cell line, AM-HLH, obtained from a patient with Epstein-Barr virus-positive (EBV+ ) nodular sclerosis-type Hodgkin lymphoma (HL). The cells were positive for CD2 and CD30 and negative for CD15. The immunoglobulin heavy- and κ light-chain genes were rearranged. The karyotype was of the triploid range. Southern blotting using the EBV terminal repeat probe detected 3 hybridizing bands that were identical to those of the parental HL material. The cells expressed EBV-encoded RNAs as well as latent genes (EBNA1, EBNA2, LMP1, and LMP2A) and lytic genes (BZLF1 and BALF2). Fluorescence in situ hybridization (FISH) with the cosmid pJB8 clone containing a fragment of EBV DNA as a probe revealed multiple hybridization signals at a marker chromosome. Additional FISH using whole chromosome painting and centromere probes in combination with multicolor FISH determined that multiple EBV copies were clustered within the chromosome 20 materials of the marker chromosome. Culture supernatants of AM-HLH contained IL-10 as measured by the bead-based immunoassay. It is possible that an integrated EBV genome and cellular genes on chromosome 20 were coamplified, leading to the enhanced expression of genes involved in cell growth control. The AM-HLH cell line will be useful to clarify the role of cytokines in the development of EBV+ HL.
1292	Neonatal Haemophagocytic Lymphohistiocytosis Associated with Maternal Adult-Onset Still's Disease.	27304999	Neonatal haemophagocytic lymphohistiocytosis (HLH) is a rare but potentially lethal condition. We recently encountered a preterm infant who developed severe HLH associated with maternal adult-onset Still's disease, which to our knowledge has not been previously reported. The infant presented with fever, generalised lymphadenopathy, transient erythematous skin rash, hepatosplenomegaly, ascites, pancytopenia, marked hyperferritinaemia, and hypofibrinogenaemia, which were features similar to maternal presentation during late pregnancy. Whole gene exome sequencing screening for familial HLH (PRF1, STX11, STXBP2, and MUNC13D genes) was negative. We postulated that factors such as auto-antibodies, antigens, or inflammatory mediators transmitted vertically from the mother could have triggered the intense inflammation in the infant. The infant responded promptly to dexamethasone, etoposide, and cyclosporin A, without the need for bone marrow transplantation. Neonatologists should be alerted to the rare diagnosis of HLH in the presence of active maternal diseases, including infection or autoimmune conditions, especially in association with fever, cytopenia, and hepatosplenomegaly.
1293	Hemophagocytic Lymphohistiocytosis in a Patient with Classical Hodgkin Lymphoma.	27803821	Introduction. Hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome that can be associated with inherited genetic mutations, malignancy, autoimmune disorders, and viral infections. Though the pathogenesis is not fully known, HLH is understood to be a reactive process in the setting of uncontrolled activation of macrophages, CD8+ cytotoxic lymphocytes, and other immune cells. Hallmark clinicopathological features of HLH include fevers, cytopenias, hepatosplenomegaly, and hemophagocytosis in the bone marrow. Case Presentation. A previously healthy 28-year-old Caucasian male presented with a one-month history of persistent fever, night sweats, and unintentional weight loss. He was diagnosed with classical Hodgkin Lymphoma (HL) by core-needle biopsy of an axillary lymph node. Both bone marrow involvement by HL and hemophagocytosis were seen on subsequent bone marrow biopsy. Other findings included pancytopenia, splenomegaly, and elevated serum ferritin. Extensive work-up for autoimmune and infectious etiologies was unremarkable. The patient had a complete response after chemotherapy with Adriamycin, bleomycin, vincristine, and dacarbazine. Conclusion. This report documents the exceedingly uncommon association between HLH and HL. HLH is a hyperinflammatory syndrome with high mortality, so it is imperative to identify and treat the underlying cause for secondary HLH. Malignancy-associated HLH should be considered in the differential diagnosis for cancer patients who present with fever, cytopenias, and splenomegaly.
1294	Diagnosis and treatment of HLH in adults.	27795515	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening syndrome of uncontrolled immune activation. It was initially recognized in children, where it occurs primarily as an inherited syndrome related to homozygous null mutations in immune response genes involved in cytotoxic T cell and NK cell function. A minority of pediatric patients develop "secondary" HLH as a consequence of infection or autoimmune disease. In the last 10-15 years, secondary HLH has been increasingly recognized in adults, where it is frequently associated with lymphoid malignancy, infection, or autoimmune disease. This relatively recently recognized diagnosis and the treatment of adult HLH have been largely shaped by observations in pediatric patients. In this brief summary, we focus on the features that distinguish pediatric from adult HLH and discuss the challenges of diagnosis and treatment of this devastating disease.
1295	A case of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with severe cardiac complications.	27793118	Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hematological disorder associated with severe systemic inflammation caused by an uncontrolled and ineffective immune response resulting in cytokine storm. Epstein-Barr virus (EBV) is the most common infectious agent in patients with the viral-associated HLH. Limited numbers of cases with cardiac complication have been demonstrated in other viral-associated HLH patients. Herein, we report a pediatric case of severe EBV-associated HLH with cardiac complications.A previously healthy 4-year-old Japanese female was admitted to a local hospital with a four day history of fever. Despite antibiotic treatment, her fever persisted to day 7 of the illness. Finally, the diagnosis of HLH was confirmed by fulfilling diagnostic criteria for HLH and pathological analysis of bone marrow aspiration. Real-time PCR detected a high copy number of EBV DNA in the peripheral blood mononuclear cells (PBMCs) at the time of hospital admission. During treatment according to HLH-2004 protocol, sudden cardiopulmonary arrest (CPA) occurred on day 30 of the illness and immediate resuscitation was successful. Acute myocarditis was considered the cause of the CPA. Although the treatment regimen was completed on day 88 of the illness, a remarkably high copy number of EBV DNA was still detected in her PBMCs. Based on our flow cytometric in situ hybridization analysis that revealed EBV infection of only B lymphocytes, we decided to administer rituximab to control the abnormal EBV infection. Afterwards the amount of EBV DNA decreased gradually to undetectable level on day 130 of the illness. Unfortunately, a coronary artery aneurysm was discovered at the left main coronary artery on day 180 of the illness. Finally, the patient was discharged from the hospital on day 203 of the illness without sequelae except for a coronary aneurysm.In this case report, EBV-HLH was complicated with cardiac symptoms such as myocarditis and coronary artery aneurysm. Although remarkably high copy number of EBV DNA was detected in PBMCs after completion of the HLH-2004 protocol, rituximab treatment resulted in a dramatic decrease of EBV DNA to undetectable levels. Rituximab treatment might have been beneficial for the patient's survival.
1296	A Case of Macrophage Activation Syndrome with Acute Respiratory Distress Syndrome.	27790499	Macrophage Activation Syndrome (MAS) is a rare form of Haemophagocytic Lymphohistiocytosis (HLH) associated with rheumatologic conditions. It is characterised by haemophagocytosis and cytokine overproduction, resulting from the activation and uncontrolled proliferation of T lymphocytes and macrophages. The patient being reported is an elderly female with Rheumatoid Arthritis (RA), who presented with fever and was found to have MAS. She later developed Acute Respiratory Distress Syndrome (ARDS) and died. Such a scenario has rarely been reported.
1297	Salvage therapy for refractory hemophagocytic lymphohistiocytosis: A review of the published experience.	27786410	Hemophagocytic lymphohistioytosis (HLH) is a severe, life-threatening hyperinflammatory disorder that requires prompt diagnosis and treatment. Approximately, 25-50% of patients with HLH fail to achieve remission with established regimens that include dexamethasone and etoposide, or methylprednisolone and antithymocyte globulin (ATG). Some of these patients may require salvage or alternative therapeutic approaches. There is a paucity of literature regarding effective salvage therapies for patients with refractory HLH. In this review, we summarize the published experience of four therapeutics reported for using at least two patients with HLH refractory to dexamethasone and etoposide or methylprednisolone and ATG.
1298	A retrospective analysis of 56 children with hemophagocytic lymphohistiocytosis.	27785117	The aim of this study was to investigate the etiological factors, clinical features, and prognostic factors in children with hemophagocytic lymphohistiocytosis (HLH).Fifty-six children with HLH in Wuhan Union Hospital, People's Republic of China, were retrospectively analyzed in recent years. We reviewed the medical records of 56 HLH children hospitalized from 2000 to 2013 to identify the possible prognostic factors.In more than half of the cases (64.29%), the etiological factor was found to be infection. Clinical characteristics such as prolonged fever (100.00%), hepatosplenomegaly (95.24%), and pancytopenia (100.00%) were observed. Characteristic laboratory values presented with increased ferritin (64.29%), triglycerides (78.57%), transaminases (80.95%), bilirubin (67.54%), lactate dehydrogenase (95.23%), and decreased fibrinogen (61.90%), natrium (40.48%), and potassium (30.94%). Bone marrow aspiration showed hemophagocytosis in 48 cases (85.71%). Forty-two patients were treated according to HLH-2004 protocol. Out of the 42 patients, 19 cases acquired remission and 13 cases died. Ten cases lost follow-up. High lactate dehydrogenase (>2,000 U/L), high bilirubin (>2 mg/mL), and younger age (<2 years) at the time of diagnosis were adverse prognostic factors.HLH is a life-threatening syndrome caused by complicated etiology. The level of lactate dehydrogenase and bilirubin accompanied with younger age were adverse factors.
1299	Clinical presentation and outcome of pediatric patients with hemophagocytic lymphohistiocytosis in China: A retrospective multicenter study.	27781387	Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous disease with major diagnostic and therapeutic difficulties. A large-scale multicenter study of pediatric HLH is still lacking in China.The Histiocytosis Study Group of the Chinese Pediatric Society conducted this retrospective study in 2014. A total of 323 patients diagnosed with HLH between 2011 and 2013 from 12 hospitals were registered.The median age at diagnosis was 2.2 years (range, 0-14.6 years), with a peak age of HLH onset at 0 to 3 years (63%). Mutations in HLH-related genes were found in 27.9% (24/86) patients who underwent genetic testing. PRF1, UNC13D, STXBP2 and LYST were the predominant genes involved. Sixteen patients (66.7%) presented with only monoallelic mutations in one gene. Epstein-Barr virus (EBV) infection was the major condition related to HLH, which was documented in 74.4% (201/270) of the patients who underwent EBV detection. Of 252 evaluable patients, 64.7% (163) achieved non-active disease at the eighth week and patients treated with a protocol containing etoposide presented higher remission rates (75.6% vs. 46.8%, P < 0.001). In multivariate analysis, a younger age at diagnosis (<12 months), platelet count less than 80×109 /L, central nervous system involvement, and initial treatment using a protocol without etoposide (not HLH-94/04) were independent prognostic factors indicating resistant disease.This study first multicenter assessment of HLH in China shows some different features in Chinese children with HLH compared with those in western countries, including older age, vulnerability to EBV infection, and a high proportion of patients with single monoallelic genetic mutations.
1300	Kikuchi-Fujimoto Disease Complicated with Reactive Hemophagocytic Lymphohistiocytosis.	27777431	Kikuchi-Fujimoto disease (KFD) is a benign cause of self-limiting subacute necrotizing lymphadenitis. KFD is rarely complicated with reactive hemophagocytic lymphohistiocytosis (HLH), and the clinical features of the simultaneous occurrence of these conditions are uncertain. A 30-year-old Japanese man with a persistent fever and sore throat presented to our hospital for treatment. Laboratory analysis showed bicytopenia, and radiological studies showed systemic lymphadenopathy accompanied by splenomegaly. A bone marrow examination showed hemophagocytic macrophages, suggesting HLH. Malignant lymphoma was suspected as a possible underlying disease, but the histology of the lymph nodes led to a final diagnosis of KFD and treatment with prednisolone (1 mg/kg/day), resulting in clinical improvement. This case highlighted the importance and difficulty of differentiating KFD from malignant lymphoma as an underlying condition of HLH. The literature review showed that patients with HLH-associated KFD may have higher serum ferritin and lactate dehydrogenase levels compared to typical KFD cases. Definite diagnosis based on pathological examination is essential for a better understanding of this rare disease. The presence of systemic lymphadenopathy does not exclude the possibility of KFD. This case serves to remind physicians that KFD is a potential etiology of HLH.
1301	Allogeneic hematopoietic stem-cell transplantation for adult and adolescent hemophagocytic lymphohistiocytosis: a single center analysis.	27431489	Myeloablative conditioning-based allogeneic hematopoietic stem-cell transplantation (allo-HSCT) in the treatment of adult and adolescent hemophagocytic lymphohistiocytosis (HLH) is rarely reported. We conducted a retrospective study of 30 adult and adolescent HLH transplanted for primary HLH (n = 4), tumor-HLH (n = 8), EBV-HLH (n = 14), and underlying disease-unknown (UDU)-HLH (n = 4). Peripheral blood stem cells (PBSCs) were the stem-cell source in all patients. Twenty-three patients were transplanted from HLA-haploidentical family donors, six from HLA-identical sibling donors, and one from a matched unrelated donor. Four patients appeared with mixed chimerism (MC), and no patient presented with graft failure. There was a high risk for EBV reactivation with an incidence of 47 %. Two patients developed post-transplant lymphoproliferative disorder (PTLD) and three were considered primary disease recurrent. With a median follow-up of 26 months, 19 patients survived and 11 patients died. The estimated 2-year overall survival (OS) was 63.3 ± 8.8 % in all patients, 100 % in primary HLH, 64.3 ± 12.8 % in EBV-HLH, 50.0 ± 17.7 % in tumor-HLH, and 50.0 ± 25.0 % in UDU-HLH. Myeloablative conditioning-based allo-HSCT is an effective treatment for adult and adolescent HLH to achieve complete remission and long-term survival.
1302	An Unusual Case of LCHAD Deficiency Presenting With a Clinical Picture of Hemophagocytic Lymphohistiocytosis: Secondary HLH or Coincidence?	27769081	There are published reports stating that some of the congenital metabolic diseases, such as lysinuric protein intolerance, multiple sulphatase deficiency, galactosemia, Gaucher disease, Pearson syndrome, and galactosialidosis, might lead to secondary hemophagocytic lymphohistiocytosis (HLH). However, to date, to our knowledge, the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency has never been investigated among patients with HLH. Here, we report on a patient who was referred to our institution for a differential diagnosis of pancytopenia, liver failure, and rhabdomyolysis. The patient was diagnosed with HLH. Further investigation revealed an underlying diagnosis of the LCHAD deficiency. Our case was reported to contribute to the literature, as well as the HLH clinic, emphasizing the consideration of LCHAD deficiency, especially in 1 to 6 months' old infants with laboratory findings of hypoglycemia, metabolic acidosis, and elevated creatine kinase.
1303	Infection Associated Secondary Hemophagocytic Lymphohistiocytosis in Sepsis Syndromes - A Tip of an Iceberg.	27766802	Hemophagocytic lymphohistiocytosis (HLH) is a rare, underdiagnosed, fatal and devastating hyperinflammatory syndrome that has gained increasing recognition over the past decade. Patients with HLH present with clinical and laboratory evidence of uncontrolled inflammation. Delay in diagnosis and management inevitably leads to a rapidly progressive and fatal course. In this case series, we present 7 cases of secondary HLH (sHLH) in adults with their presentation, course, and outcomes.We retrospectively looked at the 7 cases of secondary HLH who were diagnosed and managed in our institute between January 2013 and august 2015. Medical records were retrieved from medical records department and data analyzed and tabulated. The median age at diagnosis was 35 years (age range 26-72 years). Diagnosis was based on HLH 2004 diagnostic criteria.We report profile of 7 adult patients with sHLH. All patients had a short history of illness (<2 weeks) and presented uniformly with prolonged fever, bi or trilineage cytopenia and multiorgan dysfunction syndrome (MODS) at admission or developed MODS during the course of their illness. None of them had prediagnosed HLH. All patients fulfilled 5 to 6 of 8 criteria as per HLH 2014 diagnostic criteria. The median length of hospital stay was 12 days (range 7-50 days) and the median time to diagnosis was 5 days (range 3 to 21 days). Mortality was 57%.HLH is a rare and under-diagnosed clinical syndrome and is rapidly fatal if not diagnosed and managed timely. The cases reported in literature probably represent a tip of an iceberg of large number of undiagnosed cases mostly labeled as sepsis with MODS in critical care units. sHLH should be suspected in any patient who present with persistent and prolonged fever, transaminitis, cytopenia, and high serum Ferritin or dramatically rising serial serum Ferritin. Early diagnosis and prompt aggressive treatment are vital for patients' survival and favorable outcome.
1304	Hemophagocytic lymphohistiocytosis as an unusual cause of prolonged bicytopenia after chemotherapy.	27761260	Hemophagocytic lymphohistiocytosis (HLH) is a rare cause of cytopenia and is often associated with lymphoma. The occurrence of HLH in the course of lymphoma treatment could be an indicator for refractory disease.
1305	Symptomatic Primary Selective IgM Immunodeficiency - B Lymphoid Cell Defect in Adult Man with Secondary HLH Syndrome.	27759358	Selective immunoglobulin M deficiency(sIgMD) is a rare form of dysgammaglobulinaemia characterized by an isolated low level of serum immunoglobulin M (IgM). It was an incidence of less than 0.03% in the general population and 1% in hospitalized patients. sIgMD may occur as a primary or secondary condition. sIgMD is much more common than primary .Hemophagocytic lymphohistiocytosis (HLH) is also a rare but potentially fatal disease of normal but overactive histiocytes and lymphocytes and can be primary or secondary, characterized by the overwhelming activation of normal T lymphocytes and macrophages, invariably leading to clinical and hematologic alterations. We report an adult case of primary sIgMD with absent B lymphoid cells and secondary HLH syndrome who presented with recurrent infections, fever and pancytopenia.
1306	Successful treatment of recurrent CNS disease post-bone marrow transplant in children with familial hemophagocytic lymphohistiocytosis.	27565627	Central nervous system (CNS) involvement is a major cause of morbidity and mortality in patients with hemophagocytic lymphohistiocytosis (HLH). Current standard of care for CNS disease utilizes high-dose systemic dexamethasone plus intrathecal methotrexate and hydrocortisone prior to transplantation. However, the morbidity and mortality remains high and there are no clear guidelines posttransplantation for screening and treatment of CNS disease.We report a single-center retrospective case series of five patients with familial HLH (FHLH) who had CNS involvement post-bone marrow transplantation (BMT). All patients were monitored with monthly lumbar punctures (LPs) prior to developing neurologic symptoms. Treatment utilized systemic dexamethasone for CNS disease control.Five patients were monitored with monthly or bimonthly surveillance LPs and treated with systemic dexamethasone to control CNS relapse post-BMT. All patients are alive, a median of 34 months posttransplant (range 14-66 months). Four patients have mild neurological deficits, including mild speech delay (3) and one patient who exhibited brainstem herniation on day 0, due to CNS HLH, has made a substantial recovery of function with residual deficits of focal weakness on the right side. One patient has no deficits.Our data support vigilant screening posttransplant for occult CNS disease prior to the development of symptoms and the use of systemic dexamethasone to reverse disease progression. Future prospective trials are needed to evaluate this treatment strategy.
1307	Treatment of hemophagocytic lymphohistiocytosis.	27755125	Hemophagocytic lymphohistiocytosis (HLH) is a condition of uncontrolled immune activation with a high mortality rate. The recommended therapeutic guideline for HLH was published by the Histiocyte Society in 1994 and revised in 2004, which greatly improved the survival in patients with HLH. However, HLH is still a refractory disease for which the search for novel treatments continues. This article overviewed recent advances in treatment of HLH.Current practices in treatment extend from chemo-immunotherapy to some new cytokine-targeting biologicals, which are more effective to eliminate pathologically activated T cells and resist exaggerated cytokine storm. Preliminary results showed that some novel approaches to refractory HLH would potentially improve outcome of the fatal disease. Allogeneic hematopoietic stem cell transplantation after HLH remission represents the final solution for replacing defective cytotoxic T cells and even treating some underlying disease processes to prevent disease recurrence.A uniform protocol and algorithm for the treatment would not be appropriate for each patient given the heterogeneity of the underlying conditions. Further improvements in therapy require prospective trials to develop reasonable strategies for HLH patients in different subtypes, based on the underlying trigger, disease severity, as well as genetic background.
1308	Mitochondrial Disease as a Cause of Neonatal Hemophagocytic Lymphohistiocytosis.	27752381	Diagnosis of mitochondrial respiratory chain disorder (MRCD) is often difficult. Its pathogenesis is still unclear. We diagnosed MRCD by measuring the activity of the mitochondrial respiratory chain enzyme, and the patient also had hemophagocytic lymphohistiocytosis (HLH). A preterm female infant was born at 34 weeks of gestation. On day 6, HLH was revealed by bone marrow aspiration. She died on day 10 due to uncontrollable HLH. An autopsy was performed, and we measured the activity of the mitochondrial respiratory chain enzyme in the liver, muscle, and heart. The activity of complex I was decreased in all tissues. As we could not prove another origin of the HLH, she was diagnosed as having HLH caused by MRCD. It is useful to measure the activity of the mitochondrial respiratory chain enzyme for diagnosing MRCD. MRCD, which has a severe clinical course, may be related to HLH.
1309	Therapeutic plasma exchange in primary hemophagocytic lymphohistiocytosis: Reports of two cases and a review of the literature.	27743708	We report two children who were diagnosed as having primary hemophagocytic syndrome and who successfully underwent therapeutic plasma exchange (TPE). The first patient was a 6-month-old girl diagnosed with HLH who was admitted to the pediatric intensive care unit. The patient's clinical condition worsened on the 9th day of the HLH-2004 treatment protocol. Her ferritin level was found 50.000 ng/mL, and TPE was performed for 9 sessions, after which her clinical condition and laboratory findings improved. The patient is still on the HLH-2004 protocol and waits for a suitable stem cell transplantation donor. Case 2 involved a Syrian girl with HLH under follow-up who was receiving the HLH-2004 treatment protocol for reactivation. She presented to emergency department with fever, where her ferritin level was measured greater than 100.000 ng/mL; she was then transferred to the pediatric intensive care unit where four sessions of TPE were performed, after which her clinical condition and laboratory findings improved. However, the patient was admitted again one month later with gastrointestinal bleeding and died despite all efforts. By describing these two cases, we wish to emphasize that TPE can produce a rapid improvement until the time of stem cell transplantation in patients with hemophagocytic syndrome who do not respond to traditional treatments.
1310	Unusual Association of Hemophagocytic Lymphohistiocytosis in Systemic Lupus Erythematosus: Cases Reported at Tertiary Care Center.	27733745	BACKGROUND Hemophagocytic lymphohistiocytosis (HLH) in the background of systemic lupus erythematosus (SLE) is rare. Inability to discriminate between these two entities may be fatal for the patient. Here we report two cases of SLE with secondary HLH, one of which manifested HLH as the initial presentation, and the significance of HLH's timely diagnosis. CASE REPORT We describe two cases of SLE secondarily affected by HLH, which were diagnosed by various laboratory parameters and detection of profoundly reduced NK cell activity by using flow cytometry. Both our cases on investigation showed hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, and marked reduction or complete absence of NK cell activity. CONCLUSIONS Association of secondary HLH with SLE is rare, and when it occurs, differentiating it from lupus flare requires a high degree of suspicion and awareness of this association. Both have overlapping clinical features, but HLH is characterized by hyperferritinemia, hypofibrinogenemia, hypertriglyceridemia, and a decrease in erythrocyte sedimentation rate (ESR) and NK cell activity unlike SLE. Therefore, early diagnosis of HLH in the background of SLE facilitates timely selection of an appropriate treatment modality to prevent fatal complications.
1311	[Hemophagocytic lymphohistiocytosis: Analysis of 18 cases].	27726848	Hemophagocytic lymphohistiocytosis (HLH) is a serious condition, caused by an improper regulation of the immune response to different stimuli of the immune system. Early diagnosis and treatment are a challenge for the clinician.We conducted a retrospective study at our institution between 2010 and 2015, of adult patients diagnosed with HLH, in accordance with the criteria of the Histiocyte Society, analyzing their clinical characteristics, diagnostic and etiological studies and the outcome.Eighteen patients were analyzed. Median time to diagnosis was 24 days. We found neoplastic etiology in 8 cases (7 hematologic), while it was infection-related in 6 (4 visceral leishmaniasis), and an inflammatory disease in one. In the remaining 3, an underlying cause for the HLH was not found. Course of treatment was corticosteroids in 16 patients, associated with cyclosporine in 2 of them, one received immunoglobulins, while another received etoposide with tacrolimus.We emphasize the scarce use of etoposide therapy, the currently recommended treatment. Overall mortality was 44%, mainly associated with neoplastic etiology (67 compared to 16.6% mortality in infection-related etiology, P<.05).
1312	Hemophagocytic Lymphohistiocytosis and Bone Marrow Hemophagocytosis: A 5-Year Institutional Experience at a Tertiary Care Hospital.	27706506	The purpose of this study was to correlate the significance of bone marrow hemophagocytosis and analyze outcome data in patients with suspected hemophagocytic lymphohistiocytosis (HLH) at a tertiary care hospital during the course of 5 years.The pathology database of State University of New York Upstate Medical University, Syracuse, was searched for the terms "hemophagocytosis," "hemophagocytic syndrome," and "hemophagocytic lymphohistiocytosis" encompassing the period January 2009-December 2014. Bone marrow aspirate and biopsy specimens, along with ancillary laboratory studies, clinical course, and outcome data, were reviewed for each case.Of the 23 patients included in our study, HLH was diagnosed in 14 (60.8%). Bone marrow hemophagocytosis (HPC) was seen in a higher proportion of patients (78.5%) who were diagnosed as having HLH; however, 55.5% of the patients who were not diagnosed as having HLH also showed evidence of bone marrow HPC. Patients with malignancy-associated HLH had a markedly worse outcome compared with patients with nonmalignancy-associated HLH.Although bone marrow HPC is fairly sensitive, it is not specific to establish a diagnosis of HLH. A high index of clinical suspicion together with early diagnosis and treatment is imperative to improve outcomes in patients suspected of having HLH.
1313	Scrub typhus associated with hemophagocytic lymphohistiocytosis: A report of six pediatric patients.	27698778	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder that may be inherited or secondary to infection, malignancy or rheumatological disease. The aim of the present study was to highlight the clinical features of scrub typhus-associated HLH in children. A retrospective study was performed on 6 pediatric patients with scrub typhus-associated HLH. For each patient, medical records were reviewed and analyzed, and demographic, clinical and laboratory data and outcomes were collected. The duration of fever prior to admission ranged between 4 and 12 days. All patients exhibited persistent or intermittent fever, eschar, hepatosplenomegaly and lymphadenopathy at the time of diagnosis. Five patients experienced acute respiratory distress syndrome and disseminated intravascular coagulation (DIC) during hospitalization. Thrombocytopenia was detected in all patients with cytopenia involving two or three cell types, simultaneously. Coagulopathy with prolonged prothrombin time and/or activated partial thromboplastin time were noted in all patients. Markedly elevated serum ferritin levels (>1,500 µg/ml) were identified in all patients. Elevated lactate dehydrogenase (>1,000 U/l) was detected in 4 (66.7%) patients and elevated alanine aminotransferase was exhibited by 5 (83/3%) patients. Lung infiltrates and consolidation were the most common imaging findings. Only 1 patient succumbed, with DIC and multi-organ failure. Of the survivors, 1 patient was lost to follow-up, and the remaining patients are in remission with excellent general health, to date. In conclusion, HLH should be considered in severe pediatric cases of scrub typhus. Upon the early recognition of this syndrome, prompt and supportive treatment in the pediatric intensive care unit are vital.
1314	Mechanism of chromatin segregation to the nuclear periphery in C. elegans embryos.	2769565326607792	In eukaryotic organisms, gene regulation occurs in the context of chromatin. In the interphase nucleus, euchromatin and heterochromatin occupy distinct space during cell differentiation, with heterochromatin becoming enriched at the nuclear and nucleolar peripheries. This organization is thought to fine-tune gene expression. To elucidate the mechanisms that govern this level of genome organization, screens were carried out in C. elegans which monitored the loss of heterochromatin sequestration at the nuclear periphery. This led to the identification of a novel chromodomain protein, CEC-4 (Caenorhabditis elegans chromodomain protein 4) that mediates the anchoring of H3K9 methylation-bearing chromatin at the nuclear periphery in early to mid-stage embryos. Surprisingly, the loss of CEC-4 does not derepress genes found in heterochromatic domains, nor does it affect differentiation under standard laboratory conditions. On the other hand, CEC-4 contributes to the efficiency with which muscle differentiation is induced following ectopic expression of the master regulator, HLH-1. This is one of the first phenotypes specifically attributed to the ablation of heterochromatin anchoring.
1315	Hemophagocytic lymphohistiocytosis: A rare cause of recurrent encephalopathy.	27672548	We report an unusual case of recurrent encephalopathy due to acquired hemophagocytic lymphohistiocytosis (HLH) in a patient with propionic acidemia (PA). PA is an inherited metabolic disorder in which patients often present with encephalopathy and pancytopenia during metabolic decompensation. However, these patients may rarely develop HLH with similar presentation. This case illustrates the need to distinguish HLH induced encephalopathy from the one secondary to metabolic decompensation in these patients, as early diagnosis and treatment of HLH improves prognosis. This case also highlights the importance of considering HLH in patients presenting with unexplained encephalopathy, as early diagnosis and treatment is lifesaving in this otherwise lethal condition. To our knowledge this is the first case report of acquired HLH presenting as recurrent encephalopathy followed by complete recovery, in a metabolically stable patient with PA.
1316	Extracorporeal Membrane Oxygenation for Hemophagocytic Lymphohistiocytosis.	27670223	BACKGROUND Hemophagocytic lymphohistiocytosis (HLH) is a rare hematological disease characterized by an excessive inflammatory response to various triggers, resulting in rapid multi-organ failure. Its incidence may be underestimated due to its rarity, its variable clinical presentation, and its high mortality rate prior to diagnosis. Oftentimes, HLH is mistaken for refractory sepsis and improperly treated as such. Left untreated, the disease is universally fatal. With treatment, case series of adults with HLH report a 30-day mortality of up to 44% and an overall mortality of up to 75%. CASE REPORT We describe the use of extracorporeal membrane oxygenation (ECMO) in a previously healthy young man with HLH and acute respiratory distress syndrome (ARDS), a common sequela of HLH. ECMO was employed to provide temporary hemodynamic support, allowing for recovery of pulmonary function compromised during the initial cytokine storm. Additionally, and perhaps more importantly, implementation of ECMO provided the time necessary for the eventual diagnosis and treatment of HLH. CONCLUSIONS Although limited case reports and case series suggest that the use of ECMO in pediatric patients with HLH is associated with high mortality, our experience suggests that ECMO should not be rejected as a supportive modality in adults with HLH who have potentially recoverable cardiopulmonary function. We believe that ECMO may be appropriately instituted in select patients with HLH, or in rapidly deteriorating patients with an unknown illness refractory to conventional therapy, to allow for end-organ recovery, to reach a diagnosis, and to administer appropriate therapy.
1317	Hemophagocytic lymphohistiocytosis responding to withdrawal of gluten: a case report.	27658824	This is the first documented case of a patient with hemophagocytic lymphohistiocytosis in association with coeliac disease. There was complete clinical and biochemical remission of hemophagocytic lymphohistiocytosis following the introduction of a gluten-free diet.A 7-year-old white girl presented with fevers and maculopapular rash with a recent history of tonsillitis. Blood tests revealed thrombocytopenia (64×109/L), anemia (80 g/L), hypofibrinogenemia (1 g/L), and hyperferritinemia (71,378 μg/L). A bone marrow revealed evidence of hemophagocytosis, but the results of tests for the genetic or familial-associated hemophagocytic lymphohistiocytosis syndromes were negative. The results of screening tests for known secondary causes were negative. She was diagnosed as having hemophagocytic lymphohistiocytosis and following treatment with the hemophagocytic lymphohistiocytosis-2004 protocol these symptoms, in addition to the biochemical and hematological markers, completely resolved. She presented again 10 months later with fever, rash, and biochemical abnormalities suggestive of hemophagocytic lymphohistiocytosis. Her tissue transglutaminase was markedly raised and the results of blood tests revealed a genetic susceptibly to coeliac disease in the form of HLA-DQ2 positivity. She commenced a gluten-free diet and there was complete symptomatic and biochemical response without any further chemotherapy. She had further episodic rashes, each associated with the accidental intake of gluten.This is to the best of our knowledge the first documented case of hemophagocytic lymphohistiocytosis in association with coeliac disease. No other secondary cause found; she initially responded to chemoimmunotherapy specific for hemophagocytic lymphohistiocytosis but relapsed within a few months of cessation of treatment and then achieved complete remission on gluten withdrawal alone.
1318	Inhibitor of DNA-binding 1 promotes endothelial progenitor cell proliferation and migration by suppressing E2-2 through the helix-loop-helix domain.	27635429	Vascular endothelial damage is the major contributing factor to cardiovascular diseases. Recently, the therapeutic significance of endothelial progenitor cells (EPCs) has drawn increasing attention due to their roles in re-endothelialization following injury. The inhibitor of DNA-binding 1 (ID1) has been proven to promote EPC proliferation and migration, suggesting a critical function of ID1 in re-endothelialization. However, the underlying mechanisms remain undefined. In this study, ID1 was found to interact with E2-2 using immunoprecipitation analysis. Moreover, ID1 overexpression suppressed E2-2 expression and luciferase reporter activity; however, these effects were not observed in cells transfected with ID1 lacking the helix-loop-helix (HLH) domain (ID1ΔHLH). Further functional analysis corroborated that the upregulation of E2-2 markedly attenuated the ID1-mediated increase in EPC proliferation and migration. Furthermore, the HLH domain plays an important role in ID1-induced EPC proliferation and migration, as its deletion suppressed the positive regulatory effects of ID1 on EPC proliferation and migration. Taken together, the findings of our study confirm that ID1 promotes EPC proliferation and migration by suppressing E2-2 through the HLH domain in ID1. Therefore, ID1 may represent a potential therapeutic target for EPC-mediated re-endothelialization following vascular injury.
1319	Intravascular large B-cell lymphoma associated with silicone breast implant, HLA-DRB1*11:01, and HLA-DQB1*03:01 manifesting as macrophage activation syndrome and with severe neurological symptoms: a case report.	27634631	Silicone implants have been successfully used for breast augmentation and reconstruction in millions of women worldwide. The reaction to the silicone implant is highly variable; it can lead to local inflammatory symptoms, and sometimes to systemic symptoms and disease. Over 80 cases of anaplastic lymphoma kinase-negative anaplastic large cell lymphoma have been reported in patients with silicone breast implants and have been accepted as a new clinical entity. To the best of our knowledge, an intravascular large B-cell lymphoma associated with a silicone breast implant has not been reported previously.A 48-year-old Caucasian woman who presented with high fever was found to have splenomegaly on physical examination. A laboratory diagnosis revealed pancytopenia, hypertriglyceridemia, and hyperferritinemia. She developed signs of altered sensorium, hemiparesis, aphasia, and cauda equina syndrome. On further evaluation, she fulfilled the necessary five out of eight criteria for diagnosis of macrophage activation syndrome/hemophagocytic lymphohistiocytosis. Dexamethasone administration was followed by prompt improvement; however, 3 days later she again manifested high fever, which persisted despite administration of immunoglobulin and cyclosporine A. Her silicone breast implant was considered a possible contributor to her macrophage activation syndrome and was therefore removed. A histological examination of the capsule tissue showed an extensive lymphohistiocytic/giant cell foreign body reaction suggestive of autoimmune/inflammatory syndrome induced by adjuvants. However, the histological examination unexpectedly also revealed an intravascular large B-cell lymphoma.The genetic background of our patient with silicone breast implants might have predisposed her to three rare and difficult to diagnose syndromes/diseases: macrophage activation syndrome/hemophagocytic lymphohistiocytosis, autoimmune/inflammatory syndrome induced by adjuvants, and intravascular large B-cell lymphoma. The simultaneous manifestation of all three syndromes suggests causal interrelationships. Human leukocyte antigen testing in all women who undergo silicon breast implantation could in the future enable us to better evaluate the risk of potential side effects.
1320	Use of Extracorporeal Support in Hemophagocytic Lymphohistiocytosis Secondary to Ehrlichiosis.	27633921	Ehrlichiosis, caused by transmission of Ehrlichia chaffeensis to humans through the bite of an infected lone star tick, can lead to secondary hemophagocytic lymphohistiocytosis (HLH), a life-threatening condition caused by uncontrolled activation of the cellular immune system. We describe a child with HLH secondary to ehrlichiosis who developed multiorgan failure and was successfully managed with extracorporeal membrane oxygenation (ECMO). A 9-year-old boy developed headaches, fever, and sore throat after suspected tick exposure. He presented with pancytopenia, elevated ferritin, and soluble interleukin-2 receptor levels, all consistent with HLH. Bone marrow biopsy revealed hemophagocytosis. Polymerase chain reaction was positive for E chaffeensis He developed acute kidney injury, coagulation failure, hepatic insufficiency, and progressive respiratory failure requiring intubation. Due to refractory hypoxemia, he was cannulated for veno-venous ECMO. Continuous veno-venous hemofiltration was used to manage acute kidney injury and fluid overload. He received doxycycline and dexamethasone/etoposide for treatment of ehrlichiosis and HLH, respectively. Plasma exchange was used for thrombocytopenia-associated multiple organ failure. The patient was decannulated after 140 hours of ECMO and subsequently transferred for inpatient rehabilitation after extubation. Review of the Extracorporeal Life Support Organization Registry database identified 6 patients with tickborne diseases who received ECMO for organ support (survival in 3 of 6); ehrlichiosis was not reported in any of these cases. ECMO likely allowed a platform for stabilization and additional therapeutic interventions in this patient.
1321	Extracorporeal membrane oxygenation outcomes in children with hemophagocytic lymphohistiocytosis.	27625333	Pediatric patients with hemophagocytic lymphohistiocytosis (HLH) may develop refractory respiratory or cardiac failure that warrants consideration for extracorporeal membrane oxygenation (ECMO) support. The purposes of this study were to describe the use and outcomes of ECMO in pediatric HLH patients, to identify risk factors for hospital mortality and to compare their ECMO use and outcomes to the ECMO population as a whole.Pediatric patients (⩽ 18 years) with a diagnosis of HLH in the Extracorporeal Life Support Organization (ELSO) Registry were included.Between 1983 and 2014, data for 30 children with HLH were available in the ELSO registry and all were included in this study. All cases occurred in the last decade. Of the 30 HLH patients, 24 (80%) had a respiratory indication for ECMO and six (20%) had a cardiac indication (of which 4 were E-CPR and 2 cardiac failure). Of the 24 respiratory ECMO patients, 63% were placed on VA ECMO. Compared with all pediatric patients in the ELSO registry during the study period (n=17,007), HLH patients had worse hospital survival (non-HLH 59% vs HLH 30%, p=0.001). In pediatric HLH patients, no pre-ECMO risk factors for mortality were identified. The development of a hemorrhagic complication on ECMO was associated with decreased mortality (p=0.01). Comparing HLH patients with respiratory failure to patients with other immune compromised conditions, the overall survival rate is similar (HLH 38% vs. non-HLH immune compromised 31%, p=0.64).HLH is an uncommon indication for ECMO and these patients have increased mortality compared to the overall pediatric ECMO population. These data should be factored into decision-making when considering ECMO for pediatric HLH patients.
1322	The silent information regulator 1 (Sirt1) is a positive regulator of the Notch pathway in Drosophila.	27623778	The silent information regulator 1 (Sirt1) has been shown to have negative effects on the Notch pathway in several contexts. We bring evidence that Sirt1 has a positive effect on Notch activation in Drosophila, in the context of sensory organ precursor specification and during wing development. The phenotype of Sirt1 mutant resembles weak Notch loss-of-function phenotypes, and genetic interactions of Sirt1 with the components of the Notch pathway also suggest a positive role for Sirt1 in Notch signalling. Sirt1 is necessary for the efficient activation of enhancer of split [E(spl)] genes by Notch in S2N cells. Additionally, the Notch-dependent response of several E(spl) genes is sensitive to metabolic stress caused by 2-deoxy-d-glucose treatment, in a Sirt1-dependent manner. We found Sirt1 associated with several proteins involved in Notch repression as well as activation, including the cofactor exchange factor Ebi (TBL1), the RLAF/LAF histone chaperone complex and the Tip60 acetylation complex. Moreover, Sirt1 participates in the deacetylation of the CSL transcription factor Suppressor of Hairless. The role of Sirt1 in Notch signalling is, therefore, more complex than previously recognized, and its diverse effects may be explained by a plethora of Sirt1 substrates involved in the regulation of Notch signalling.
1323	PEG-aspargase and DEP regimen combination therapy for refractory Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	27613189	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is the most frequent subtype of secondary HLH triggered by infections. Previous studies have shown that ~30 % or more of patients with EBV-HLH do not respond to standard therapy. This study investigated the efficacy and safety profile of a modified DEP regimen in combination with PEG-aspargase (L-DEP) as a salvage therapy for refractory EBV-HLH.In this study from October 2014 to October 2015, 28 patients with refractory EBV-HLH received a L-DEP regimen at the Beijing Friendship Hospital, Capital Medical University. Treatment efficacy and adverse events were evaluated at 2 and 4 weeks after L-DEP treatment.Median EBV-DNA concentrations before and 2 weeks after receiving the L-DEP regimen were 9.6 × 10(5) (1.5 × 10(4) - 1 × 10(9)) copies/mL and 2.2 × 10(5) (3.8 × 10(2) - 1.2 × 10(7)) copies/mL, respectively; the post-treatment values were significantly lower than that of the pretreatment (P = 0.048). Nine of the 28 study patients achieved complete response (CR) and 15 partial response (PR), resulting in an overall response rate of 85.7 % (CR+PR). Four patients who did not achieve response died within 4 weeks of receiving L-DEP. Thirteen of the 24 patients who achieved partial or complete response received subsequent allogenic hematopoietic stem cell transplantation (allo-HSCT). Ten of these 13 patients survived until 1 March 2016. The major adverse effects of the L-DEP regimen were high serum amylase concentrations, abnormal liver function, and coagulation disorders.This study suggests that L-DEP is a safe and effective salvage therapy prior to allo-HSCT for refractory EBV-HLH and increases the possibility of such patients receiving allo-HSCT. A prospective multicenter large-scale clinical trial that aims to validate the L-DEP regimen for refractory EBV-HLH is currently underway (ClinicalTrails.gov Identifier: NCT02631109).
1324	[Hemophagocytic lymphohistiocytosis : A diagnostic challenge on the ICU].	27612865	Hemophagocytic lymphohistiocytosis (HLH) has well been studied as a genetic disorder in children (primary HLH). Mutations in the regulatory complex of the cellular immune synapse lead to a loss of function of cytotoxic T‑cells and natural killer cells with excessive inflammation based on a cytokine storm. During the last decade, an increasing number of adult HLH patients without a family history of HLH (secondary or acquired HLH) have been reported. Various triggers - infections, malignancies or autoimmune diseases - result in an acquired loss of function of these cells and a sepsis-like disease. Missed or late diagnosis is believed to be a major cause of the high mortality.To describe the current knowledge on HLH and to raise awareness.Analysis of case reports, current studies, and expert recommendations.Increased vigilance in identifying the adult form of HLH resulted in an increasing number of case reports over the past few years. HLH patients typically present with a clinical phenotype resembling severe sepsis or septic shock with fever, cytopenia, and organomegaly, which do not or insufficiently respond to anti-infective treatment. Early recognition of HLH distinction from sepsis, and prompt initiation of treatment - which is fundamentally different from sepsis - are crucial for improved outcome. A promising diagnostic parameter is ferritin, which has gained sufficient specificity, but only in the context of the triad of fever, cytopenia, and organomegaly. Treatment of adult HLH patients requires immunosuppression, with strict therapeutic guidance derived from the triggering disease.Because of the similar clinical presentation to that of sepsis, HLH is often not recognized, resulting in a fatal outcome. In "sepsis" patients on the ICU with deterioration despite a standard of care, HLH needs to be considered by testing for ferritin when considering differential diagnoses. The complexity of the illness requires interdisciplinary patient care with specific integration of the hematologist in the diagnostic workup and therapeutic management, because of the frequent use of chemotherapy-based immunosuppression.
1325	Correlation of Antiglobulin Reactivity and Severity of Pancytopenia in a Patient with Hemophagocytic Lymphohistiocytosis: A Case Report and Review of Literature.	27610283	A 46‑year‑old obese male with a medical history of thalassemia minor presented to the emergency room with complaints of severe fatigue and jaundice worsening over two weeks. On further evaluation, the patient was found to have significant hyperbilirubinemia and transaminitis. The hospital course was further complicated by pancytopenia requiring multiple transfusions, worsening hyperbilirubinemia, severe hyperferritinemia, hypofibrinogenemia, and hypertriglyceridemia. He was also found to have splenomegaly and evidence of hemophagocytosis on bone marrow biopsy. On further testing, the patient was also found to have evidence of hemolysis along with a positive direct Coomb's test consistent with autoimmune hemolytic anemia (AIHA), and elevated soluble IL-2 receptor level. The patient was subsequently diagnosed with hemophagocytic lymphohistiocytosis (HLH). He was treated with HLH-94 protocol along with rituximab for AIHA which resulted in improvement of patient's condition. We present a case of HLH with no prior history of autoimmune disease, associated with Coomb's positive AIHA that resolved after therapy for HLH. Our case also delineates how the intensity of antiglobulin reactivity, if present, may correlate with severity of the disease, its progression, and response to treatment.
1326	Successful allogeneic hematopoietic stem cell transplantation in a boy with X-linked inhibitor of apoptosis deficiency presenting with hemophagocytic lymphohistiocytosis: A case report.	27602064	X-linked inhibitor of apoptosis (XIAP) deficiency, also known as X-linked lymphoproliferative syndrome type 2 (XLP2), is a rare inherited primary immunodeficiency resulting from the XIAP (also known as BIRC4) mutation. XIAP deficiency is mainly associated with familial hemophagocytic lymphohistiocytosis (HLH) phenotypes, and genetic testing is crucial in diagnosing this syndrome. Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only successful strategy for the treatment of this disease; however, a limited number of studies has been published concerning the outcomes of allogeneic HSCT in patients with XIAP deficiency. The present study reported a successful allogeneic HSCT performed to treat XIAP deficiency in a Chinese boy presenting with HLH. Polymerase chain reaction and DNA sequencing were performed to confirm the diagnosis of XIAP deficiency, and allogeneic HSCT was performed. Genetic tests revealed a two-nucleotide deletion (c.1021_1022delAA) in the patient, which was inherited from his mother, and resulted in frameshift mutation and premature stop codon (p.N341fsX348); this is considered to be a disease-causing mutation. The XIAP deficiency patient underwent allogeneic HSCT, receiving busulfan-containing reduced intensity myeloablative conditioning regimen, with a good intermediate follow-up result obtained. Therefore, genetic testing is essential to confirm the diagnosis of XIAP deficiency and detect the carrier of mutation. The present case study may promote the investigation of allogeneic HSCT in patients with XIAP deficiency.
1327	Human monocytic ehrlichiosis complicated by hemophagocytic lymphohistiocytosis and multi-organ dysfunction syndrome.	27575459	Human monocytic ehrlichiosis can manifest as a disease causing multi-organ failure. Rarely, it can cause secondary hemophagocytic lymphohistiocytosis (HLH). Early diagnosis and initiation of treatment for both ehrlichiosis and HLH is lifesaving. Therefore, clinical suspicion of HLH must remain high in the setting of an ehrlichiosis infection.
1328	Hemophagocytic lymphohistiocytosis associated with Epstein-Barr virus in the central nervous system.	27574808	Hemophagocytic lymphohistiocytosis (HLH) is a rare immune hyperactivation syndrome which may be primary (genetic) or secondary to various immune-related conditions including infection, immunodeficiency, and malignancies. Rapid diagnosis and treatment are essential because it can be associated with significant morbidity and mortality. Epstein-Barr virus (EBV) is a known infectious cause of acquired HLH, but EBV-associated HLH involving the central nervous system is rare and not well characterized neuropathologically. We report a case of fatal EBV-associated HLH with severe involvement of the central nervous system showing florid hemophagocytosis in the choroid plexus, with extensive neuron loss and gliosis in the cerebrum, cerebellum, and brainstem.
1329	Hemi-meningitis with hemophagocytic lymphohistiocytosis.	27570395	Hemophagocytic lymphohistiocytosis (HLH) is a rare lymphoproliferative disorder. HLH may occur as a complication of Epstein-Barr virus (EBV), particularly in patients with immunodeficiencies. Herein, we describe a 16-year-old girl with neurological complications associated EBV-induced HLH. Her cerebral magnetic resonance imaging (MRI) showed contrast-enhanced axial T1-weighted images with enhancement of meningeal surface in the right hemisphere that was consistent with right hemi-meningitis. Hydrocephalus, dilated subdural spaces, delayed myelination, edema, diffuse parenchymal atrophy, calcifications, diffuse/patchy white matter abnormalities have all been previously described with HLH. To the best of our knowledge, this is the first case of hemi-meningitis associated with HLH. We suggest that clinicians should consider HLH with vascular disorders when they determine unilateral meningitis on a brain MRI.
1330	A Rare Cause of Hemophagocytic Lymphohistiocytosis: Fusobacterium Infection-A Case Report and Review of the Literature.	27563473	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome characterized by excessive activation of the immune system. Bacterial infections are very rare precipitants of this disease. A 19-year-old gentleman presented with headache, fatigue, and malaise. He was found to be hypotensive, tachycardic, and febrile. Broad spectrum antibiotics were initiated, and a lumbar puncture ruled out meningitis. Patient progressively developed shock that required use of vasopressors, as well as renal and respiratory failure. Blood cultures grew Fusobacterium necrophorum. Given continued fevers despite appropriate antimicrobials, a bone marrow biopsy was performed revealing increased histiocytes with hemophagocytosis. Dexamethasone was added with dramatic clinical improvement. Our case highlights Fusobacterium as a rare precipitant of HLH and proves that a high index of clinical suspicion is crucial for early diagnosis of HLH, allowing for prompt initiation of HLH-specific immunosuppressive therapy that can be life-saving.
1331	Use of a mouse model to identify a blood biomarker for IFNγ activity in pediatric secondary hemophagocytic lymphohistiocytosis.	27559680	Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.
1332	Incidence and Risk Factors for Developing Dengue-Associated Hemophagocytic Lymphohistiocytosis in Puerto Rico, 2008 - 2013.	27556807	Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal disorder characterized by fever, pancytopenia, hepatosplenomegaly, and increased serum ferritin. HLH is being increasingly reported as a complication of dengue, a common tropical acute febrile illness.After a cluster of pediatric dengue-associated HLH patients was identified during the 2012-2013 dengue epidemic in Puerto Rico, active surveillance and a case-control investigation was conducted at four referral hospitals to determine the incidence of HLH in children and identify risk factors for HLH following dengue. Patients with dengue-associated HLH (cases) were matched by month of illness onset and admission hospital to dengue patients that did not develop HLH (controls). During 2008-2013, a total of 33 HLH patients were identified, of which 22 (67%) were associated with dengue and 1 died (dengue-associated HLH case-fatality rate: 4.5%). Two patients with dengue-associated HLH had illness onset in 2009, none had illness onset during the 2010 dengue epidemic, and 20 had illness onset during the 2012-2013 epidemic. Frequency of infection with either dengue virus (DENV)-1 or DENV-4 did not differ between cases and controls. Cases were younger than controls (median age: 1 vs. 13 years, p < 0.01), were hospitalized longer (18 vs. 5 days, p < 0.01), and were admitted more frequently to pediatric intensive care units (100% vs. 16%, p < 0.01). Cases had co-infection (18.2% vs. 4.5%, p = 0.04), recent influenza-like illness (54.5% vs. 25.0%, p = 0.01), and longer duration of fever (7 vs. 5 days; p < 0.01). Cases were more likely to have lymphadenopathy, hepatomegaly, splenomegaly, anemia, and elevated liver transaminases (p ≤ 0.02).During this cluster of dengue-associated HLH cases that was temporally associated with the 2012-2013 epidemic, most patients with dengue-associated HLH were infants and had higher morbidity than dengue inpatients. Physicians throughout the tropics should be aware of HLH as a potential complication of dengue, particularly in patients with anemia and severe liver injury.
1333	18F-FDG PET/CT for identifying the potential causes and extent of secondary hemophagocytic lymphohistiocytosis.	27537853	We aimed to evaluate the value of 18F-FDG positron emission tomography/computed tomography (PET/CT) for identifying the possible causes of secondary hemophagocytic lymphohistiocytosis (HLH).Forty-five cases (17 female, 28 male; age, 17-79 years) with secondary HLH were included. The standard of reference for diagnosis in all patients was a combination of histology, clinical results (medical history, physical examination, and laboratory test results), and follow-up imaging for at least 12 months. All cases underwent 18F-FDG PET/CT to identify the possible trigger in HLH.Of 45 secondary HLH cases 10 (22.2%) were associated with infection, seven (15.6%) with rheumatic disease, and 28 (62.2%) with lymphoma. PET/CT images of 22 secondary HLH cases (48.9%) showed true positive results. PET/CT images demonstrated obvious tracer uptake in five of 10 secondary HLH cases with infection, one of three cases with lupus, two of two cases with rheumatoid arthritis, one of two cases with adult-onset Still disease, and 13 of 28 cases with lymphoma.PET/CT is helpful for identifying the possible trigger (infection or malignant disease) and extent of secondary HLH. However, PET/CT alone is not sufficient to make a correct differential diagnosis.
1334	[The significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary hemophagocytic lymphohistiocytosis].	27535855	To investigate the significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary hemophagocytic lymphohistiocytosis (HLH).Four cases of primary HLH patients with PRF1, UNC13D and SH2D1A gene mutations were conducted pedigree investigation, including family genetic screening and detections of immunological parameters (NK cell activity, CD107a degranulation and expression of HLH related defective protein), to evaluate the significance of these different indicators in the diagnosis of primary HLH and explore their correlations.The DNA mutations of the four families included missense mutation c.T172C (p.S58P) and non- frameshift deletions c.1083_1094del (p.361_365del), missense mutation c.C1349T (p.T450M) and frameshift mutation c.1090_1091delCT (p.T364fsX93) in PRF1 gene, missense mutation c.G2588A (p.G863D) in UNC13D gene and hemizygous mutation c.32T>G (p.I11S) in SH2D1A gene. The patients and their family members presented decreased NK cell activities. Individuals who carried mutations of PRF1 gene and SH2D1A gene showed low expression of perforin (PRF1) and signaling lymphocytic activation molecule associated protein (SAP). And the patient with UNC13D gene mutation and his family member with identical mutation showed significant reducing cytotoxic degranulation function (expression of CD107a).Pedigree genetic screening and rapid detection of immunological parameters might play an important role in the diagnosis of primary HLH, and both of them had good consistency. As an efficient detection means, the rapid immunological detection indicators would provide reliable basis for the early diagnosis of the primary HLH.
1335	Hyperferritinemic Sepsis: An Opportunity for Earlier Diagnosis and Intervention?	27532033	We describe a case of an infant with HSV meningitis and septic shock who demonstrated a remarkably high serum ferritin level. Aggressive pediatric intensive care and the administration of high-dose glucocorticoids were not able to reverse the multiple organ dysfunctions. Subsequent autopsy identified the presence of hemophagocytosis, thus the patient fulfilled hemophagocytic lymphohistiocytosis (HLH) criteria post-mortem. This case highlights that serum ferritin may be an important early indicator of mortality in sepsis due to a cytokine storm similar to macrophage activation syndrome and HLH.
1336	Hemophagocytic lymphohistiocytosis: a rare complication of autologous stem cell transplantation.	27516033	Hemophagocytic lymphohistiocytosis (HLH) is a very severe and rare syndrome of pathologic immune activation characterized by cytopenia and clinical signs and symptoms of extreme inflammation. HLH is usually fatal without treatment so that accurate and timely diagnosis is very important. The syndrome occurs as a familial disorder (familial HLH - FLH) or as an acquired condition (secondary - sHLH) in association with a variety of pathologic states: infections, rheumatologic, malignant or metabolic diseases. Malignancy associated HLH is primarily reported in T÷NK (natural killer)-cell malignancies but also in B-cell neoplasms and other types of cancer. HLH has also been reported in rare cases as a highly fatal and difficult to diagnose complication of stem cell transplantation (SCT). In this paper, we present the case of a young male patient who underwent autologous SCT as consolidation therapy for a T÷NK-cell lymphoma, complicated with graft failure due to HLH. The patient was successfully treated with corticosteroids, Etoposide, Cyclosporine and immunoglobulins. As a particularity, he developed a second B-cell neoplasia a few months after SCT.
1337	Hemophagocytic Lymphohistocytosis in the Chinese Han Population May Be Associated with an STXBP2 Gene Polymorphism.	27513731	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease of severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages. In this study, we aimed to explore the genetic factors involved in the pathogenesis of both acquired and familial type HLH.The ION TORRENT semi-conductor sequencing method was used to sequence samples from 10 patients who were diagnosed or highly suspected of HLH. Then SNP rs2303116 of STXBP2 genotyping was performed by Sanger sequencing method on samples from 24 patients with HLH and 182 normal controls. Genotype frequencies were then compared and tested by multivariate logistic regression. Finally, the potential impact of rs2303116 on splicing factor binding ability was evaluated using the ESEfinder 3.0 online tool.A total of 92 variants were identified in 10 HLH patients, of which 24 variants were rare variants (MAF<0.01), while the remaining 68 variants were common variants (MAF>0.01). Among them, 8 different genetic variations in the STXBP2 sequence were identified. We focused on the synonymous SNP rs2303116, as 30% of patients had CT/TT genotype. SNP genotyping was further performed on 24 HLH patients and 182 healthy control cohorts, and the results indicated a significantly elevated CT/TT genotype frequency of rs2303116 in HLH patients compared with healthy controls (patients 37.5% VS. controls 13.2%, P = 0.009, OR = 3.900, 95% CI 1.537-9.899). Multivariate logistic regression analysis indicated that being female (OR 0.350, 95% CI 0.143-0.861, P = 0.018) and of an older age (>43y, OR 0.312, 95% CI 0.118-0.822, P = 0.014) were independent protective factors, and the rs2303116 CT/TTgenotype (OR 3.900, 95% CI 1.537-9.899, P = 0.009) was an independent risk factor for HLH pathogenesis. By comparing the clinical parameters between HLH patients with CT/TT and CCgenotypes, we found that the patients with CT/TT genotype had significantly lower levels of fibrinogen, indicating more aggravated macrophage activation. In silico analysis of splice factor binding to rs2303116 CT/TT genotypes showed significant decrease for SRSF1 but increase for SRSF6, which suggested abnormal splicing machinery was associated with HLH pathogenesis.Our study demonstrated for the first time that HLH patients had significantly higher frequencies of the STXBP2 gene polymorphism rs2303116 variant compared with a healthy Chinese Han population, through clinical comparisons and further predictions we suggested regulation of alternative splicing by alleles of SNP rs2303116 could be involved in HLH pathogenesis.
1338	[Hemophagocytic lymphohistiocytosis in adults].	27509340	Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome with established diagnostic criteria, and well characterized gene mutations in children. In contrast, acquired HLH in adults (aHLH) due to the heterogeneity of trigger diseases (infections, malignancies and autoimmune diseases) is less clear defined. There is both a risk of under- and overtreatment. Due to the lack of validated diagnostic criteria for HLH in adults, diagnostic and therapeutic delay are significant risk factors for patient outcome. This article presents the current status and new developments in epidemiology, pathophysiology, diagnosis and treatment of aHLH.
1339	Cytomegalovirus-Induced Hemophagocytic Lymphohistiocytosis in an Extreme Preterm Infant: Recognition and Therapy Leading to Recovery.	27501069	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease that can be triggered by cytomegalovirus, a relatively common infectious exposure to neonates. The clinical presentation is common to many acute illnesses seen in extreme premature infants; however, there are key clinical and laboratory findings that can lead to the diagnosis.We present a case of an extreme premature infant of 25 weeks' gestation who developed cytomegalovirus-induced HLH. Using the current published protocols that are used in pediatric cancer can be adapted for use in a premature infant, which led to remission of HLH and eventual discharge from the neonatal intensive care unit.There are published treatment protocols used in pediatric oncology that when initiated early can lead to favorable outcomes and remission in even the most fragile neonates.Additional studies are needed on the pharmacokinetics, dosing, and side effects on medications used for treatment of HLH in preterm infants. Additional research is needed to improve the clinician's ability to reach the diagnosis as well as define treatment strategies that provide optimal outcomes.
1340	Domestic dengue infection with hemophagocytic lymphohistiocytosis successfully treated by early steroid therapy.	27498730	A 34-year-old man, working at a park in Tokyo, Japan, was repeatedly bitten by mosquitoes while cutting grass. He was hospitalized with sudden fever, fatigue, and weakness. He was eventually diagnosed with dengue virus infection, detected using reverse transcription polymerase chain reaction for the genome and by the presence of nonstructural protein 1 in his peripheral blood. Symptomatic treatments such as acetaminophen for the fever were not effective. Moreover, peripheral blood examination showed drastically decreased white blood cells and platelets, as well as marked elevations of ferritin and soluble interleukin 2 receptor. Furthermore, bone marrow examination revealed increased macrophages with hemophagocytosis. Dengue infection with hemophagocytic lymphohistiocytosis (HLH) was ultimately diagnosed. Half-dose steroid pulse therapy for three days dramatically reduced his temperature, thereby ameliorating physical symptoms and restoring normal peripheral blood data. He was discharged 12 days after admission. Dengue infection with HLH is rare and this is the first report, to our knowledge, of domestic dengue infection with HLH in Japan. Early steroid therapy may be effective in such cases.
1341	The Histologic Classifications of Lung Adenocarcinomas Are Discriminable by Unique Lineage Backgrounds.	27496649	Lung adenocarcinomas are a heterogeneous set of diseases with distinct genetic and histologic characteristics. Besides the discovery of oncogenic mutations and introduction of the histologic classifications (2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society and 2015 WHO), increasing evidence has linked this intertumor heterogeneity to the lung lineage-specific pathways and lineage genes. Therefore, in this study, we assessed the gene expression of identified lung lineage genes to study their role in distinguishing lung adenocarcinoma diversities.A total of 278 surgically resected lung adenocarcinomas were included. Each case was evaluated for genetic mutations and histologic classification. Lineage genes associated with respiratory tract differentiation (NK2 homeobox 1 gene [NKX2-1], GATA protein binding 6 gene [GATA6], foxhead box J1 gene [FOXJ1], and SAM pointed domain containing ETS transcription factor gene [SPDEF]) and stem/basal-like status (inhibitor of DNA binding 2, HLH protein gene [ID2], POU class 5 homeobox 1 gene [POU5F1], SRY-box 2 gene [SOX2], and v-myc avian myelocytomatosis viral oncogene homolog gene [MYC]) were selected. mRNA expression of these genes in each tumor sample was assessed by quantitative real-time polymerase chain reaction and normalized to paired normal lung tissue.Distinct lineage gene expressions were found on the basis of genetic and histologic diversities. Expression of NKX2-1, GATA6, FOXJ1, and POU5F1 exhibited a significant linear relationship across histologic subgroups that was independent of genetic mutation status. Expression levels of NKX2-1 and POU5F1 were also associated with EGFR mutation status, independent of histologic subtypes. Further analysis revealed that the overexpression of SPDEF defined longer relapse-free survivals, especially in stage I disease.For the first time, we showed the unique lineage backgrounds of different histologic subtypes and oncogenic mutations. Assessing this added parameter might be beneficial in discriminating intertumor heterogeneity, advancing target exploration, developing theranostic/prognostic biomarkers, and designing clinical trials.
1342	Hemophagocytic lymphohistiocytosis associated with SFTS virus infection: A case report with literature review.	27495089	Severe fever with thrombocytopenia syndrome (SFTS) is a new emerging zoonosis. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome caused by hyperinflammation. Here, we report the case of SFTS-associated HLH.A 62-year-old man was admitted to local hospital with 8 days of fever and chill. He had leukopenia, thrombocytopenia, and developed seizure. An attending physician examined bone marrow to rule out hematologic malignancy. He was transferred to tertiary referral hospital for suspicious HLH. We decided to confirm its histologic feature for sure. Bone marrow and liver biopsy showed hemophagocyotic histiocytes. Serological tests for other infections were all negative except SFTS virus polymerase chain reactions (PCRs) as positive from serum, bone marrow, bronchoalveolar lavage fluid, and liver biopsy specimen. A definitive diagnosis was SFTS-associated HLH. During 2 weeks of conservative treatment, he succeeded in recovery from multiple organ failure.SFTS should be considered one of differential diagnosis of HLH. In certain endemic areas, SFTS infection deserves clinicians' attention because it can be presented hematologic diseases as HLH.
1343	X-linked Inhibitor of Apoptosis Complicated by Granulomatous Lymphocytic Interstitial Lung Disease (GLILD) and Granulomatous Hepatitis.	27492372	The X-linked inhibitor of apoptosis (XIAP) deficiency is a primary immunodeficiency characterized by Epstein-Barr virus (EBV)-driven hemophagocytic lymphohistiocytosis (HLH), splenomegaly, and colitis. Here, we present, for the first time, granulomatous hepatitis and granulomatous and lymphocytic interstitial lung disease (GLILD) as manifestations of XIAP deficiency. We report successful treatment of GLILD in XIAP deficiency with rituximab and azathioprine and discuss the role of XIAP deficiency in immune dysregulation.
1344	Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors.	27487365	Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.
1345	5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4.	27485769	We evaluated DNA binding of the B-HLH family members TCF4 and USF1 using protein binding microarrays (PBMs) containing double-stranded DNA probes with cytosine on both strands or 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) on one DNA strand and cytosine on the second strand. TCF4 preferentially bound the E-box motif (CAN|NTG) with strongest binding to the 8-mer CAG|GTGGT. 5mC uniformly decreases DNA binding of both TCF4 and USF1. The bulkier 5hmC also inhibited USF1 binding to DNA. In contrast, 5hmC dramatically enhanced TCF4 binding to E-box motifs ACAT|GTG and ACAC|GTG, being better bound than any 8-mer containing cytosine. Examination of X-ray structures of the closely related TCF3 and USF1 bound to DNA suggests TCF3 can undergo a conformational shift to preferentially bind to 5hmC while the USF1 basic region is bulkier and rigid precluding a conformation shift to bind 5hmC. These results greatly expand the regulatory DNA sequence landscape bound by TCF4.
1346	Anti-aging treatments slow propagation of synucleinopathy by restoring lysosomal function.	27485532	Aging is the major risk factor for neurodegenerative diseases that are also associated with impaired proteostasis, resulting in abnormal accumulation of protein aggregates. However, the role of aging in development and progression of disease remains elusive. Here, we used Caenorhabditis elegans models to show that aging-promoting genetic variations accelerated the rate of cell-to-cell transmission of SNCA/α-synuclein aggregates, hallmarks of Parkinson disease, and the progression of disease phenotypes, such as nerve degeneration, behavioral deficits, and reduced life span. Genetic and pharmacological anti-aging manipulations slowed the spread of aggregates and the associated phenotypes. Lysosomal degradation was significantly impaired in aging models, while anti-aging treatments reduced the impairment. Transgenic expression of hlh-30p::hlh-30, the master controller of lysosomal biogenesis, alleviated intercellular transmission of aggregates in the aging model. Our results demonstrate that the rate of aging closely correlates with the rate of aggregate propagation and that general anti-aging treatments can slow aggregate propagation and associated disease progression by restoring lysosomal function.
1347	Hemophagocytic lymphohistiocytosis with leukoencephalopathy in a patient with dermatomyositis accompanied with peripheral T-cell lymphoma: a case report.	27484255	Hemophagocytic lymphohistiocytosis associated with autoimmune diseases is seen in patients with systemic juvenile idiopathic arthritis, adult-onset Still's disease, and systemic lupus erythematosus, whereas it is rarely seen in patients with dermatomyositis. In addition, central nervous system involvement with dermatomyositis is rare. To the best of our knowledge, this is the first case of hemophagocytic lymphohistiocytosis complicated by leukoencephalopathy in a patient with dermatomyositis accompanied with peripheral T-cell lymphoma.A 17-year-old Asian male adolescent with dermatomyositis and hemophagocytic lymphohistiocytosis that were controlled with corticosteroid therapy presented to our hospital with high fever and altered consciousness. Brain magnetic resonance imaging revealed multiple cerebral lesions. We diagnosed the central nervous system lesions as leukoencephalopathy secondary to dermatomyositis and hemophagocytic lymphohistiocytosis. Because corticosteroid and cyclophosphamide pulse therapy was ineffective, he was treated with a modified hemophagocytic lymphohistiocytosis-2004 protocol, which resulted in the disappearance of the lesions of his central nervous system.Our findings suggest that the hemophagocytic lymphohistiocytosis-2004 protocol including etoposide should be initiated immediately in patients with hemophagocytic lymphohistiocytosis who respond poorly to treatment for the underlying disease. Moreover, irrespective of the underlying disease, patients with hemophagocytic lymphohistiocytosis with central nervous system lesions might require bone marrow transplantation.
1348	Investigation of hemophagocytic lymphohistiocytosis in severe sepsis patients.	27481757	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition characterized by uncontrolled inflammation and has common clinical and laboratory features with sepsis. The aim of this study was to investigate patients treated with severe sepsis who had bicytopenia for the presence of HLH.Patients with severe sepsis who were non-responsive to treatment and developed at least bicytopenia were included. Peripheral blood samples were collected and stored for later evaluation for natural killer (NK) activity and soluble interleukin-2 receptor levels. Diagnostic criteria of HLH were retrospectively analyzed.Seventy-five of 382 patients (20%) were followed as severe sepsis and septic shock. Among them, 40 patients had bicytopenia. Twenty-six of 40 patients were excluded due to the presence of active solid or hematological malignancies. Three patients died before fulfillment of HLH criteria and one patient denied to give consent. All of the remaining 10 patients had at least five of the eight criteria according to criteria of the Histiocyte Society. Only one of 10 patients was diagnosed as HLH and received treatment during intensive care unit stay. None of the 10 patients survived.This study emphasizes to consider the possibility of HLH and the need of rapid assessment of patients with severe sepsis who had bicytopenia and were resistant to treatment in intensive care.
1349	Acute myeloid leukemia following etoposide therapy for EBV-associated hemophagocytic lymphohistiocytosis: a case report and a brief review of the literature.	27473573	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disorder characterized by prolonged fever, cytopenia, hepatosplenomegaly, rash, icterus, and other neurological symptoms. Successful treatment of HLH by etoposide has improved outcomes for children with HLH. However, the development of treatment-related acute myeloid leukemia (t-AML) after the usage of this drug is a concern.We report a case of acquired EBV-triggered HLH with progression to t-AML following etoposide therapy with cytogenetic abnormality for t (11; 19) (q23; p13) resulting in MLL gene fusion. The development of t-AML was detected 23 months after HLH diagnosis.Although the successful treatment of HLH by etoposide has improved outcomes for children with HLH, t-AML is a serious complication of topoisomerase II inhibitor therapy and is characterized by the presence of gene rearrangement. This study suggests that HLH patients undergoing therapy with HLH-2004 protocol need monitoring for future malignancy, especially in the initial 2 to 3 years.
1350	High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis.	27471778	Perforin-1 mutations result in a potentially fatal hemophagocytic lymphohistiocytosis (HLH) with heightened immune activation, hypercytokinemia, pancytopenia, and end-organ damage. At present, hematopoietic stem cell (HSC) transplantation is curative, but limited by donor availability and associated mortality, making gene therapy an attractive alternative approach for HLH. We reported that perforin expression driven by cellular promoters in lentiviral (LV) vectors resulted in significant, albeit partial, correction of the inflammatory features in a murine model of HLH. We hypothesized that the level of perforin expression achieved per cell from ectopic moderate-strength cellular promoters (phosphoglycerate kinase gene/perforin-1 gene) is inadequate and thus engineered an LV vector using a viral promoter (MND; a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer) containing microRNA126 target sequences to restrict perforin expression in HSCs. We show here that the MND-LV vector restored perforin expression to normal levels in a perforin-deficient human natural killer cell line and perforin gene-corrected Perforin1-/- transplant recipients, whereas cellular promoters drove only partial correction. On lymphocytic choriomeningitis virus challenge, the clinical scores and survival improved only with the MND-LV vector, but inflammatory markers and cytotoxicity were improved with all LV vectors. Our studies suggest that although moderate levels of expression can result in partial amelioration of the HLH phenotype, high levels of perforin expression per cell are required for complete correction of HLH.
1351	Risk of hemophagocytic lymphohistiocytosis in adults with fevers of unknown origin: the clinical utility of a new scoring system on early detection.	27465587	The diagnosis of hemophagocytic lymphohistiocytosis (HLH) is delayed by most physicians. This study aimed to identify early parameters and suitable scoring systems for the risk of HLH. Clinical and laboratory data collected ≤3 days after admission were defined as early parameters and used to calculate the number of HLH-2004 criteria met and bone marrow (BM) score. Between January 2006 and February 2016, 233 immunocompetent adults with naïve fever of unknown origin who underwent a BM study were enrolled to mimic patients at risk of HLH and randomly assigned into the developmental or validation cohort. Hemophagocytic lymphohistiocytosis was finally diagnosed in 47 patients, with non-Hodgkin lymphoma as the major etiology (51.1%). Upon admission, four-fifths of patients who developed subsequent HLH fulfilled ≤3 of 8 HLH-2004 criteria, and 6 early parameters were independent predictors of HLH: anemia (hemoglobin < 10 g/dL), thrombocytopenia (platelet count < 100 × 103 /μL), leukoerythroblastosis, hyperbilirubinemia (total bilirubin > 2 × upper normal limit), hyperferritinemia (ferritin > 1000 ng/mL), and splenomegaly. Compared with the HLH criteria met upon admission, the BM score was an independent predictor (odds ratio = 1.621; 95% confidence interval, 1.355-1.940) with excellent discrimination (area under the receiver operating characteristic curve = 0.920; 95% confidence interval, 0.883-0.958). The sensitivity and specificity for a BM score cutoff of 10 points were 95% and 75%, respectively. When approaching immunocompetent adults with a continuously high fever, the BM score at initial admission assists with early identification of patients at risk of HLH.
1352	The Conserved MAPK Site in E(spl)-M8, an Effector of Drosophila Notch Signaling, Controls Repressor Activity during Eye Development.	27428327	The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to 'cis'-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader applicability than has been previously recognized.
1353	LRRC14 attenuates Toll-like receptor-mediated NF-κB signaling through disruption of IKK complex.	27426725	Activation of NF-κB signaling plays pivotal roles in innate immune responses against pathogens. It requires strict control to avert inflammatory diseases. However, the mechanisms underlying this tight regulation are not completely understood. Here, we identified LRRC14, a novel member of LRR (leucine-rich repeat) protein family, as a negative regulator in TLR signaling. Expression of LRRC14 resulted in decreased activation of NF-κB, whereas knockdown of LRRC14 enhanced NF-κB activation as well as the production of inflammatory cytokines. Mechanistically, LRRC14 bound to HLH domain of IKKβ to block its interaction with NEMO and thereby inhibiting the phosphorylation of IKKβ and NF-κB activation. In addition, our data showed that TLR signaling led to lower expression of LRRC14. Together, LRRC14 may function as a checkpoint to prevent overzealous inflammation.
1354	Secondary hemophagocytic lymphohistiocytosis induced by cholecystitis: A case report and a review of the literature.	27423920	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease resulting from excessive activation and non-malignant proliferation of macrophages and T lymphocytes. Whether it can be caused by cholecystitis has not yet been reported in the world.A 4-year-old girl was admitted to hospital with cholecystitis. The patient was diagnosed with hemophagocytic lymphohistiocytosis after 3days of admission based on the results of laboratory tests showing hypofibrinogenemia, hypertriglyceridemia, thrombocytopenia, anemia and leukopenia.From this case experience, if a timely symptomatic treatment is given, the condition of the patient with secondary HLH can be alleviated. This is the first report of cholecystitis-induced hemophagocytic syndrome in the world also.
1355	Hemophagocytic Lymphohistiocytosis Associated with Visceral Leishmaniasis: Varied Presentation.	27408434	Visceral leishmaniasis (VL) is endemic in many parts of India. Rarely, it may be complicated by hemophagocytic lymphohistiocytosis (HLH) that has varied presentation and course. We describe two cases of VL complicated by HLH that were markedly different in clinical presentation, course and management. First case presented with Fever of unknown origin whereas second case had fever with severe bleeding manifestations. VL was diagnosed by bone marrow aspiration and serum rk39 immunodiagnostic test respectively in these cases. HLH was diagnosed by HLH 2004 diagnostic criteria. VL was treated by intravenous amphotericin B in both cases. HLH was managed by treating primary disease in the first case whereas steroid was given for management in the second case. High index of suspicion is crucial for early diagnosis of HLH to reduce morbidity and mortality.
1356	Familial Hemophagocytic Lymphohistiocytosis due to Mutation of UNC13D Gene.	27408432	Hemophagocytic lymphohistiocytosis (HLH) are basically a heterogenous group of clinical syndromes, characterised by activation and non-malignant proliferation of benign histiocytes i.e. lymphocytes and macrophages, leading to a cytokine storm that accounts for the fever, organomegaly and multi-organ dysfunction. Two types of HLH are described, either due to known genetic defect (familial HLH/FHL) or due to some acquired cause either infection or rheumatological diseases. Here we present a case of a 3 months old baby, admitted with fever, hepatosplenomegaly and cytopenia and ultimately was diagnosed to be a case of Familial HLH type 3 due to defect in UNC13D gene as a result of compound heterozygous for two nonsense mutation resulting in the Munc13-4 protein defect.
1357	Haemophagocytic lymphohistiocytosis presenting as neonatal liver failure: A case series.	27397412	Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening clinical syndrome with liver involvement varying from mild dysfunction to severe fulminant failure. The aim of this study was to present a case series of four HLH patients presenting with acute liver failure (ALF) in the neonatal period.All four patients were neonates at the onset of symptoms. They presented to Cairo University Pediatric Hospital with ALF; they underwent prompt investigations including determination of ferritin, fibrinogen, and triglyceride levels as part of our ALF workup. Further investigations were tailored according to the associated clinical features and the results of preliminary investigations.HLH was diagnosed according to HLH-2004 criteria. Three patients fulfilled at least five out of eight criteria. Fever, splenomegaly, elevated ferritin levels, and low fibrinogen levels were present in all patients. The fourth patient had a serum ferritin level >10,000ng/ml, favouring the diagnosis of HLH, despite fulfilling only four out of eight criteria. For three patients, positive consanguinity and previous sibling death were reported, suggesting a genetic aetiology of HLH.ALF can be the presenting feature of HLH; thus, a high index of suspicion is necessary. Fever is a hallmark, especially in neonates. Diagnosis is important for this potentially treatable condition.
1358	Your critical care patient may have HLH (hemophagocytic lymphohistiocytosis).	27389585	NaN
1359	The role of 18F-FDG PET/CT in the management of patients with secondary haemophagocytic lymphohistiocytosis.	27387105	To investigate the ability of combined 2-[18F]-fluoro-2-deoxy-d-glucose (18F-FDG) positron-emission tomography (PET)/computed tomography (CT) to determine potential causes of secondary haemophagocytic lymphohistiocytosis (sHLH) and to predict prognosis.Forty-three patients (male/female 20/23, median age 48.5 years), who were diagnosed with sHLH and underwent FDG-PET/CT before treatment, were retrospectively reviewed. The clinical characteristics were compared to identify the predictors of high-yield FDG-PET/CT. Univariate and multivariate analyses were conducted to identify factors associated with survival. Statistical analysis was performed using SPSS version 19.0.PET results were helpful in 65.1% (28/43), whilst non-contributory in 34.9% (15/43) of patients with regard to the final diagnosis. Lymphoma was the most common (25/43) reason for sHLH, and patients with focal FDG uptake were more likely to be diagnosed with underlying diseases. C-reactive protein (CRP) was found to be a good indicator for the usefulness of PET/CT in HLH patients. Multivariate analysis showed that therapy regimen (hazard ratio [HR]=4.99, p=0.026), fibrinogen (FBG) <1.5 g/l (HR=3.87, p=0.049) and spleen:mediastinum ratio (SP/M) (HR=7.44, p=0.006) were independent prognostic factors for survival.FDG-PET/CT could be a useful technique for detecting underlying diseases causing sHLH. CRP was a useful predictor of FDG-PET/CT effectiveness. Therapy regimen, FBG level, and SP/M were independent prognostic factors for HLH survival.
1360	Hemophagocytic Lymphohistiocytosis in a Young Child.	27376605	Hemophagocytic lymphohistiocytosis (HLH) is a multisystem disorder mediated by cytokine storm and is characterized by fever, pancytopenia and organomegaly coupled with laboratory features like hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia and transaminitis. Etiology can be genetic or acquired such as infections, malignancy and autoimmune disorders. Diagnosis, identification of underlying etiology and management of HLH remain tough clinical puzzles to sort out for the managing physician. We report a clinico-pathological conference of a three-year-old boy who had such a presentation and succumbed during the hospital stay.
1361	Hemophagocytic Lymphohistiocytosis in an Adult: A Rapidly Progressive Disease with Grave Outcome.	27376224	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal syndrome seen primarily in children. It is characterized by pathologic systemic hyper inflammation which in adults is easily overlooked due to non-specific clinical features. Most of the data available are on paedriatic population, making the diagnosis of HLH in adults challenging for the clinician. Here we report a case of HLH in a 48-year male who presented with pyrexia of unknown origin for 2 months but remained undiagnosed despite extensive workup. Due to a high index of suspicion, re-evaluation of bone marrow biopsy was done which showed hemophagocytosis, earlier reported as normal. It led to specific investigations, needed for establishing the diagnostic criteria of HLH. Even though chemotherapy was initiated, the patient did not survive. The aggressive nature of this disease makes it crucial for the physician to be aware of its signs and symptoms for the early diagnosis and immediate introduction of adequate treatment.
1362	Low reversibility of intracellular cAMP accumulation in mouse Leydig tumor cells (MLTC-1) stimulated by human Luteinizing Hormone (hLH) and Chorionic Gonadotropin (hCG).	27373440	In order to study the intracellular cAMP response kinetics of Leydig cells to hormones with LH activity, we used MLTC-1 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be followed using oxiluciferin luminescence produced from catalyzed luciferin oxidation. The potencies of the different LHs and CGs were evaluated using areas under the curves (AUC) of their kinetics over 60 min stimulation. All mammalian LHs and CGs tested were found to stimulate cAMP accumulation in these cells. The reversibility of this stimulation was studied by removing the hormone from the culture medium after 10 min of incubation. The ratios of kinetics AUC after removing or not the hormone were used to evaluate the stimulation reversibility of each hormone. Natural and recombinant hLHs and hCGs were found to exhibit slowly reversible activation compared to pituitary rat, ovine, porcine, camel and equine LHs, serum-derived eCG (PMSG) and recombinant eLH/CGs. Carbohydrate side chains are not involved in this phenomenon since natural and recombinant homologous hormones exhibit the same reversibility rates. It is still unknown whether only one human subunit, α or β, is responsible for this behaviour or whether it is due to a particular feature of the hLH and hCG quaternary structure.
1363	Assessment of the HScore for reactive haemophagocytic syndrome in patients with rheumatic diseases.	27359073	Reactive haemophagocytic syndrome (RHS) is a hyperinflammatory disorder often occurring in the background of several disorders such as infections, malignancies, and rheumatic diseases. Recently, a score known as the HScore was developed for the diagnosis of RHS. In the original study, most of the patients had underlying haematological malignancy or infection and the best cut-off value for the HScore was 169 (sensitivity 93%; specificity 86%). In this study we aimed to analyse the performance of the HScore in rheumatic disease-related RHS.The patients with rheumatic disorders evaluated in the Departments of Rheumatology and Paediatric Rheumatology at Hacettepe University, Ankara, Turkey between 2002 and 2014 were reviewed retrospectively. The first group (n = 30) consisted of patients with RHS; the control group (n = 64) included patients with active rheumatic diseases without RHS.In the RHS group, 14 (46.7%) had adult-onset Still's disease (AOSD), 10 (33.3%) systemic juvenile idiopathic arthritis (SJIA), and six (20%) systemic lupus erythematosus (SLE). The control group (n = 64) consisted of 32 (50%) AOSD, 13 (20.3%) SJIA, and 19 (29.7%) SLE patients. Applying the HScore to the RHS patients, the best cut-off value was 190.5 with a sensitivity of 96.7% and specificity of 98.4%. When we excluded the patients from the control group who had not had bone marrow aspiration (n = 23), the same cut-off (190.5) performed best (sensitivity 96.7%; specificity 97.6%). Applying the 2004 haemophagocytic lymphohistiocytosis (HLH-2004) criteria gave a sensitivity of 56.6% and a specificity of 100% in the whole study group.In our study, a cut-off value for the HScore different from the original study performed better. Further studies are warranted to determine optimum cut-off values in different studies.
1364	Hemophagocytic lymphohistiocytosis as a paraneoplastic syndrome associated with ovarian dysgerminoma.	27354999	•Ovarian dysgerminoma associated with paraneoplastic fever, cytopenia and splenomegaly•Complete symptom resolution resulted from tumor resection and medical management•Non-hematolymphoid neoplasms are part of differential diagnosis in secondary HLH.
1365	Haploidentical transplantation as a promising therapy for relapsed hemophagocytic lymphohistiocytosis in an older adult patient.	27352259	Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe and often overwhelming systemic hyper-inflammatory syndrome generally presenting with unexplained fevers, hepatosplenomegaly, and progressive multi-organ dysfunction. Treatment of HLH has two major goals: Halting the triggering event and controlling the overactive immune system. However, patients with primary or recurrent secondary HLH should subsequently undergo allogeneic HCT for long lasting disease remission. Hereby we present the case of a 69 years old man with recurrent HLH who underwent a reduced intensity conditioning of fludarabine, cyclophosphamide and low dose total body irradiation followed by a haploidentical marrow graft and post-transplantation cyclophosphamide (PTCy), tacrolimus and mycophenolate mofetil as GVHD prophylaxis. He achieved a durable remission of HLH symptoms despite persistent myeloid mixed chimerism. The use of haploidentical donors and PTCy as tolerance inducing regimen is feasible in HLH. The achievement of mixed donor chimerism may be enough to control the clinical manifestations and to cure HLH.
1366	Extreme hyperferritinemia in the setting of acute myeloid leukaemia: a case report of hemophagocytic lymphohistiocytosis.	27346972	Major hyperferritinemia is a rare feature in clinical laboratories associated with a wide variety of disorders, including hemophagocytic lymphohistiocytosis (HLH). The diagnosis of HLH is based on clinical and biological criteria, such as those proposed by the Histiocyte Society. However, several of these criteria are not relevant in the specific setting of hematologic malignancies.A 69-year-old male was treated for an acute myeloid leukaemia. On day 15 after the start of chemotherapy, he developed severe sepsis with high fever, low blood pressure and hepatosplenomegaly.Blood tests were marked by extreme hyperferritinemia (191,000 µg/L, reference range: 26-388 µg/L) with increased C-reactive protein (87.0 mg/L) and procalcitonin (1.94 µg/L) and aspartate aminotransferase (499 U/L 37 °C) in the setting of chemotherapy-induced aplasia. This unusual extreme ferritinemia led to suspect HLH triggered by an invasive infection. Under intensive treatment, the clinical status improved and ferritin levels significantly decreased.The diagnosis of HLH is usually based on clinical and biological criteria, mainly fever, splenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis and hyperferritinemia. In this patient, the diagnosis of HLH was challenging because several criteria, such as hypertriglyceridemia, hemophagocytosis and hypofibrinogenemia, were absent. In addition, some criteria of HLH are not relevant in the setting of hematologic malignancy, in which fever, splenomegaly, cytopenias and elevated lactate dehydrogenase are commonly observed independently of HLH. This unusual case of extremely high ferritinemia emphasizes the important weight of the ferritin level for the diagnosis of HLH in adult patients in the setting of hematologic malignancies.
1367	[Expression of Serum HMGB-1 in Patients with Secondary Hemo-phagocytic Lymphohistiocytosis and Its Clinical Significance].	27342526	To investigate the expression levels and clinical significance of serum high mobility group box 1 (HMGB-1) in patients with secondary hemophagocytic lymphohistiocytosis (sHLH).Serum HMGB-1 levels were determined by using enzyme linked immunosorbent assays (ELISA) in 51 sHLH patients and 15 healthy contrlols. Other laboratory data, including soluble interleukin-2 receptor (sCD25), white blood cells (WBC), hemoglobin (Hb), platelet (Plt), fibrinogen (FIB), lactate dehydrogenase (LDH), triglyceride (TG), alanine transaminase (ALT), aspartate aminotransferase (AST), serum ferritin (SF), C reactive protein (CRP), and blood sedimentation rate (ESR) were also collected.Serum HMGB-1 levels in the newly diagnosed group were significantly higher than that in the control group (P<0.01). Serum HMGB-1 levels in the newly diagnosed lymphoma-associated HLH (LHLH) group were significantly higher than that in non-HLH group, including infection-associated HLH (IHLH) and autoimmune-associated HLH (AHLH) group (P<0.05); The serum HMGB-1 levels in the clinical remission group were significantly lower than that in the newly diagnosed group (P<0.05), however, serum HMGB-1 was not decreased significantly in the progression/relapsed group, compared with the newly diagnosed group (P>0.05). Serum HMGB-1 levels in newly diagnosed sHLH patients positively correlated with sCD25 (r=0.62, P<0.01) and ESR (r=0.55, P<0.05). The receiver operating characteristic curves (ROC) for serum HMGB-1 levels of sHLH patients and healthy controls produced a cutoff value at 15.3 µg/L, with its 90% sensitivity and 99% specificity, respectively. In addition, an optimal cutoff value for HMGB-1 was 27.4 µg/L in the patients LHLH and non-HLH (AHLH+IHLH) with 96% sensitivity and 81% specificity, separately.Serum HMGB-1 levels possesses an important clinically significance for disease diagnosis, differential diagnosis, evaluation of nosographic activity and treatment efficacy in the patients with sHLH.
1368	Are There Head Volume Alterations at 11 to 14 Weeks in Fetuses with Congenital Heart Defects? A First Trimester Case Series.	27308099	Objective This study aims to assess head volume (HV) alterations at 11 to 14 weeks in fetuses with congenital heart defects (CHD). Methods A retrospective case-control study on 100 normal and 26 CHD fetuses was conducted. The fetuses had a first trimester scan with volume data sets stored from which HV was calculated. The mean HV and HV as a function of crown-rump length (CRL) in normal fetuses were compared with established normograms. Mean HV, HV as a function of CRL, and HV/CRL were compared between normal and CHD fetuses. Nonparametric Kruskal-Wallis H test was used with p < 0.05 considered significant. Results Overall, 83 normal and 19 CHD fetuses were included. The mean HV and HV as a function of CRL in the normal fetuses were comparable to what has been established (p = 0.451 and 0.801, respectively). The mean HV was statistically smaller in fetuses with CHD, particularly those with hypoplastic left heart (HLH): 10.7 mL in HLH versus 13.0 mL in normal fetuses (p = 0.043). The HV/CRL was statistically smaller in fetuses with CHD (p = 0.01). Conclusion Despite the small sample size, our case series suggests that alterations in HV may potentially be apparent as early as 11 to 14 weeks in CHD fetuses, particularly those with HLH. Larger prospective studies are needed to validate our findings.
1369	Prognostic factors of early outcome in pediatric hemophagocytic lymphohistiocytosis: an analysis of 116 cases.	27307280	Early mortality remains a major challenge for the treatment of hemophagocytic lymphohistiocytosis (HLH), which warrants the need for prompt risk stratification in the early phase of the disease. We retrospectively analyzed clinical features of a cohort of pediatric patients managed at a tertiary hospital in southern China from 2005 to 2015. A total of 116 patients (median age 27.5 months) with predominantly secondary HLH were included. In a multivariate Cox regression model, neutrophils <0.5 × 10(9)/L (risk ratio (RR) = 5.01; 95 % confidence interval (CI) 1.55-16.20; P = 0.007), total bilirubin over twofold upper limit of normal value (RR = 2.86; 95 % CI 0.83-9.88; P = 0.097), and albumin ≤20 g/L (RR = 5.79; 95 % CI 1.70-19.73; P = 0.005) at diagnosis were independent risk factors for 30-day mortality. The 30-day overall survival rate (OS) of patients with three risk factors was significantly lower than that of patients with zero to two risk factors (0 vs 90.7 %; P<0.001). Patients with three risk factors were 64-fold more likely to have early adverse outcome as compared to patients with zero to two risk factors (RR = 64.45; 95 % CI 18.35-226.33; P<0.001). Platelet count normalization in 2 weeks was an independent predictor for resolution after initial therapy with an odds ratio (OR) of 18.4 (95 % CI 2.7-122.9; P = 0.003). Our results indicate that severe neutropenia and liver function damage are prognostic factors for early death in HLH and platelet count normalization in 2 weeks is a critical predictor for resolution after initial therapy.
1370	Performances of the H-Score for Diagnosis of Hemophagocytic Lymphohistiocytosis in Adult and Pediatric Patients.	27298397	In this study, we compared the performances of adapted hemophagocytic lymphohistiocytosis (HLH)-2004 guidelines with those of the new diagnostic H-score to identify patients with HLH in a multicenter cohort consisting of adult and pediatric cases of suspected HLH.The study sample consisted of 147 cases, including 20 adults and 16 children with HLH. Two sets of biological data were evaluated: at presentation and the maximal values reached during the episode.At presentation, for both children and adults, the H-score was more efficient than adapted HLH-2004 guidelines to identify HLH. The diagnostic sensitivity and specificity were respectively 100% and 80% for children and 90% and 79% for adults. However, for adults, performances became comparable between adapted HLH-2004 guidelines and H-score as patient clinical status worsened. The specificity decreased to 73% for the same sensitivity.The adapted HLH-2004 guidelines seem less powerful and H-score seems to be more appropriate for children, which may be due to less significantly marked biological features. For adults, H-score performances are better when determined at presentation. The cutoff value of the H-score should be adapted depending on the target population to obtain optimal specificity.
1371	Haemophagocytic lymphohistiocytosis (HLH): a rare but potentially fatal association with Plasmodium vivax malaria.	27298293	Haemophagocytic lymphohistiocytosis (HLH) is a potentially fatal syndrome that is caused by an abnormal activation of the immune system. It can present as the primary syndrome or occur secondary to a variety of conditions such as malignancy, autoimmune diseases and infections. We present a case of a man who developed HLH secondary to Plasmodium vivax infection. He presented with symptoms of fever, chills and myalgias. Physical examination revealed significant hepatosplenomegaly. The presence of pancytopaenia, elevated ferritin levels and haemophagocytosis on bone marrow biopsy confirmed the diagnosis of HLH (based on HLH-2004 criteria). There was a significant improvement after the initiation of intravenous antimalarials. No relapses were documented on follow-up. It is imperative that physicians should promptly recognise and treat this rare condition, as a timely intervention can be lifesaving.
1372	Elevated circulating levels of interferon-γ and interferon-γ-induced chemokines characterise patients with macrophage activation syndrome complicating systemic juvenile idiopathic arthritis.	27296321	Interferon-γ (IFNγ) is the pivotal mediator in murine models of primary haemophagocytic lymphohistiocytosis (pHLH). Given the similarities between primary and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), we investigate the involvement of the IFNγ pathway in MAS by evaluating levels of IFNγ and of the induced chemokines, and their relation with laboratory parameters of MAS in systemic juvenile idiopathic arthritis (sJIA) patients with MAS and in a murine MAS model.The Luminex multiplexing assay was used to assess serum levels of interleukin (IL)-1β, IL-6, IFNγ and of the IFNγ-induced chemokines CXCL9, CXCL10 and CXCL11 in patients with sec-HLH (n=11) and in patients with sJIA (n=54), of whom 20 had active MAS at sampling. Expression of IFNγ-induced chemokines was assessed in IL-6 transgenic mice in which MAS is induced by TLR4 stimulation with lipopolysaccharide.Levels of IFNγ and of IFNγ-induced chemokines were markedly elevated during active MAS and sec-HLH and were significantly higher in patients with MAS compared with active sJIA without MAS. Levels in patients with active sJIA without MAS were comparable to those of patients with clinically inactive sJIA. During MAS, ferritin and alanine transferase levels and neutrophil and platelet counts were significantly correlated with serum levels of IFNγ and CXCL9. In murine MAS, serum levels of ferritin were significantly correlated with mRNA levels of Cxcl9 in liver and spleen.The high levels of IFNγ and of IFNγ-induced chemokines and their correlation with the severity of laboratory abnormalities of MAS suggest a pivotal role of IFNγ in MAS.
1373	Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation.	27295348	The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.
1374	Understanding the spectrum of haemophagocytic lymphohistiocytosis: update on diagnostic challenges and therapeutic options.	27292929	The cytokine storm syndrome 'haemophagocytic lymphohistiocytosis' (HLH) is an under-recognized hyperinflammatory disorder, causing high morbidity and mortality risk in children and adults. It can be subdivided into a primary, genetic form and a secondary, acquired form that complicates diverse infections, malignancies and autoimmune or autoinflammatory disorders. Both subtypes present with the same spectrum of non-specific symptoms, making accurate diagnosis and rapid treatment initiation challenging. In the last decade, increased awareness and international collaborative efforts fuelled a marked progress in diagnostic protocols and novel treatment strategies for HLH and new diagnostic guidelines are being tailored to specific secondary HLH subtypes. Therapy is gradually shifting its focus from overall immunosuppression towards targeting specific cytokines, cell types or signalling pathways underlying pathophysiology. Nevertheless, continued research efforts remain indispensable to customize therapy to individual patient needs.
1375	Hemophagocytic lymphohistiocytosis: Pathogenesis, diagnosis, and management.	27289085	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome that is classified into primary and secondary HLH. Primary HLH consists of monogenic disorders that mainly affect the perforin-mediated cytotoxicity of cytotoxic T lymphocytes and natural killer cells. Secondary HLH occurs as a complication in various settings such as infection, malignancy, autoimmune disease, and post-allogeneic hematopoietic stem cell transplantation. Both primary and secondary HLH are characterized by uncontrolled hypercytokinemia that results in myelosuppression and vascular endothelium damage. More than 10% of patients with HLH die within 2 months of diagnosis due to bleeding in the visceral organs, opportunistic infection due to neutropenia, or multiple organ failure. The most obvious presentations of HLH are persistent fever refractory to antimicrobial agents and hyperferritinemia due to hypersecretion of various cytokines. The first rule is not to overlook signs of hypercytokinemia and to settle the hyperactivated immunological state as soon as possible. In addition, to improve outcome, it is essential to identify the disorders underlying HLH and provide disorder-appropriate treatment.
1376	Variable clinical phenotypes of X-linked lymphoproliferative syndrome in China: Report of five cases with three novel mutations and review of the literature.	27288720	X-linked lymphoproliferative disease (XLP) is a rare life-threatening syndrome. Rapid recognition and definitive diagnosis are critical to improve the prognosis and survival of patients with XLP. Nowadays, little is known about patients with XLP in China.We report the characterization of five Chinese XLP patients with three novel mutations and review the literature related to this syndrome. Male patients with fulminant infectious mononucleosis (FIM), Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) or persistent EBV viraemia were enrolled in this study. The patients' clinical features were assessed by retrieval of data from medical records. Immunological function included analysis of lymphocyte subsets and the detection of immunoglobulins G, A, M and/or E were evaluated by flow cytometry and nephelometry. Direct sequencing was used to detect SH2D1A/XIAP gene mutations.Twenty-two male patients with FIM, EBV-associated HLH or persistent EBV viraemia were evaluated among 421 PID patients in our centre. Four patients had SH2D1A mutations, and one patient had an XIAP mutation. The onset age of the 5 patients range from 1month to 4years which was earlier than that in the western world. The diagnosis age was between 16months and 9years with a long diagnosis lag (1-97months). Two of them had positive family history. The clinical phenotypes varied in different patients among which two patients with FHLH and hypogammaglobulinaemia, one with hypogammaglobulinaemia, lymphoma and aplastic anaemia (AA) which is the first case with AA in China, one with hypogammaglobulinaemia only and the other one with FHLH. For immunological function, three exhibited reduced CD4/CD8 ratios. Arg55stop mutations as well as splice mutation in intron 1 were most frequently found and exon 2 was the hottest exon in China. Two patients died at the time of diagnosis for severe infection or hepatic coma. Three were alive and waiting for haematopoietic stem cell transplantation (HSCT).For patients with severe EBV-associated HLH, hypogammaglobulinaemia, lymphoma and aplastic anaemia, possibility of XLP should be considered and if confirmed, HSCT should be performed as soon as possible.
1377	Virus-associated haemophagocytic lymphohistiocytosis in a young Filipino man.	27284096	Haemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of pathological immune activation characterised by extreme inflammation. We present a case of a young Filipino man consulting for non-specific symptoms of fever, body malaise and weight loss. Prominent physical examination findings included gross pallor, cachexia and hepatosplenomegaly. Laboratory results revealed pancytopaenia, while bone marrow examination revealed haemophagocytosis. Further workup for HLH showed hypertriglyceridaemia, hypofibrinogenaemia and hyperferritinaemia (fulfilling 6 of 8 diagnostic criteria). Exhaustive serological and haematological examinations showed chronic hepatitis B virus infection and past evidence of Epstein-Barr virus infection as possible triggers. The patient was started on antiviral therapy, high-dose steroids and chemotherapy. He initially improved, but eventually succumbed to severe fungal sepsis and pulmonary haemorrhage. An autopsy confirmed the diagnosis of HLH.
1378	Associations between inflammatory cytokines and organ damage in pediatric patients with hemophagocytic lymphohistiocytosis.	27269180	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal disease characterized by overwhelming inflammation response and multiple organ damage. Most of the clinical and laboratory manifestations of HLH are thought to be related to hypercytokinemia and organ infiltration with lymphocytes and histiocytes. The aim of this study was to investigate the associations between cytokines and various manifestations of HLH. A total of 105 patients diagnosed with HLH were enrolled in this retrospective study. The information including the patients' demographic characteristics, clinical and laboratory findings at presentation and cytokine data were collected. The median age at diagnosis was 2.8years, with 74 patients (70.4%) documented Epstein-Barr virus infection. Hepatomegaly (88.6%), splenomegaly (81.9%), cytopenia (68.6%), elevated ferritin level (93.3%), hypofibrinogenemia (61.9%) and hemophagocytosis (77.3%) were found in more than half of the patients. Interleukin (IL)-6, IL-10 and interferon (IFN)-γ were found to be moderately or significantly elevated in most patients. In the correlation analysis, IFN-γ was closely related to the concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, lactate dehydrase (LDH), triglyceride and fibrinogen, while IL-10 was associated with platelet count. When split the patients into two groups according to the cytokine levels, patients with high IFN-γ presented higher level of ALT, AST, bilirubin, LDH, triglyceride, and fibrinogen, while patients with high IL-10 presented much lower hemoglobin and platelet count. In conclusion, the present study put forward clinical evidence that hypercytokinemia is related to organ damage in HLH. IFN-γ may contribute to liver impairment and coagulation disease, while IL-10 is a cytokine related to cytopenias.
1379	Advances in the pathogenesis of primary and secondary haemophagocytic lymphohistiocytosis: differences and similarities.	27264204	Haemophagocytic lymphohistiocytosis (HLH) comprises a heterogeneous spectrum of hyperinflammatory conditions that are inherited (primary HLH) or acquired in a context of infections, malignancies or autoimmune/autoinflammatory disorders (secondary HLH). Genetic defects in the cytotoxic machinery of natural killer and CD8(+) T cells underlie primary HLH, with residual cytotoxicity determining disease severity. Improved sequencing techniques have expanded the range of causal mutations and have redefined many cases of secondary HLH as primary HLH and vice versa, blurring the distinction between both subtypes. These insights allow HLH to be conceptualized as a threshold disease, in which interplay between various genetic and environmental factors causes progressive inflammation into a critical point, beyond which uncontrolled activation of immune cells and excessive cytokine production give rise to the cardinal symptoms of HLH. Various pathogenic pathways may thus converge to a common end stage of fulminant HLH.
1380	Luteinizing Hormone is an effective replacement for hCG to induce ovulation in Xenopus.	27263125	Injection of human Chorionic Gonadotropin (hCG) directly into the dorsal lymph sac of Xenopus is a commonly used protocol for induction of ovulation, but recent shortages in the stocks of commercially available hCG as well as lack of a well tested alternative have resulted in frustrating experimental delays in laboratories that predominantly use Xenopus in their research. Mammalian Luteinizing Hormones (LH) share structural similarity, functional equivalency, and bind the same receptor as hCG; this suggests that LH may serve as a good alternative to hCG for promoting ovulation in Xenopus. LH has been found to induce maturation of Xenopus oocytes in vitro, but whether it can be used to induce ovulation in vivo has not been examined. Here we compared the ability of four mammalian LH proteins, bovine (bLH), human (hLH), ovine (oLH), porcine (pLH), to induce ovulation in Xenopus when injected into the dorsal lymph sac of sexually mature females. We find that both ovine and human LH, but not bovine or porcine, are good substitutes for hCG for induction of ovulation in WT and J strain Xenopus laevis and Xenopus tropicalis.
1381	Adult onset hemophagocytic lymphohistiocytosis prognosis is affected by underlying disease and coexisting viral infection: analysis of a single institution series of 35 patients.	27255829	Adult onset hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome, which can develop as a complication of many disorders. Early diagnosis is essential in order to avoid a fatal outcome. To confirm the diagnosis of acquired HLH made in a single institution series of adult patients with HLH-04 criteria, we applied the HScore and evaluated prognostic factors associated with clinical outcome. The median age of 35 patients was 54 (range 17-81), M/F ratio was 20/15. In 26/35 (74.3%) patients, an underlying haematological disease was present (2 Multicentric Castleman Disease, 10 B-cell Non-Hodgkin Lymphoma [NHL] and 14 T/NK-cell NHL); an autoimmune disorder was observed in four (11.4%) patients (one Still Disease, one undifferentiated connective tissue disease and two haemolytic anaemia); in five (14.3%), no underlying disease was identified. A concomitant infection by EBV was observed in 10 patients (28.6%), CMV in 8 (22.9%), HHV8 in 6 (17.1%) and HIV in 1 (2.9%). Hyperferritinemia, fever and splenomegaly were present in more than 90% of patients, whereas bone marrow hemophagocytosis in 51% of cases only. According to HScore, 34/35 patients had a >75% and 32/35 >93% probability of HLH. Four-year overall survival and HLH-free survival were 17.8% (CI 1.9-33.8) and 23.8% (CI 7.3-40.3), respectively. By multivariate analysis, presence of oedema and hyperbilirubinemia were predictors of death, whereas there was a statistically significant trend for viral infection as predictor of poor prognosis. B-NHL diagnosis was confirmed as associated to a better prognosis in comparison with T/NK lymphoma (4-year HFS 53.3% vs. 0%, p = 0.09) and similar to other aetiologies. Copyright © 2016 John Wiley & Sons, Ltd.
1382	A rare fatal case of a novel bunyavirus-associated hemophagocytic lymphohistiocytosis.	27249531	Herein we describe a rare fatal case of a novel bunyavirus-associated hemophagocytic lymphohistiocytosis (HLH) in a 62-year-old female patient. The novel bunyavirus infects patients with or without HLH who have similar clinical features such as fever, thrombocytopenia, and leukocytopenia. Therefore, the diagnosis of HLH can be easily missed. When HLH occurs, the disease worsens and the fatality rate rises. Our finding highlights the importance of bone marrow biopsy performed as soon as possible for patients suspected of having a novel bunyavirus infection and showing marked cytopenia in three cell lines.
1383	Extreme Hyperferritinemia:  Causes and Impact on Diagnostic Reasoning.	27247369	Hyperferritinemia can be a result of inflammation, infection, chronic iron overload, or other uncommon pathologies including hemophagocytic lymphohistiocytosis (HLH). There is a historical association between extreme hyperferritinemia and HLH, but in reality HLH is associated with a minority of hyperferritinemic states.We identified conditions most associated with hyperferritinemia by identifying 65,536 serum ferritin levels at the University of Minnesota Hospital over a five-year period, with 86 values higher than 10,000 ng/mL. Pediatric patients comprised 22% of this population, and adults, 78%.The majority of cases in both populations with hyperferritinemia were due to chronic transfusion (35%), followed by liver disease (27%), and hematologic malignancy (16%). Solid malignancies, infection, macrophage activation syndrome, and primary and secondary HLH comprised the remaining (22%).Although this supports the relationship between extreme hyperferritinemia and HLH, it maintains that the positive predictive value of hyperferritinemia for HLH is quite low, and one should consider more common explanations before suspecting HLH.
1384	Malignancy-associated hemophagocytic lymphohistiocytosis in adults: Relation to hemophagocytosis, characteristics, and outcomes.	27244347	Malignancy-associated hemophagocytic lymphohistiocytosis (HLH) in adults is a highly lethal disorder. Knowledge gaps have resulted in under diagnosis or delayed diagnosis.The University of Texas MD Anderson Cancer Center pathology database (1991-2014) was retrospectively interrogated for the keywords "hemophagocytosis" and/or "lymphohistiocytosis." Seventy-seven adult patients were identified. All had an underlying malignancy. Sixteen patients who had insufficient documentation were excluded.The majority of patients who had pathologic evidence of hemophagocytosis/lymphohistiocytosis had an incomplete workup to confirm or refute HLH using the 2004 HLH criteria (HLH-2004; n = 8 variables), which is a common problem in adult HLH. Only 13 of 61 patients (21%) met the HLH-2004 diagnostic criteria based on available retrospective data. To identify potentially missed cases of HLH, the published literature was reviewed, and selected additional variables known to be associated with adult HLH were selected, resulting in extended diagnostic criteria of 18 variables. Thirty-five patients met the extended criteria, and 33 had follow-up data available. The median overall survival of the 13 patients who met both the extended criteria and the HLH-2004 criteria was similar to that of the 20 patients who met the extended criteria but NOT the HLH-2004 criteria (1.43 vs 1.76 months, respectively; P = .34) indicating a similar underlying, aggressive, systemic process. Twenty-six patients did not meet either criteria, and 17 had follow-up data available. The median overall survival of the 17 patients who had pathologic hemophagocytosis or lymphohistiocytosis but met neither criteria was significantly superior to the survival of those who met both the extended criteria and the HLH-2004 criteria and those who met the extended criteria but not the HLH-2004 criteria (17.27 vs 1.43 vs 1.76, respectively; P = .002).The addition of diagnostic laboratory variables that are more easily and rapidly available in smaller institutions and primary care settings than the HLH-2004 variables may be a good surrogate to raise early suspicion of malignancy-associated HLH. Prospective validation is warranted. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2857-2866. © 2016 American Cancer Society.
1385	Hemophagocytic Lymphohistiocytosis in Children: Pathogenesis and Treatment.	27242976	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder in children that is characterized by persistent fever, splenomegaly with cytopenia, hypertriglyceridemia, and hypofibrinogenemia. Increased levels of various cytokines and soluble interleukin-2 receptor are biological markers of HLH. HLH can be classified into two major forms: primary and secondary. Familial hemophagocytic lymphohistiocytosis (FHL), a type of primary HLH, is an autosomal recessive disorder that typically occurs in infancy and can be classified into five different subtypes (FHL types 1-5). In Japan, >80% of patients with FHL have either PRF1 (FHL type 2) or UNC13D (FHL type 3) defects. FHL is considered to be a disorder of T-cell function because the activity of NK cells or cytotoxic T lymphocytes as target cells is usually impaired. Moreover, Epstein-Barr virus-associated HLH (EBV-HLH) is considered a major subtype of secondary HLH. Any genetic background could have an effect on the pathogenesis of secondary HLH because EBV-HLH is considered to be particularly prevalent in Asian countries. For primary HLH, hematopoietic stem cell transplantation is the only accepted curative therapy, although cord blood transplantation with a reduced-conditioning regimen has been used with superior outcomes. For secondary HLH, including EBV-HLH, immunochemotherapy based on the HLH-2004 protocol has been used. In the near future, the entire mechanism of HLH should be clarified to establish less toxic therapies, including cell therapy and gene targeting therapy.
1386	Hemophagocytic syndromes (HPSs) including hemophagocytic lymphohistiocytosis (HLH) in adults: A systematic scoping review.	27238576	Most knowledge of hemophagocytic syndromes (HPSs) including hemophagocytic lymphohistiocytosis (HLH) is derived from pediatric studies; literature on adult HPS/HLH predominantly consists of small retrospective studies with clinical and methodological heterogeneity. The aims of this systematic scoping review were to provide an overview of existing literature on adult HPS/HLH, describe current practices in diagnosis and treatment, and propose priorities for future research. Articles from Ovid Medline, Embase and Pubmed (1975-2015) describing 10 or more unique adults (age>15years) with HPS/HLH were included. 82 publications were eligible: 10 were prospective and 72 were retrospective. Of the six distinct diagnostic criteria, the HLH-2004 criteria were by far the most commonly used. A minority of studies tested for genetic abnormalities (12), soluble interleukin-2 receptor (11), and/or NK function (11) in a subset of patients. Most centers used steroids and either etoposide-based (HLH-94/HLH-2004) or doxorubicin-based (CHOP) initial therapy regimens. Allogeneic hematopoietic cell therapy for treatment of adult HLH has rarely been reported. Mortality in larger treatment focused studies ranged from 20 to 88%. Developing adult-specific diagnostic criteria based on widely evaluable features of secondary HPS/HLH and establishing standard initial therapies are priorities for future research.
1387	Ccndbp1 is a new positive regulator of skeletal myogenesis.	27235421	Skeletal myogenesis is a multistep process in which basic helix-loop-helix (bHLH) transcription factors, such as MyoD (also known as MyoD1), bind to E-boxes and activate downstream genes. Ccndbp1 is a HLH protein that lacks a DNA-binding region, and its function in skeletal myogenesis is currently unknown. We generated Ccndbp1-null mice by using CRISPR-Cas9. Notably, in Ccndbp1-null mice, the cross sectional area of the skeletal tibialis anterior muscle was smaller, and muscle regeneration ability and grip strength were impaired, compared with those of wild type. This phenotype resembled that of myofiber hypotrophy in some human myopathies or amyoplasia. Ccndbp1 expression was upregulated during C2C12 myogenesis. Ccndbp1 overexpression promoted myogenesis, whereas knockdown of Ccndbp1 inhibited myogenic differentiation. Co-transfection of Ccndbp1 with MyoD and/or E47 (encoded by TCF3) significantly enhanced E-box-dependent transcription. Furthermore, Ccndbp1 physically associated with MyoD but not E47. These data suggest that Ccndbp1 regulates muscle differentiation by interacting with MyoD and enhancing its binding to target genes. Our study newly identifies Ccndbp1 as a positive modulator of skeletal myogenic differentiation in vivo and in vitro, providing new insights in order to decipher the complex network involved in skeletal myogenic development and related diseases.
1388	Therapeutic effect of JAK1/2 blockade on the manifestations of hemophagocytic lymphohistiocytosis in mice.	27222478	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome, characterized by severe hyperinflammation and immunopathological manifestations in several tissues. These features result from organ infiltration by overactivated CD8 T-cells and macrophages, which produce high levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, and IL-18. Recently, several Janus kinase 1/2 (JAK1/2) inhibitors, such as ruxolitinib, have been developed as immunosuppressive agents. They have proven beneficial effects in the treatment of myeloproliferative disorders and inflammatory conditions. To determine whether pharmacological inhibition of the JAK1/2 not only prevents the onset of HLH immunopathology but also is effective against existing HLH, cytotoxicity-impaired Prf1(-/-) and Rab27a(-/-) mice with full-blown HLH syndrome were treated with a clinically relevant dose of ruxolitinib. In vivo, ruxolitinib treatment suppressed signal transducer and activator of transcription 1 activation and led to recovery from HLH manifestations in both murine models. In the Prf1(-/-) mice, these beneficial effects were evidenced by a greater survival rate, and in both murine models, they were evidenced by the correction of blood cytopenia and a rapid decrease in serum IL-6 and TNF-α levels. During ruxolitinib treatment, liver tissue damage receded concomitantly with a decrease in the number of infiltrating inflammatory macrophages and an increase in the number of alternatively activated macrophages. In Rab27a(-/-) mice, central nervous system involvement was significantly reduced by ruxolitinib therapy. Our findings demonstrate that clinically relevant doses of the JAK1/2 inhibitor ruxolitinib suppresses the harmful consequences of macrophage overactivation characterizing HLH in 2 murine models. The results could be readily translated into the clinic for the treatment of primary, and perhaps even secondary, forms of HLH.
1389	Liver transplantation for children with acute liver failure associated with secondary hemophagocytic lymphohistiocytosis.	27216884	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening systemic disease, characterized by overwhelming stimulation of the immune system and categorized as primary or secondary types. Occasionally, acute liver failure (ALF) may dominate the clinical presentation. Given the systemic nature of HLH and risk of recurrence, HLH is considered by many a contraindication to liver transplantation (LT). The aim of this study is to review our single-center experience with LT in children with secondary HLH and ALF (HLH-ALF). This is a cross-sectional, retrospective study of children with secondary HLH-ALF that underwent LT in 2005-2014. Of 246 LTs, 9 patients (3 males; median age, 5 years; range, 0.7-15.4 years) underwent LT for secondary HLH-ALF. Disease progression was rapid with median 14 days (range, 6-27 days) between first symptoms and LT. Low fibrinogen/high triglycerides, elevated ferritin, hemophagocytosis on liver biopsy, and soluble interleukin 2 receptor levels were the most commonly fulfilled diagnostic criteria; HLH genetic studies were negative in all patients. Immunosuppressive therapy after LT included corticosteroids adjusted to HLH treatment protocol and tacrolimus. Thymoglobulin (n = 5), etoposide (n = 4), and alemtuzumab (n = 2) were used in cases of recurrence. Five (56%) patients experienced HLH recurrence, 1 requiring repeat LT, and 3 died. Overall graft and patient survival were 60% and 67%, respectively. Six patients are alive and well at a median of 24 months (range, 15-72 months) after transplantation. In conclusion, LT can be beneficial in selected patients with secondary HLH-ALF and can restore good health in an otherwise lethal condition. Liver Transplantation 22 1245-1253 2016 AASLD.
1390	Comparison of Th1/Th2 cytokine profiles between primary and secondary haemophagocytic lymphohistiocytosis.	27209435	Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation, and HLH patients with mutations in genes including PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK were reported to be primary HLH. Due to the different treatment options, the differentiation between primary and secondary HLH is critical. Our previous studies have showed that a Th1/Th2 cytokine profile is diagnostic for HLH, yet the cytokine profiles between primary and secondary HLH have not been compared. The aim of the study was to test whether the Th1/Th2 cytokine profile could be used as a tool to differentiate between primary and secondary HLH.A total of 45 hospitalized Chinese children with HLH during the period of February 2010 through September 2012 were enrolled in the study. Fifty healthy children were enrolled as controls. Primary HLH related genes were sequenced using genomic DNA samples. The Th1/Th2 cytokine levels including interferon-γ (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10, IL-6, IL-4 and IL-2 were quantitatively determined by cytometric bead assay techniques.Primary HLH group (n = 4) included one patient with biallelic heterozygous mutations in PRF1 gene, and three patients with hemizygous mutation in SH2D1A gene. Based on the available genetic data, the other 41 patients were classified into the secondary HLH group. When compared the cytokine levels between the two groups, IL-4 level in primary-HLH was significantly lower than that in secondary HLH (P = 0.025), while IFN-γ level in primary HLH had a tendency of statistically lower than that in secondary HLH (P = 0.051). Area under receiver operating characteristic (ROC) curves of IL-4 and IFN-γ, IL-10, TNF-α, IL-2, and IL-6 levels were 0.841, 0.799, 0.506, 0.494, 0.457, and 0.250, respectively. ROC curves showed that 1.7 pg/ml of IL-4 had sensitivity and specificity for differentiation between primary and secondary HLH as 70.7 and 100.0 %, while 433.9 pg/ml of IFN-γ had sensitivity and specificity as 51.2 and 100.0 %, respectively.HLH patients with lower IL-4 and IFN-γ levels have higher possibility to be primary HLH. The cytokine profile may be used as an additional tool for the quick differential diagnosis between primary and secondary HLH.
1391	Weathering a Cytokine Storm: A Case of EBV-Induced Hemophagocytic Lymphohistiocytosis.	27200381	Hemophagocytic lymphohistiocytosis (HLH) is a rare but life-threatening disease caused by excessive immune activation. Acquired HLH is seen in adults and is often caused by infection or malignancy. Diagnosis is difficult and usually missed as clinical and laboratory findings are nonspecific. Moreover, the pathophysiology of the systemic inflammatory response syndrome and/or sepsis is remarkably similar to HLH. Thus, in patients presenting with presumed severe sepsis or septic shock complicated by multiorgan failure without a clear infectious source, HLH should be considered. A disproportionately high ferritin may be one of the earlier laboratory findings to suggest HLH. We discuss a case of a young male who presented with presumed septic shock with multiorgan failure who was eventually found to have Epstein-Barr virus-induced HLH.
1392	PARTIAL OCULOCUTANEOUS ALBINISM AND IMMUNODEFICIENCY SYNDROMES: TEN YEARS EXPERIENCE FROM A SINGLE CENTER IN TURKEY.	27192893	Partial oculocutaneous albinism and immunodeficiency (OCA-ID) diseases are autosomal recessive syndromes characterized by partial hypopigmentation and recurrent infections. Moreover, some OCA-ID syndromes confer susceptibility to develop a life-threatening hyperinflammatory condition called hemophagocytic lymphohistiocytosis (HLH). We investigated the genetic, clinical and immunological characteristics of 20 OCA patients.Herein, we present the clinical and immunological characteristics of 20 OCA patients who referred to the Department of Pediatric Immunology, Erciyes University Medical Faculty in Kayseri, Turkey between 2004 and 2014.Of the 20 OCA patients, 7 fulfilled diagnostic criteria for HLH, 9 showed defective functions of CD8 T cells and natural killer cells, and 8 received a definitive molecular diagnosis. Among the patients, we also report a patient diagnosed with two different genetic defects, in TYR and JAK3 genes, causing, respectively, OCA and ID.Our results illustrate the variability of clinical presentations and disease severity in OCA-ID patients, with consequent challenges in diagnosing and treating these patients.
1393	Clinicopathological categorization of Epstein-Barr virus-positive T/NK-cell lymphoproliferative disease: an analysis of 42 cases with an emphasis on prognostic implications.	27157296	Epstein-Barr virus-positive T/NK-cell lymphoproliferative diseases (EBV-T/NK-LPDs) include several overlapping EBV-related conditions with variably aggressive courses. For prognostic categorization, we retrospectively analyzed 42 EBV-T/NK-LPD cases. Male (79% [33/42]), young (≤40 years; 83% [35/42]) patients and T-cell lineage (81% [34/42]; CD8/CD4 = 1.8) were predominant. Clinicopathologically, three systemic and one cutaneous category were developed: hemophagocytic lymphohistiocytosis (HLH; 26% [11/42]), chronic active EBV infection (CAEBV; 31% [13/42]), systemic unclassifiable disease (24% [10/42]), and hydroa vacciniforme/hydroa vacciniforme-like lymphoma (HV/HVL; 19% [8/42]). Prognostically, cutaneous disease (HV/HVL) was better than systemic disease (p = 0.014; median, 285 vs. 10 months). In systemic diseases, HLH was worst (p = 0.002; 3[HLH] vs. 4[unclassifiable] vs. not reached [CAEBV]). Univariate survival analysis (n = 42) revealed cytopenia (≥one lineage; p < 0.001), onset age (>40 years; p = 0.001), T-cell lineage (p = 0.041), hemophagocytic histiocytes (p = 0.031), elevated lactate dehydrogenase (p = 0.020), and liver dysfunction (p = 0.023) predicted shorter survival. In multivariate analysis, T-cell lineage (p = 0.025 [HR =11.3]) and cytopenia (p = 0.028 [HR =5.4]) were independent prognostic factors. Therefore, EBV-T/NK-LPD could be classified into four prognostic categories.
1394	Concomitant Presentation of Hemophagocytic Lymphohistiocytosis and Posttransplant Lymphoproliferative Disease-Like Lymphoma in a Mildly Immunosuppressed Leukemia Patient: An Unusual Association.	27148941	We describe a 4-year-old female with pre-B-cell acute lymphoblastic leukemia on maintenance chemotherapy, who developed hemophagocytic lymphohistiocytosis (HLH) secondary to Epstein-Barr virus (EBV) infection, complicated by an aggressive lymphoproliferative disorder. Although there was no history of bone marrow transplant or underlying immunodeficiency, EBV triggered a post-transplant lymphoproliferative disease (PTLD)-like lymphoma. Multiple regimens of chemotherapy failed to induce remission and patient developed multiorgan failure. The association of HLH with EBV-related PTLD-like lymphoproliferative disorder is rare. We present this case to highlight this unusual association so that this highly fatal disease can be recognized and promptly addressed.
1395	Disseminated Histoplasmosis with Haemophagocytic Lymphohistiocytosis in an Immunocompetent Host.	27134914	Haemophagocytic lymphohistiocytosis (HLH) is a devastating syndrome due to uninhibited immune activation. Disseminated histoplasmosis is a rare cause of HLH. There have been few case reports and series demonstrating a relation between the two disease entities in immunosuppressed hosts. HLH secondary to disseminated histoplasmosis is even rarer in an immunocompetant host. We report a rare case of HLH triggered by disseminated histoplasmosis in an immunocompetant patient.
1396	HLH susceptibility: genetic lesions add up.	2712729926864340	NaN
1397	The Prognostic Significance of Beta2 Microglobulin in Patients with Hemophagocytic Lymphohistiocytosis.	27110054	To determine the prognostic significance of beta2 microglobulin (β2-m) concentrations in patients with hemophagocytic lymphohistiocytosis (HLH), a rare disorder caused by pathologic activation of the immune system.The study population consisted of 74 patients diagnosed with HLH and 35 healthy controls. Serum β2-m levels were measured using a latex agglutination photometric immunoassay.Median serum β2-m levels were significantly higher in HLH patients than in healthy controls (4.05 versus 1.5 mg/L; P < 0.001) and were significantly higher in patients with lymphoma associated hemophagocytic syndrome (LAHS) than in patients with benign disease-associated HLH (4.2 versus 3.3 mg/L; P < 0.001). Higher serum β2-m levels were positively correlated with LAHS (P = 0.005), abnormal lactate dehydrogenase concentrations (P = 0.009), and hypoalbuminemia (P = 0.003). ROC analysis showed that overall survival (OS) was significantly shorter in LAHS patients with serum β2-m levels ≥4.03 mg/L compared to <4.03 mg/L (P < 0.001). Moreover, multivariate analysis showed that serum β2-m level was an independent prognostic of OS (P = 0.034) in patients with LAHS.High serum β2-m levels and LAHS were associated with markedly poorer OS in patients with HLH. Serum β2-m concentration was a powerful and independent prognostic factor for OS in patients with LAHS.
1398	"Irony" of managing refractory anemia with transfusional support in hemophagocytic lymphohistiocytosis.	27102761	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening derangement of the immune system in which host macrophages phagocytose the patient's own blood cells. Herein, we present the case of a patient with HLH and associated refractory anemia who developed rapid iron deposition in the liver after transfusion of sixteen units of packed red blood cells (RBCs). Before transfusion, neither a liver biopsy nor computed tomography scan demonstrated iron deposition in the organ parenchyma. After receiving sixteen units of packed RBCs, liver iron concentration rose to 6.7 mg/g dry weight, which is highly unusual in other diseases requiring transfusional support.
1399	Synchronous brain and intravascular B-cell lymphoma after remission of an adult hemophagocytic syndrome.	27099719	Hemophagocytic lymphohistiocytosis (HLH) should be considered in the differential diagnosis of adult patients with white matter disease. Brain involvement can be life-threatening and should prompt aggressive therapy. Even after HLH remission, the possibility of subsequent deterioration due to emergence of an aggressive intravascular lymphoma is highlighted here.
1400	The minimum required level of donor chimerism in hereditary hemophagocytic lymphohistiocytosis.	27099148	Reduced-intensity conditioning has improved survival after hematopoietic stem cell transplantation (HSCT) for hemophagocytic lymphohistiocytosis (HLH) at the cost of more frequent mixed chimerism. The minimum level of donor chimerism (DC) required to prevent HLH reactivation in humans remains to be determined. In a multicenter retrospective study, 103 patients transplanted for hereditary HLH (2000-2013) and DC permanently or transiently <75% (overall, CD3(+), CD56(+)) were analyzed regarding DC, specific immunologic function, occurrence of systemic reactivations (≥5/8 HLH criteria), partial systemic flares (<5 criteria and HLH-directed treatment), isolated central nervous system reactivations, and management. Recurrence was reported in 18 patients (systemic reactivation n = 11, partial flare n = 3, isolated central nervous system reactivation n = 4). Ten events occurred during profound immune suppression before day 180 (median DC, 10%; range, 1-100%; CD3(+) if available, otherwise overall DC), which renders a differentiation between secondary post-HSCT HLH and HLH related to the genetic defect difficult. Eight events occurred between 0.5 and 6.7 years post-HSCT (median DC, 13%; range, 0-30%). In 5 patients, overall and lineage-specific DC were ≤10% for >6 months (median, 5.1; range, 1.1-10 years) without reactivation. A second HSCT was performed in 18 patients (median, DC 4%; range, 0-19%). Death from reactivation occurred in 4 patients (22% of recurrences). Six patients died of transplant complications following a second HSCT (33% of second HSCT). We conclude that a DC >20%-30% is protective against late reactivation. Lower levels do not, however, inescapably result in recurrences. The decision for or against second HSCT must be based on a thorough risk assessment.
1401	Hemophagocytic lymphohistiocytosis: an update for nephrologists.	27098410	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome caused by defective lytic capability of cytotoxic T lymphocytes and NK cells, which results in proliferation of benign hemophagocytic histiocytes. A cytokine storm ensues, and a severe systemic inflammatory response syndrome, multiorgan dysfunction syndrome, and death frequently follow. It may occur as a primary (inherited) form, or be acquired secondary to malignancy, infection, rheumatologic disease, or immunosuppression. Cardinal manifestations include fever, cytopenias, hepatosplenomegaly, and dysfunction of liver, kidney, CNS, and/or lung. Additional laboratory findings include marked hyperferritinemia, hypofibrinogenemia, hypertriglyceridemia, abnormal LFTs, coagulopathy, and hyponatremia. Nephrologists need to be aware of this syndrome owing to the frequent occurrence of acute kidney injury in these severely ill patients. Glomerulopathy and nephrotic syndrome may develop. Kidney transplant recipients are at increased risk of HLH due to immunosuppression, and most such cases are triggered by infection with over 50 % mortality. Effective treatment of HLH usually requires chemoimmunotherapy to acutely suppress inflammation, specific treatment of underlying infection or malignancy, and in certain cases hematopoietic stem cell transplantation. The pathogenesis, clinical manifestations, diagnosis, and treatment of HLH are discussed.
1402	Neonate with haemophagocytic lymphohistiocytosis secondary to dengue infection: a case report.	27083493	We report the first case of haemophagocytic lymphohistiocytosis (HLH) in a neonate secondary to primary Dengue virus infection. This neonate presented in the third week of life with fever, shock and hepatosplenomegaly and was diagnosed to have Dengue infection by serology and HLH was confirmed on bone marrow.
1403	Hemophagocytic Lymphohistiocytosis: Single-Center Series of 12 Cases from Saudi Arabia.	27081327	Hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal disease that commonly appears in infancy, although it has been reported in adults. Chemoimmunotherapy-based treatments have improved the survival of patients with HLH; however, overall survival is still poor. We retrospectively analyzed the data of 12 HLH patients who were admitted between 2005 and 2014. All patients were Saudi Arabia in origin with a female predominance (75%) and a median age of onset of 9.5 months. The consanguinity rates were significantly high (75%) with a positive family history in 41% of cases. Of the 12 patients, nine were defined as primary HLH patients and three were confirmed to be secondary HLH patients. All patients fulfilled the 2004 diagnostic criteria for HLH and received HLH-2004 treatment. Six of these patients showed a good response to chemotherapy, while the remainder of the patients showed partial or no response to chemotherapy. Five patients in this cohort received stem cell transplant, and these patients are currently in remission. The mortality rate of this cohort is currently 50%. Genetic mutational analysis showed a positive STX11 mutation in five patients and a PRF1 (perforin) mutation in two patients. To the best of our knowledge, this is the first case series of HLH from Saudi Arabia.
1404	Hemophagocytic lymphohistiocytosis caused by systemic herpes simplex virus type 1 infection: Successful treatment with dexamethasone palmitate.	27076380	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory condition resulting from an uncontrolled and ineffective immune response. Here, we report a case of HLH caused by disseminated herpes simplex virus (HSV)-1 infection. The patient was initially treated with prednisolone and high-dose acyclovir. Although liver enzymes, coagulation abnormalities, and inflammatory markers were remarkably improved, the platelet count remained low. Prednisolone was therefore switched to dexamethasone palmitate. Thereafter, the platelet count normalized. Inflammatory markers normalized 30 days after admission and serum HSV-DNA became undetectable on day 41. The patient was discharged on day 91 and no developmental delay was evident at 7 months of age. These findings suggest that dexamethasone palmitate is effective for neonatal HLH.
1405	A Negative Feedback Loop between PHYTOCHROME INTERACTING FACTORs and HECATE Proteins Fine-Tunes Photomorphogenesis in Arabidopsis.	27073231	The phytochrome interacting factors (PIFs), a small group of basic helix-loop-helix transcription factors, repress photomorphogenesis both in the dark and light. Light signals perceived by the phytochrome family of photoreceptors induce rapid degradation of PIFs to promote photomorphogenesis. Here, we show that HECATE (HEC) proteins, another small group of HLH proteins, antagonistically regulate PIFs to promote photomorphogenesis. HEC1 and HEC2 heterodimerize with PIF family members. PIF1, HEC1, and HEC2 genes are spatially and temporally coexpressed, and HEC2 is localized in the nucleus. hec1, hec2, and hec3 single mutants and the hec1 hec2 double mutant showed hyposensitivity to light-induced seed germination and accumulation of chlorophyll and carotenoids, hallmark processes oppositely regulated by PIF1. HEC2 inhibits PIF1 target gene expression by directly heterodimerizing with PIF1 and preventing DNA binding and transcriptional activation activity of PIF1. Conversely, PIFs directly activate the expression of HEC1 and HEC2 in the dark, and light reduces the expression of these HECs possibly by degrading PIFs. HEC2 is partially degraded in the dark through the ubiquitin/26S-proteasome pathway and is stabilized by light. HEC2 overexpression also reduces the light-induced degradation of PIF1. Taken together, these data suggest that PIFs and HECs constitute a negative feedback loop to fine-tune photomorphogenesis in Arabidopsis thaliana.
1406	Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling.	27070787	The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.
1407	Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence.	27068092	Aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis, A. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened the Neurospora crassa gene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of the A. fumigatus rhomboid homolog rbdA resulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation-accurately phenocopying deletion of srbA. In vivo, rbdA deletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdA mutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease in A. fumigatus that mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation.
1408	Exploratory Study to Identify Radiomics Classifiers for Lung Cancer Histology.	27064691	Radiomics can quantify tumor phenotypic characteristics non-invasively by applying feature algorithms to medical imaging data. In this study of lung cancer patients, we investigated the association between radiomic features and the tumor histologic subtypes (adenocarcinoma and squamous cell carcinoma). Furthermore, in order to predict histologic subtypes, we employed machine-learning methods and independently evaluated their prediction performance.Two independent radiomic cohorts with a combined size of 350 patients were included in our analysis. A total of 440 radiomic features were extracted from the segmented tumor volumes of pretreatment CT images. These radiomic features quantify tumor phenotypic characteristics on medical images using tumor shape and size, intensity statistics, and texture. Univariate analysis was performed to assess each feature's association with the histological subtypes. In our multivariate analysis, we investigated 24 feature selection methods and 3 classification methods for histology prediction. Multivariate models were trained on the training cohort and their performance was evaluated on the independent validation cohort using the area under ROC curve (AUC). Histology was determined from surgical specimen.In our univariate analysis, we observed that fifty-three radiomic features were significantly associated with tumor histology. In multivariate analysis, feature selection methods ReliefF and its variants showed higher prediction accuracy as compared to other methods. We found that Naive Baye's classifier outperforms other classifiers and achieved the highest AUC (0.72; p-value = 2.3 × 10(-7)) with five features: Stats_min, Wavelet_HLL_rlgl_lowGrayLevelRunEmphasis, Wavelet_HHL_stats_median, Wavelet_HLL_stats_skewness, and Wavelet_HLH_glcm_clusShade.Histological subtypes can influence the choice of a treatment/therapy for lung cancer patients. We observed that radiomic features show significant association with the lung tumor histology. Moreover, radiomics-based multivariate classifiers were independently validated for the prediction of histological subtypes. Despite achieving lower than optimal prediction accuracy (AUC 0.72), our analysis highlights the impressive potential of non-invasive and cost-effective radiomics for precision medicine. Further research in this direction could lead us to optimal performance and therefore to clinical applicability, which could enhance the efficiency and efficacy of cancer care.
1409	ITK Gene Mutation: Effect on Survival of Children with Severe Hemophagocytic Lymphohistiocytosis.	27056244	Hemophagocytic lymphohistiocytosis (HLH) is characterized by deadly hyperinflammatory syndrome, but data on severe HLH with multi-organ dysfunction in children are scant. The authors report a retrospective study of 8 cases with severe HLH from a pediatric intensive care unit (PICU) over a 1-y period and found that Epstein barr virus (EBV) -infection was the most common etiology. All patients had genetic analysis, which showed that four patients with EBV -infection had one homozygous mutation, c.985+75G>A (at position chr5:156667232) in exon10 of the ITK gene with poor survival rates. ITK + mutation group had higher percentages of CD3+CD8+ T cells (36.0 ± 8.4 %) than those in ITK - mutation group (28.8 ± 5.5 %), while they had similar levels of CD3+CD4+ T cells. ITK + mutation group had lower proportion of CD3-CD19+ B cells (16.3 ± 2.9 %) and CD16+CD56+ NK cells (8.4 ± 2.6 %) than ITK - mutation group (29.6 ± 5.1 % and 15.9 ± 9.0 % respectively). Most importantly, patients with EBV infection with c.985+75G>A mutation in ITK had lower survival rates than ITK - mutation group which it may be related with cellular immune dysfunction.
1410	[X-linked lymphoproliferative syndrome type 1 complicated with secondary hemophagocytic lymphohistiocytosis and ileal perforation: case report and literature review].	27055430	To analyze and summarize the clinical characteristics, laboratory tests and treatment of X-linked lymphoproliferative syndrome type 1 (XLP-1).A retrospective study was done in 2012 on an XLP-1 patient to collect the data on clinical manifestation, laboratory examination, gene and protein expression, complications and prognosis. Literatures were reviewed in Pubmed with the key word"X-linked lymphoproliferative syndrome".The patient with persistent high fever, jaundice, abdominal distension, hepatosplenomegaly and lymphadenectasis, rash and suspicious positive family history; the patient eventually died of hemophagocytic lymphohistiocytosis (HLH), with intestinal perforation, intestinal infection and bleeding after being infected with EB virus. This patient with SH2D1A gene exon 1 large fragment of the coding region of the nucleotide deletion and insertion mutations causing missense mutations (p.Leu25Lys) and nonsense mutations (stop codon TAG was inserted after missense mutation so that the protein encoded by the early termination of the 25 amino acids), which led to SAP protein missing. The expression of SAP in his mother was also partly missing. Retrieval of reports on XLP-1 was conducted through literature search (included totally 157 cases) at home and abroad, positive family history accounted for 60.6%(40/66); lymphoma incidence accounted for 49.7%(72/145); low gamma globulin occurred in 24.8%(39/157) of cases; secondary HLH ratio accounted for 43.3%(68/157); XLP-1 in patients with hemorrhagic enteritis and gastritis was low, accounted for only 2.6%(3/116).XLP-1 patients occasionally develop necrotic enteritis complicated with ileal perforation.XLP-1 with large fragment deletion of SH2D1A gene might be associated with serious gastrointestinal manifestations.
1411	The Rapid Effectiveness of Minocycline against Scrub Typhus Meningoencephalitis.	27041169	Scrub typhus is associated with various clinical symptoms. However, the pathogenesis of scrub typhus infection remains to be elucidated. A 73-year-old man was admitted to our hospital with consciousness disturbance and suspected meningoencephalitis. The patient's laboratory data showed deterioration and were indicative of hemophagocytic lymphohistiocytosis (HLH). A whole body examination to detect the trigger disease revealed an eschar, which is a characteristic of scrub typhus, on his back. His symptoms showed dramatic improvement after the administration of minocycline (MINO). This case report highlights that the clinical course of a case of scrub typhus meningoencephalitis that was cured with MINO.
1412	Alleviating the storm: ruxolitinib in HLH.	2703441726825707	In this issue of Blood, Das and colleagues report their results on the use of the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib in murine models of hemophagocytic lymphohistiocytosis (HLH), and the HLH-sibling macrophage activation syndrome (MAS).
1413	Murine Models of Epstein-Barr Virus-Associated Lymphomagenesis.	27034395	The Epstein-Barr virus (EBV) is a B-lymphotropic gamma herpes virus associated with a number of malignancies. Most EBV-related cancers present complex medical management challenges; thus it has been essential to develop preclinical in vivo models allowing for the study of pathogenesis, prevention, and treatment of these diseases. Early in vivo models used nonhuman primates; however, such models were limited by the inability of EBV to achieve viral latency, availability, and cost. Immunodeficient mouse strains emerged as efficient models that allow for engraftment of human mononuclear cells and controlled evaluation of EBV-driven lymphoproliferative disease (EBV-LPD). By using highly immunodeficient strains of mice such as severe combined immune deficiency (SCID) and NOD/LtSz-scid ILrg(-/-)(NOG) mice, investigators have developed efficient platforms for evaluating pathogenesis of benign (HLH) and malignant (EBV-LPD) diseases associated with EBV. Humanized murine chimeric models have been essential tools for evaluating preventive strategies with vaccine and adoptive cellular approaches, as well as development of experimental therapeutic strategies. Manipulation of the human immune cells before engraftment or mutation of viral lytic and latent genes has enhanced our understanding of the oncogenic nature of EBV and the complexity of human immune responses to EBV. In this review, we discuss how the EBV murine models have evolved to become essential tools for studying the virology of EBV as it relates to human EBV-LPD pathogenesis, the immunobiology of innate and adaptive responses, and limitations of these models.
1414	[Successful Treatment of Seasonal Influenza A (H3N2) infection-related Hemophagocytic Lymphocytosis in an Elderly Man].	27032178	A 79-year-old man experienced severe chronic obstructive pulmonary disease (COPD) and was receiving treatment for ischemic heart disease. Starting from dizziness and chilliness, he lost consciousness after few days. He was taken to our emergency department. On initial evaluation, he complained of dyspnea and was afebrile with a pulse rate, blood pressure, and respiratory rate of 105 beats/min, 112/98mmHg, and 28 breath/min, respectively. His respiratory sounds were clear and chest radiography did not show any abnormal shadows, but his arterial blood gas examination showed type II respiratory failure. Because the nasopharyngeal seasonal influenza A virus (IAV) test was positive, the patient was admitted with the diagnosis of acute exacerbation of COPD due to IAV. We administered peramivir, a specific anti-influenza drug, and started mechanical ventilation. Over time, he started to show signs of disseminated intravascular coagulation, such as multiple organ failure and thrombocytopenia. Subsequently, blood tests showed elevation of ferritin and soluble interleukin 2 receptor (sIL2R); microscopic examination of the peripheral blood revealed hemophagocytosis. Secondary hemophagocytic lymphohistiocytosis (HLH) due to IAV was diagnosed and together with corticosteroid therapy, intravenous gamma globulin was administered from the 3rd clinical day. The patint was saved with our early diagnosis and treatment of HLH and was discharged on the 92nd clinical day. Viral-induced HLH, formerly known as virus-associated hemophagocytic syndrome (VAHS), leads to multiple organ failure due to a cytokine storm scattered by viral-infected pathogenic inflammatory cells. It is well known that pandemic swine flu causes secondary HLH leading to poor outcomes. Currently, not much is known about HLH due to seasonal flu; particularly, IAV (H3N2)-related HLH cases are rare and reported cases showed poor outcomes as well. With an early diagnosis and minimum immunotherapy, we report herein on a case of IAV (H3N2)-related HLH which was treated successfully.
1415	[Prokaryotic expression and purification of Aquilaria sinensis(Lour.) Gilg AsMYC2 protein].	29860754	The MYC2 transcription factor is a member of the important plant b HLH transcription factor families, and it is also the core regulatory elements in jasmonate（JA） signaling pathway. However, there is a little information about AsMYC2 gene in Aquilaria sinensis. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length coding sequence (CDS) of AsMYC2 gene was amplified using RT-PCR method and subcloned into pGEX-4T-1 vector by the gene recombination technique. The recombinant vector pGEX-4T-1-AsMYC2 was verified by restriction enzyme digestion and nucleotide sequencing, and was transformed into E. coli BL21（DE3） to express the protein. A maximum expression of soluble protein was observed with induction by 0.1 mmol·L(-1) IPTG at 37 ℃ for 4 hours. The fusion protein was purified through a Sepharose-Glutathione column, and verified by SDS-PAGE and Western blotting using an anti-GST polyclonal antibody. We successfully constructed the GST-AsMYC2 plasmid, produced and purified the GST-AsMYC2 fusion protein, which would provide the basic material for polyclonal antibody preparation, interactive factors screening and gene function research. According to the tissue-specific expression pattern analysis by q RT-PCR method, the AsMYC2 gene in A. sinensis tissues is mainly expressed in roots and stems, the main agarwood formation parts, and lowest expressed in leaves. These results indicate that AsMYC2 gene likely play some roles in agarwood formation in A. sinensis.
1416	Macrophage activation syndrome in the era of biologic therapy.	27009539	Macrophage activation syndrome (MAS) refers to acute overwhelming inflammation caused by a 'cytokine storm'. Although increasingly recognized as a life-threatening complication of various rheumatic diseases, clinically, MAS is strikingly similar to primary and secondary forms of haemophagocytic lymphohistiocytosis (HLH). Not surprisingly, many rheumatologists prefer the term secondary HLH rather than MAS to describe this condition, and efforts to change the nomenclature are in progress. The pathophysiology of MAS remains elusive, but observations in animal models, as well as data on the effects of new anticytokine therapies on rates and clinical presentations of MAS in patients with systemic juvenile idiopathic arthritis (sJIA), provide clues to the understanding of this perplexing clinical phenomenon. In this Review, we explore the latest available evidence and discuss potential diagnostic challenges in the era of increasing use of biologic therapies.
1417	Mondo complexes regulate TFEB via TOR inhibition to promote longevity in response to gonadal signals.	27001890	Germline removal provokes longevity in several species and shifts resources towards survival and repair. Several Caenorhabditis elegans transcription factors regulate longevity arising from germline removal; yet, how they work together is unknown. Here we identify a Myc-like HLH transcription factor network comprised of Mondo/Max-like complex (MML-1/MXL-2) to be required for longevity induced by germline removal, as well as by reduced TOR, insulin/IGF signalling and mitochondrial function. Germline removal increases MML-1 nuclear accumulation and activity. Surprisingly, MML-1 regulates nuclear localization and activity of HLH-30/TFEB, a convergent regulator of autophagy, lysosome biogenesis and longevity, by downregulating TOR signalling via LARS-1/leucyl-transfer RNA synthase. HLH-30 also upregulates MML-1 upon germline removal. Mammalian MondoA/B and TFEB show similar mutual regulation. MML-1/MXL-2 and HLH-30 transcriptomes show both shared and preferential outputs including MDL-1/MAD-like HLH factor required for longevity. These studies reveal how an extensive interdependent HLH transcription factor network distributes responsibility and mutually enforces states geared towards reproduction or survival.
1418	Impairment of Immune Function in Children with Familial Hemophagocytic Lymphohistiocytosis.	26987330	Hemophagocytic lymphohistiocytosis (HLH) is a severe systemic syndrome associated with hyperactivation of macrophages and impaired regulation of the immune system. Two forms of HLH are currently recognized: genetically determined or familial (FHLH), and secondarily developed in the course of primary diseases, like autoimmune disorders, rheumatoid disorders, cancers, or infections. In the Polish population, FHLH is rather rare. The aim of the present study was to assess the immune function in a group of children with clinical symptoms suggesting FHLH. Forty five children with suspected HLH of the median age of 4 years and 15 healthy children, taken as a control group, were enrolled into the study. All presented results were obtained with the use of flow cytometry. In the HLH group, there were only three cases identified with the UNC13D gene mutation responsible for the FHLH3 phenotype. Another four children, without known mutation, were classified as FHLH because of frequent recurrence of the disease. In all cases of FHLH, cell cytotoxicity was impaired compared with healthy children (p = 0.003). Perforin expression in FHLH was normal or higher than that observed in controls (p = 0.09). In case of patients with mutation in the Munc13 protein, degranulation was lower than that in healthy children (<5 %). The findings of this study demonstrate that children with known mutations responsible for the FHLH development are immunocompromised. However, it requires further elucidation whether the presence of currently unknown mutations could lead to a similar phenotype.
1419	Cord Blood Transplantation Following Reduced-intensity Conditioning for Adult-onset Inherited Hemophagocytic Lymphohistiocytosis.	26984088	Inherited hemophagocytic lymphohistiocytosis (HLH) is a genetic anomaly disorder in which abnormally activated cytotoxic T lymphocytes cannot induce the apoptosis of target cells and antigen-presenting cells, leading to hemophagocytosis, pancytopenia, and a variety of symptoms such as a high fever. The present patient with adult-onset HLH developed refractory disease despite receiving immunosuppressive treatments. He underwent a reduced-intensity conditioning (RIC) regimen that comprised antithymocyte globulin (ATG) followed by cord blood transplantation (RIC-CBT). He achieved and maintained a complete donor type. The incorporation of ATG into RIC-CBT may prevent graft failure and control hemophagocytosis, however, further efforts are necessary to reduce infectious complications.
1420	An Unusual Case of Systemic Lupus Erythematosus and Hemophagocytic Syndrome.	26981305	Hemophagocytic syndrome (HS) or hemophagocytic lymphohistiocytosis (HLH) is an immune mediated phenomenon that can occur in the setting of an autoimmune disease, chronic immunosuppression, malignancy, or infection. It has been more commonly described in the pediatric population and less commonly in adults. We describe a case of a 52-year-old male who presented with a rash. He simultaneously met the Systemic Lupus International Collaborating Clinics (SLICC) criteria for the diagnosis of systemic lupus erythematosus (SLE) and the diagnostic criteria of HS as described in the hemophagocytic lymphohistiocytosis (HLH) 2004 trial. The bone marrow on autopsy showed the presence of abundant hemosiderophages with focal hemophagocytosis. SLE-associated HS might be underdiagnosed due to the overlap in clinical findings. This case represents the importance of prompt diagnosis and treatment of such a potentially fatal clinical syndrome.
1421	Poor growth, thyroid dysfunction and vitamin D deficiency remain prevalent despite reduced intensity chemotherapy for hematopoietic stem cell transplantation in children and young adults.	26974276	Myeloablative conditioning regimens for hematopoietic stem cell transplant (HSCT) are known to affect endocrine function, but little is known regarding reduced intensity conditioning (RIC) regimens. We retrospectively reviewed 114 children and young adults after single RIC HSCT. The analysis was grouped by age (<2 and ⩾2 years) and diagnosis (hemophagocytic lymphohistiocystosis/X-linked lymphoproliferative syndrome (HLH/XLP), other immune disorders, metabolic/genetic disorders). All groups displayed short stature by mean height-adjusted Z-score (HAZ) before (-1.29) and after HSCT (HAZ -1.38, P=0.47). After HSCT, younger children with HLH/XLP grew better (HAZ -3.41 vs -1.65, P=0.006), whereas older subjects had decline in growth (HAZ -0.8 vs -1.01, P=0.06). Those with steroid therapy beyond standard GVHD prophylaxis were shorter than those without (P 0.04). After HSCT, older subjects with HLH/XLP became thinner with a mean body mass index (BMI) Z-score of 1.20 vs 0.64, P=0.02, and similar to metabolic/genetic disorders (BMI-Z= 0.59 vs -0.99, P<0.001). BMI increased among younger children in these same groups. Thyroid function was abnormal in 24% (18/76). 25-OH vitamin D levels were insufficient in 73% (49/65), with low bone mineral density in 8 of 19 evaluable subjects. Despite RIC, children and young adults still have significant late endocrine effects. Further research is required to compare post-transplant endocrine effects after RIC to those after standard chemotherapy protocols.
1422	A Diagnostic Dilemma: Similarity of Neuroradiological Findings in Central Nervous System Hemophagocytic Lymphohistiocytosis and Aspergillosis.	26970537	Central nervous system (CNS) involvement in the context of hemophagocytic lymphohistiocytosis (HLH) is not uncommon. Given the immunosuppressive nature of HLH therapy, infectious complications are also seen. We describe a 9-year-old male who developed acute neurological decline secondary to aspergillosis while undergoing HLH therapy. The significant overlap observed in CNS neuroimaging of HLH and aspergillosis and the subtleties that may help differentiate the two are discussed. The importance of obtaining tissue for definitive diagnosis is underscored.
1423	IL-2 consumption by highly activated CD8 T cells induces regulatory T-cell dysfunction in patients with hemophagocytic lymphohistiocytosis.	26947179	Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition driven by excessive CD8(+) T-cell activation. HLH occurs as both acquired and familial hemophagocytic lymphohistiocytosis (FHL) forms. In both conditions, a sterile or infectious trigger is required for disease initiation, which then becomes self-sustaining and life-threatening. Recent studies have attributed the key distal event to excessive IFN-γ production; however, the proximal events driving immune dysregulation have remained undefined.We sought to investigate the role of regulatory T (Treg) cells in the pathophysiology of experimental FHL.Because mutation in perforin is a common cause of FHL, we used an experimental FHL mouse model in which disease in perforin-deficient mice is triggered by lymphocytic choriomeningitis virus (LCMV). We assessed Treg and CD8(+) T-cell homeostasis and activation during the changing systemic conditions in the mice. In addition, human blood samples were collected and analyzed during the HLH episode.We found no primary Treg cell defects in perforin-deficient mice. However, Treg cell numbers collapsed after LCMV inoculation. The collapse of Treg cell numbers in LCMV-triggered perforin-deficient, but not wild-type, mice was accompanied by the combination of lower IL-2 secretion by conventional CD4(+) T cells, increased IL-2 consumption by activated CD8(+) T cells, and secretion of competitive soluble CD25. Moreover low Treg cell numbers were observed in untreated patients experiencing HLH flares.These results demonstrate that excessive CD8(+) T-cell activation rewires the IL-2 homeostatic network away from Treg cell maintenance and toward feed-forward inflammation. These results also provide a potential mechanistic pathway for the progression of infectious inflammation to persistent inflammation in patients with HLH.
1424	Extreme Hypertriglyceridemia in an Infant with Hemophagocytic Lymphohistiocytosis and Hydroxycobalamin Deficiency.	26946773	Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition characterized by fever, cytopenias, hepatosplenomegaly and hemophagocytosis. HLH may be primary or secondary to infection, autoimmune disease or malignancy. Hypertriglyceridemia is a common abnormality in HLH and one of the HLH-2004 diagnostic criteria.We present an infant with severe hypotonia and hypoproteinemic edema who also had extreme hypertriglyceridemia (21 mmol/l) and was diagnosed with HLH based on six of eight HLH-2004 criteria (fever, hepatosplenomegaly, bicytopenia, hypertriglyceridemia with hypofibrinogenemia, slL-2R > 2400 IU/ml, hemophagocytosis). The presence of IgM antibodies to Epstein-Barr virus and cytomegalovirus indicated a probable infectious trigger. The child was cured by the HLH-2004 protocol for secondary HLH (consisting of dexamethasone and cyclosporine). He was also found to have low serum hydroxycobalamin levels, promptly corrected upon hydroxycobalamin administration.The presented case history underlines the need to ascertain the presence or absence of each of the eight HLH-2004 criteria in any patient suspected to suffer from HLH.
1425	IDO1 Deficiency Does Not Affect Disease in Mouse Models of Systemic Juvenile Idiopathic Arthritis and Secondary Hemophagocytic Lymphohistiocytosis.	26914138	Indoleamine 2,3-dioxygenase-1 (IDO1) is an immune-modulatory enzyme that catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn) and is strongly induced by interferon (IFN)-γ. We previously reported highly increased levels of IFN-γ and corresponding IDO activity in patients with hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory syndrome. On the other hand, IFN-γ and IDO were low in patients with systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory syndrome. As HLH can occur as a complication of sJIA, the opposing levels of both IFN-γ and IDO are remarkable. In animal models for sJIA and HLH, the role of IFN-γ differs from being protective to pathogenic. In this study, we aimed to unravel the role of IDO1 in the pathogenesis of sJIA and HLH.Wild-type and IDO1-knockout (IDO1-KO) mice were used in 3 models of sJIA or HLH: complete Freund's adjuvant (CFA)-injected mice developed an sJIA-like syndrome and secondary HLH (sHLH) was evoked by either repeated injection of unmethylated CpG oligonucleotide or by primary infection with mouse cytomegalovirus (MCMV). An anti-CD3-induced cytokine release syndrome was used as a non-sJIA/HLH control model.No differences were found in clinical, laboratory and hematological features of sJIA/HLH between wild-type and IDO1-KO mice. As IDO modulates the immune response via induction of regulatory T cells and inhibition of T cell proliferation, we investigated both features in a T cell-triggered cytokine release syndrome. Again, no differences were observed in serum cytokine levels, percentages of regulatory T cells, nor of proliferating or apoptotic thymocytes and lymph node cells.Our data demonstrate that IDO1 deficiency does not affect inflammation in sJIA, sHLH and a T cell-triggered cytokine release model. We hypothesize that other tryptophan-catabolizing enzymes like IDO2 and tryptophan 2,3-dioxygenase (TDO) might compensate for the lack of IDO1.
1426	Mouse Cytomegalovirus Infection in BALB/c Mice Resembles Virus-Associated Secondary Hemophagocytic Lymphohistiocytosis and Shows a Pathogenesis Distinct from Primary Hemophagocytic Lymphohistiocytosis.	26903481	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the β-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8(+) T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV-infected IFN-γ-deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ-deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH.
1427	Previously undiagnosed fatal familial haemophagocytic lymphohistiocytosis in a 24-year-old woman.	26903364	We present a case of a 24-year-old woman with previously undiagnosed familial haemophagocytic lymphohistiocytosis (HLH). The patient presented with fevers and cough and was found to have pancytopaenia. She underwent an extensive work up and initially met only 3 of 8 criteria for HLH. Owing to high clinical suspicion, soluble CD25 level was sent and HLH2004 protocol initiated. The soluble CD25 level returned elevated with other laboratory work and the patient met criteria for diagnosis of HLH. Genetic studies revealed a homozygous mutation in PRF1 with absent perforin in cytotoxic cells, consistent with familial HLH. The patient expired before intrathecal chemotherapy could be initiated. This case illustrates the potential for familial HLH to present at an older age, and highlights the importance of early recognition and initiation of treatment of HLH, as patients may not initially fulfil the diagnostic criteria for HLH, and mortality is high if left untreated.
1428	The diagnosis of adult-onset haemophagocytic lymphohistiocytosis: lessons learned from a review of 29 cases of bone marrow haemophagocytosis in two large academic institutions.	26896491	Haemophagocytic lymphohistiocytosis (HLH) is divided into paediatric (primary) and adult (secondary) types. While paediatric-HLH has been extensively characterised, similar studies in adults are limited. This study aims to evaluate the significance of the HLH diagnostic criteria as well as other clinical parameters in adults with bone marrow evidence of haemophagocytosis.We conducted a 10-year retrospective search of the pathology archives of two institutions for cases with bone marrow haemophagocytosis. We included those cases that fulfilled the currently established HLH diagnostic criteria. For the 29 cases that met inclusion criteria, we assessed clinical features, co-morbidities, therapy and clinical outcome. The effect of 19 clinical variables on mortality outcomes was assessed using logistic and hazard regression analyses.Of cases for which an aetiology could be identified, infectious diseases were the most common association (14 of 19, 74%). Fever and elevated ferritin were the most frequently available criteria used to establish HLH. The overall mortality rate was 61% despite HLH-specific therapy, which had been initiated in 48% of the cases. The remaining cases were treated with supportive therapy and antibiotics. The most statistically significant marker of mortality was an elevated absolute neutrophil count (ANC), a feature not typical of HLH.Since elevated ANC correlates with poor outcomes in sepsis, and not HLH, we postulate that many of the patients fulfilling HLH diagnostic criteria in this study likely had sepsis/systemic inflammatory response syndrome rather than HLH. Our results highlight the need to define HLH diagnostic criteria specific to the adult population.
1429	[A multicenter retrospective etiological analysis of 601 patients with hemophagocytic lymphohistiocytosis in China].	26887367	To investigate the prevalence and etiology of hemophagocytic lymphohistiocytosis (HLH) in different age groups.Clinical data of patients with HLH were retrospectively collected from June 2005 to March 2014 in 49 hospitals in China. These patients were divided into child, youth, middle-aged and elderly groups according to the age of onset; meanwhile divided into primary HLH group, infection-associated HLH group, tumor-associated HLH group, rheumatic disease-associated HLH group according to the etiology. Prevalence rates, gender and underlying diseases of each group were retrospectively analyzed.A total of 601 patients were included in the study, age ranging from 1 month to 82 years. The median age of onset was 27 years and 26 years respectively in males(316 cases) and females (285 cases) without statistical significance (P=0.622). There were 171 in child group (28.5%); 262 in youth group(43.6%); 104 in middle-aged group(17.3%); 64 in elderly group(10.6%). The most common causes were infections in child group, malignancies and infections in youth group, malignancies in middle-aged group as well as elderly group. There were 48 patients in primary HLH group(8.0%), 197 in infection-associated HLH group(32.78%), 208 in tumor-associated HLH group(34.61%), 56 in rheumatic disease-associated HLH group(9.32%). Patients with primary HLH were significantly younger than those with secondary HLH (P<0.001), however the tumor-associated HLH group was the eldest. Primary HLH, infection-associated HLH and tumor-associated HLH had more male patients but without statistical significance (P=0.196), while rheumatic disease-associated HLH was mostly female patients (P<0.001).HLH is not a disease peculiar to children, instead, it may occur in all ages. There are a variety of etiology in different age groups, and a possible link between sex and HLH development. In clinical practice, due attention should be paid to the patients with suspected HLH. Positive relevant inspections may aid in the final diagnosis.
1430	A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis.	26880764	Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in NK and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report two unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (c.259G→C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant- and wild-type-transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, respectively. Overexpression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein decreased binding to Munc13-4 and delayed granzyme B polarization toward the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.
1431	Current Updates on Classification, Diagnosis and Treatment of Hemophagocytic Lymphohistiocytosis (HLH).	26872683	Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hyperinflammatory syndrome characterized by excessive activation of macrophages and T cells resulting from defective cytotoxicity. Severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and histiocytes (macrophages) secreting high amounts of inflammatory cytokines threatens the life of the patient and may lead to death unless arrested by appropriate treatment. HLH can be caused either by certain underlying genetic diseases (familial HLH), or may also occur due to particular triggers in patients with no known inherited disorder (acquired HLH). Due to life threatening nature of the disease, early diagnosis and initiation of immunosuppressive therapy is extremely important. HLH diagnosis is based on constellation of clinical manifestations and laboratory parameters which often overlap with those of severe infection or sepsis. Identification of patients with familial HLH and their underlying genetic defects requires specialized laboratory tests and is important for predicting relapses and planning early therapeutic hematopoietic stem cell transplantation (HSCT). A high suspicion and thorough clinical, immunological and genetic work-up is required for diagnosis of HLH. Prompt initiation of adequate treatment is essential for the survival. Substantial progress has been made in exploring the complex cause and pathophysiology of HLH and also in management of HLH patients.
1432	An Unusual Cause of Acute Liver Failure: Three Cases of Hemophagocytic Lymphohistiocytosis Presenting at a Transplant Center.	26870936	Acquired hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disorder of the immune system. Hemophagocytic lymphohistiocytosis has been associated with infections, autoimmune disorders, and malignancy. This case series describes three patients admitted to an academic liver transplant center from February 2014 to February 2015 with acute liver failure (ALF) who were ultimately diagnosed with HLH. All cases were female patients (44 to 53 years of age) transferred for workup of ALF. All developed fevers and cytopenias and underwent rapid evaluation for liver transplant by a multidisciplinary team. A complete workup for ALF was negative for intrinsic liver disease and none had significant alcohol or toxin exposure. The patients had liver biopsies showing diffuse lobular necroinflammation, of which two had evidence of hemophagocytosis on histopathology. The diagnosis of HLH was made by bone marrow biopsy featuring histiocytes with hemophagocytosis. All cases were treated with chemotherapy, but died during their hospitalization. Hemophagocytic lymphohistiocytosis can present as ALF in adult patients. Given the low success rate of treatment, early diagnosis is critical. Therefore, a high degree of suspicion should be exercised in patients with unexplained ALF.
1433	Polygenic mutations in the cytotoxicity pathway increase susceptibility to develop HLH immunopathology in mice.	2686434027127299	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disease. Inherited forms of HLH are caused by biallelic mutations in several effectors of granule-dependent lymphocyte-mediated cytotoxicity. A small proportion of patients with a so-called "secondary" form of HLH, which develops in the aftermath of infection, autoimmunity, or cancer, carry a monoallelic mutation in one or more HLH-associated genes. Although this observation suggests that HLH may have a polygenic mode of inheritance, the latter is very difficult to prove in humans. In order to determine whether the accumulation of partial genetic defects in lymphocyte-mediated cytotoxicity can contribute to the development of HLH, we generated mice that were doubly or triply heterozygous for mutations in HLH-associated genes, those coding for perforin, Rab27a, and syntaxin-11. We found that the accumulation of monoallelic mutations did indeed increase the risk of developing HLH immunopathology after lymphocytic choriomeningitis virus infection. In mechanistic terms, the accumulation of heterozygous mutations in the two degranulation genes Rab27a and syntaxin-11, impaired the dynamics and secretion of cytotoxic granules at the immune synapse of T lymphocytes. In addition, the accumulation of heterozygous mutations within the three genes impaired natural killer lymphocyte cytotoxicity in vivo. The genetic defects can be ranked in terms of the severity of the resulting HLH manifestations. Our results form the basis of a polygenic model of the occurrence of secondary HLH.
1434	Adult hemophagocytic lymphohistiocytosis causing multi organ dysfunction in a patient with multiple autoimmune disorders: when the immune system runs amok.	26862416	We report a case of several autoimmune disorders eventually presenting as severe multi organ dysfunction syndrome caused by adult hemophagocytic lymphohistiocytosis (HLH). Clinical and laboratory tests might lead to fatal misinterpretation without awareness of its diagnostic evaluation, as HLH shares common features with sepsis and immune-mediated systemic inflammatory response syndromes.
1435	The role of natural killer cells in pathogenesis of autoimmune diseases.	26862312	There is growing evidence that NK cell-mediated immunoregulation plays an important role in the control of autoimmunity. NK cells are a subset of lymphocytes that generally contribute to innate immunity but have also a great impact on the function of T and B lymphocytes. The major role of NK cells is cytotoxic reaction against neoplastic, infected and autoreactive cells, but they regulatory function seems to play more important role in the pathogenesis of autoimmune diseases. Numerous studies suggested the involvement of NK cells in pathogenesis of such a common autoimmune diseases as juvenile rheumatoid arthritis, type I diabetes and autoimmune thyroid diseases. The defects of NK cells regulatory function as well as cytotoxic abilities are common in patients with autoimmune diseases with serious consequences including HLH hemophagocytic lymphocytosis (HLH) and macrophage activation syndrome (MAS). The early diagnosis of NK cells defect responsible for the loss of the protective abilities is crucial for the prevention of life-threatening complications and implementation of necessary treatment.
1436	Successful treatment of cytomegalovirus associated hemophagocytic lymphohistiocytosis with the interleukin 1 inhibitor - anakinra.	26839691	Hemophagocytic lymphohistiocytosis (HLH) is a rare, frequently under-recognized condition associated with multi-organ failure and very high mortality. A 44-year-old woman was admitted with a 4-day history of fever, headache, delirium, and dyspnea. She progressed rapidly to type 1 respiratory failure and required intubation and mechanical ventilation. Laboratory tests showed pancytopenia, abnormal liver enzyme levels, elevated triglyceride level, and elevated ferritin level. Bone marrow biopsy showed features of HLH. Computed tomography scan showed bilateral consolidation. Bronchoalveolar lavage was positive for cytomegalovirus. She was treated with ganciclovir, methylprednisolone, broad spectrum antibiotics, and cytomegalovirus hyperimmunoglobulin without clinical response. Given the poor prognosis and reports of success in pediatric HLH, anakinra 100 µg subcutaneously daily was commenced. There was rapid defervescence, resolution of delirium, and improvement in gas exchange, leading to complete recovery. This case illustrates successful treatment of HLH associated with cytomegalovirus pneumonitis with the interleukin 1 inhibitor anakinra.
1437	Pulmonary Involvement in Patients With Hemophagocytic Lymphohistiocytosis.	26836913	Acquired hemophagocytic lymphohistiocytosis (HLH) is a life-threatening event that usually occurs as a complication of immunodeficiency. Lung involvement in HLH has received little attention. This article describes lung involvement in HLH and assesses whether it affects the prognosis.We retrospectively studied 219 patients with HLH admitted to a national reference center over a 14-year period, including 118 (54%) with lung involvement.Dyspnea and cough were the most common onset symptoms. Radiographs revealed interstitial infiltrates with centrilobular nodules, ill-defined consolidation, or localized ground-glass opacities. Pleural effusions and mediastinal lymphadenopathies were found in approximately one-half of the patients. One or more causes of lung involvement were documented in 91 of 118 patients (77.1%) and included infection (n = 52), pulmonary edema (n = 34), and malignancies (n = 22 [mostly lymphoma]). HLH-specific treatment combined with treatment of the cause of lung involvement improved respiratory function in only 67 of the 188 patients (56.7%). Hospital mortality was higher in patients with lung involvement (52.5% vs 20%). Infection as the cause of lung involvement was the only determinant of death (56% vs 30%; P = .004).Lung involvement is common and of poor prognosis in patients with HLH. Studies should assess whether specific diagnostic and therapeutic strategies are warranted in patients with HLH and lung involvement.
1438	Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemophagocytic lymphohistiocytosis.	2682570727034417	Hemophagocytic lymphohistiocytosis (HLH) comprises an emerging spectrum of inherited and noninherited disorders of the immune system characterized by the excessive production of cytokines, including interferon-γ and interleukins 2, 6, and 10 (IL-2, IL-6, and IL-10). The Janus kinases (JAKs) transduce signals initiated following engagement of specific receptors that bind a broad array of cytokines, including those overproduced in HLH. Based on the central role for cytokines in the pathogenesis of HLH, we sought to examine whether the inhibition of JAK function might lessen inflammation in murine models of the disease. Toward this end, we examined the effects of JAK inhibition using a model of primary (inherited) HLH in which perforin-deficient (Prf1(-∕-)) mice are infected with lymphocytic choriomeningitis virus (LCMV) and secondary (noninherited) HLH in which C57BL/6 mice receive repeated injections of CpG DNA. In both models, treatment with the JAK1/2 inhibitor ruxolitinib significantly lessened the clinical and laboratory manifestations of HLH, including weight loss, organomegaly, anemia, thrombocytopenia, hypercytokinemia, and tissue inflammation. Importantly, ruxolitinib treatment also significantly improved the survival of LCMV-infectedPrf1(-∕-)mice. Mechanistic studies revealed that in vivo exposure to ruxolitinib inhibited signal transducer and activation of transcription 1-dependent gene expression, limited CD8(+)T-cell expansion, and greatly reduced proinflammatory cytokine production, without effecting degranulation and cytotoxic function. Collectively, these findings highlight the JAKs as novel, druggable targets for mitigating the cytokine-driven hyperinflammation that occurs in HLH. These observations also support the incorporation of JAK inhibitors such as ruxolitinib into future clinical trials for patients with these life-threatening disorders.
1439	Clinical characteristics and follow-up analysis of adult-onset Still's disease complicated by hemophagocytic lymphohistiocytosis.	26809798	We evaluated clinical characteristics and prognosis for adult-onset Still's disease (AOSD) complicated by hemophagocytic lymphohistiocytosis (HLH). We retrospectively identified cases of AOSD with (n = 10) and without (n = 305) HLH complications. We reviewed their medical records, completed follow-up through outpatient clinic and telephone interviews, and analyzed their clinical symptoms, signs, laboratory test results, treatments, and prognosis. More AOSD patients with HLH developed hepatomegaly, bleeding, serositis, and neurologic symptoms than those without HLH, and they more commonly presented with leukopenia, thrombocytopenia, severe anemia, severe liver function abnormalities, decreased fibrinogen, elevated immunoglobulin, and bone marrow hemophagocytosis. The ten patients with AOSD complicated by HLH were treated with high-dose steroids or pulse steroid therapy, and eight of them also received cytotoxic drugs, while biological agents showed poor response. Follow-up results indicated that AOSD patients overall had good prognosis, while those with HLH showed worse prognosis, including higher relapse and readmission rates and increased mortality. In patients with AOSD, unexplained decreased blood cells, severe liver dysfunction, and/or hemophagocytosis in the bone marrow should be considered as signs of HLH complication. Patients with AOSD complicated by HLH have worse prognosis and higher relapse rates compared to AOSD patients without HLH complications. Thus, these patients should undergo frequent and careful follow-up.
1440	Joint-linkage mapping and GWAS reveal extensive genetic loci that regulate male inflorescence size in maize.	26801971	Both insufficient and excessive male inflorescence size leads to a reduction in maize yield. Knowledge of the genetic architecture of male inflorescence is essential to achieve the optimum inflorescence size for maize breeding. In this study, we used approximately eight thousand inbreds, including both linkage populations and association populations, to dissect the genetic architecture of male inflorescence. The linkage populations include 25 families developed in the U.S. and 11 families developed in China. Each family contains approximately 200 recombinant inbred lines (RILs). The association populations include approximately 1000 diverse lines from the U.S. and China. All inbreds were genotyped by either sequencing or microarray. Inflorescence size was measured as the tassel primary branch number (TBN) and tassel length (TL). A total of 125 quantitative trait loci (QTLs) were identified (63 for TBN, 62 for TL) through linkage analyses. In addition, 965 quantitative trait nucleotides (QTNs) were identified through genomewide study (GWAS) at a bootstrap posterior probability (BPP) above a 5% threshold. These QTLs/QTNs include 24 known genes that were cloned using mutants, for example Ramosa3 (ra3), Thick tassel dwarf1 (td1), tasselseed2 (ts2), liguleless2 (lg2), ramosa1 (ra1), barren stalk1 (ba1), branch silkless1 (bd1) and tasselseed6 (ts6). The newly identified genes encode a zinc transporter (e.g. GRMZM5G838098 and GRMZM2G047762), the adapt in terminal region protein (e.g. GRMZM5G885628), O-methyl-transferase (e.g. GRMZM2G147491), helix-loop-helix (HLH) DNA-binding proteins (e.g. GRMZM2G414252 and GRMZM2G042895) and an SBP-box protein (e.g. GRMZM2G058588). These results provide extensive genetic information to dissect the genetic architecture of inflorescence size for the improvement of maize yield.
1441	Useful Parameters for Distinguishing Subcutaneous Panniculitis-like T-Cell Lymphoma From Lupus Erythematosus Panniculitis.	26796503	Some cases of subcutaneous panniculitis-like T-cell lymphoma (SPTCL) and lupus erythematosus panniculitis (LEP) demonstrate clinical and histopathologic overlap, raising the possibility that they represent opposite ends of a disease spectrum. SPTCL, however, is typically associated with greater morbidity and risk for hemophagocytic lymphohistiocytosis (HLH); therefore, diagnostic distinction is clinically important. We present the histopathologic, immunophenotypic, and molecular findings with long-term clinical follow-up of 13 patients with SPTCL (median, 64 mo follow-up) and 7 with LEP (median, 50 mo follow-up) in our multidisciplinary cutaneous oncology clinic. Six SPTCL patients developed HLH, including 2 under the age of 21 years. In the SPTCL group, 2 of 13 patients died of disease. In contrast, we had no mortality or development of HLH in our LEP cohort. We demonstrate that a limited panel (Ki-67, CD3, CD4, and CD8 immunostains) reveals foci of "Ki-67 hotspots" enriched in cytotoxic atypical CD8+ T cells in SPTCL. Ki-67 hotspots were not identified in LEP, thus aiding the distinction of SPTCL from LEP. Lymphocyte atypia combined with adipocyte rimming of CD8+ T cells within Ki-67 hotspots was also highly specific for the diagnosis of SPTCL. Hyaline lipomembranous change, B-cell aggregates, plasmacytoid dendritic cell clusters, and plasma cell aggregates favored the diagnosis of LEP but were identified in some cases of SPTCL including patients with HLH. We confirm that SPTCL and LEP can show significant histologic overlap, suggest a role for high-throughput sequencing in confirming neoplastic clones, and introduce the concept of SPTCL "Ki-67 hotspots" in evolving disease.
1442	Hemophagocytic Syndrome in Children With Visceral Leishmaniasis.	26780020	Hemophagocytic lymphohistiocytosis (HLH) is a serious complication of visceral leishmaniasis (VL). The aim of this study is to describe demographical, clinical and laboratory features of HLH in children with VL.This is a retrospective cohort of children with HLH and VL admitted to a tertiary hospital in Northeast, Brazil, from January 2012 to April 2014. Clinical and laboratory data at admission and during hospital stay were reviewed. Acute kidney injury (AKI) was defined according to the pediatric Risk, Injury, Failure, Loss, End-stage kidney disease criteria.A total 127 VL children were admitted, and 35 children had diagnosis of HLH. Mean age was 4.2 ± 4.3 years, with 62.9% males. Mean hospital stay was 29 ± 12 days. Main signs and symptoms were fever (100%), splenomegaly (94.2%) and hepatomegaly (60%). Laboratory findings showed pancytopenia, albumin 3.03 ± 0.77 g/dL, fibrinogen 236.1 ± 117.2 mg/dL, total calcium 8.2 ± 1.2 mEq/L, lactate dehydrogenase 1804 ± 1019 mg/dL, alkaline phosphatase 1275.4 ± 2160.5 IU/L, total bilirubin 1.9 ± 2.4 mg/dL, direct bilirubin 0.67 ± 1.02 mg/dL, indirect bilirubin 1.2 ± 2.2 mg/dL, aspartate aminotransferase 140.0 ± 145.3 IU/L, alanine aminotransferase 71.4 ± 81.1 IU/L, ferritin 4296.5 ± 8028.8 ng/dL and triglycerides 333 ± 141 mg/dL. AKI was observed in 16 children (45.7%), predominantly mild forms (93.75% "risk"). AKI group presented lower levels of platelets (69,131 ± 40,247 vs. 138,678 ± 127,494/mm, P = 0.035) than non-AKI. No patient required dialysis and there was no death.HLH was not a rare complication of VL. Main symptoms were compatible with both VL and HLH. Main laboratory findings reflected HLH pathophysiology. Mild forms of AKI were a common complication of HLH. Despite the disease severity and complications, mortality was low.
1443	Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin.	26762975	The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition.
1444	[Significance of soluble CD163 and soluble CD25 in diagnosis and treatment of children with hemophagocytic lymphohistiocytosis].	26758320	To explore significance of serum soluble CD163(sCD163) and soluble CD25(sCD25) in diagnosis and guiding treatment of children with hemophagocytic lymphohistiocytosis (HLH).Data of 42 cases of children with HLH, 32 cases of non-HLH children with infection presented to First Affiliated Hospital of Zhengzhou University pediatric clinic and ward were collected from December 2013 to December 2014. Twenty-four healthy children were enrolled into a normal control group in the same period.Peripheral venous blood specimens (3 ml) were taken from the children with HLH after fasting before treatment, two weeks after treatment and eight weeks after treatment.Peripheral venous blood specimens (3 ml) were also taken from children of non-HLH infected group and normal control group after fasting at the initial visit. Serum sCD163 and sCD25 levels in the peripheral blood in three groups were determined by ELISA. According to cause of disease, children with HLH were divided into infection-related HLH, tumor-related HLH, primary HLH and others; relationship between serum sCD163 and sCD25 level and cause of disease was analyzed.Serum sCD163 of HLH group ((6 094 ± 2 769) µg/L) and serum sCD163 of non-HLH infection group ((2 174 ± 950) µg/L) were significantly higher than that of normal control group ((777 ± 256) µg/L), F=71.396, P<0.05), and the differences among groups were statistically significant (P<0.05); serum sCD25 of HLH group ((41 963 ± 31 821) ng/L) and serum sCD25 of non-HLH infection group ((6 700 ± 4 105) ng/L) were significantly higher than that of normal control group ((2 440 ± 1 870) ng/L, F=37.513, P<0.05).There was no statistically significant difference between the non-HLH infection group with the normal control group (P>0.05), and the difference between the remaining groups was statistically significant (P<0.05). And serum sCD163 and sCD25 level of HLH group had a positive linear correlation, and Pearson correlation coefficient r=0.742 (t=7.000, P<0.05). The difference of serum sCD163 and sCD25 level among the different cause of disease in HLH group was significant (P<0.05).Pairwise comparison showed that serum sCD163 and sCD25 level of tumor-associated HLH group significantly increased as compared with infection-associated HLH group (P<0.05), but the difference was not statistically significant between the other groups (all P>0.05). Serum sCD163 and sCD25 level of HLH group before treatment, 2 weeks and 8 weeks after treatment showed a statistically significant tendency of decrease (P<0.05). Seen from the ROC curve, when sCD163 cut-off point was 2 359.08 µg/L, the diagnostic sensitivity was 83.3%, and specificity was 83.9%.When sCD25 cut-off point was 14 901.024 ng/L, the diagnosis sensitivity was 76.2%, and specificity was 98.2%.Serum sCD163 and sCD25 levels may be used for diagnosis of HLH.Dynamically monitoring of serum sCD163 and sCD25 level can help to determine deterioration of HLH and guide treatment.
1445	Haemophagocytic lymphohistiocytosis: a cause of unresponsive malaria in a 5-year-old girl.	26744159	A 5-year-old immunocompetent girl presented with fever, jaundice, hepatosplenomegaly and pancytopenia. The peripheral blood smear demonstrated mixed malaria infection (Plasmodium vivax and Plasmodium falciparum). Fever was persistent despite antimalarials in the absence of any coexisting bacterial or viral infection. Laboratory findings included cytopaenia, hyperbilirubinaemia, hyperferritinaemia, hypertriglyceridaemia, hyponatraemia, deranged partial thromboplastin time, decreasing ESR and megaloblastic changes on bone marrow aspiration. A final diagnosis of haemophagocytic lymphohistiocytosis (HLH) with megaloblastic anaemia associated with severe mixed malaria was made. There was a dramatic response to corticosteroid treatment with improvement in her clinical condition. This report endorses the use of corticosteroids in malaria-associated HLH whenever there is no clinical improvement with antimalarials alone.
1446	Acute kidney injury induced by thrombotic microangiopathy in a patient with hemophagocytic lymphohistiocytosis.	26739581	Hemophagocytic lymphohistiocytosis (HLH) is a fatal clinical syndrome characterized by excessive immune activation and inflammation. It is frequently complicated by acute kidney injury (AKI) that often develops as acute tubular necrosis (ATN). Meanwhile, renal thrombotic microangiopathy (TMA) is a rare pathologic finding that mostly occurs in hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. There are only few reports on TMA developing in patients with HLH. We present here a rare case of TMA associated HLH.A 60-year-old woman was admitted for a fever of unknown origin that had persisted for several weeks. She presented with AKI and pancytopenia. Clinical, laboratory and bone marrow biopsy findings met the criteria of HLH. Kidney biopsy showed TMA and minimal ATN, which suggested that the primary cause of AKI was TMA in this case. Because of sustained oliguria, we initiated hemodialysis (HD) and also decided to use chemotherapy composed of dexamethasone, etoposide and cyclosporine for treatment of HLH. Six months after the initiation of chemotherapy, pancytopenia was completely resolved, indicating the resolution of HLH. At the same time, serum creatinine decreased to a normal range without the need for HD, suggesting the resolution of TMA.We report a case of renal TMA associated HLH. This case suggests that renal TMA should be considered as a primary cause of AKI in patients with underlying HLH.
1447	A Hemophagocytic Lymphohistiocytosis Patient that Presented with Unilateral Panuveitis.	26731243	To describe a case of hemophagocytic lymphohistiocytosis (HLH) with ocular changes prior to the systemic changes.A 53-year-old man presented with the chief complaint of decreased vision in his right eye. The patient was examined by ocular examination, slit lamp examination, optical coherence tomography, laboratory examination, abdominal ultrasound, and bone marrow biopsy.Ocular examination revealed uveitis OD and optical coherence tomography revealed macular edema OD. Laboratory examination demonstrated cytopenia in two cell lines, hypofibrinogenemia, and elevated serum ferritin. Abdominal ultrasound findings indicated hepatosplenomegaly. The bone marrow biopsy specimen demonstrated histiocytes and significant hemophagocytosis, leading to a diagnosis of HLH.Ophthalmic manifestation can be the first sign of HLH and progress to fatal systemic changes.
1448	[Lung injury associated with severe Epstein-Barr virus (EBV) infection].	26717656	Severe Epstein-Barr (EB) virus infection is potentially a devastating process that often leads to death encountered in pediatrics recently. Inappropriate control of EB virus replication may cause severe infection resulting in multiple organ dysfunction. However, little information is available on pulmonary complications associated with EB virus infection. The aim of the present study was to investigate severe EB virus (EBV) infection complicated with lung injury in pediatric intensive care unit (PICU), including clinical characteristics, laboratory or imaging feature and outcomes.A total of 45 children with severe EBV infection seen in PICU of Shanghai Children's Hospital between January 2011 and December 2014 were retrospectively reviewed. According to clinical characteristics and imaging feature, 45 children were divided into non-lung injury group (n =27), lung injury without pulmonary fibrosis group(n = 12) and pulmonary fibrosis group (n = 6).In totally 45 cases of severe EBV infection, 21 (46.7%) were male and 24 (53. 3%) were female, mean age was 2. 4 years; 18 cases were complicated with lung injury, including 8 male and 10 female, median age was 31. 2 months. All of 18 cases presented with fever and cough, 15 of them exhibited dyspnea,12 cases were complicated with gasping, and 6 cases with ARDS. Eight cases accepted mechanical ventilation for acute respiratory distress; 6 cases who developed pulmonary fibrosis had tachypnea, refractory hypoxemia and hypercapnia, severe pulmonary air leak. The average EBV-DNA level in peripheral blood was 4. 42 x 10(6) copies/ml (range: 3. 25 x 10(3) - 6.59 x 10(7) copies/ml). Anti-EBV antibodies were positive in 41 cases, 18 cases were positive (+) for VCA-IgM, 15 cases were VCA-IgG and EA-IgG (+), 8 cases VCA-IgM and VCA-IgG (+). The radiographic findings revealed pulmonary interstitial infiltrates in all 18 cases with lung injury, 4 cases with segmental consolidation and 2 cases showed pleural effusions. HRCT scanning found EBV associated fibrosis including multifocal patches and diffuse ground-glass attenuation in both lungs, reticular opacities and honeycombing changes were observed 4 weeks after illness onset. There were significant differences in respiratory failure, PICU stay (days), Pediatric risk of mortality III (PRISM III) and pediatric clinical illness score(PCIS), serum TNF-α, EBV-DNA levels, percentage of NK cells and CD4+/CD8+ T cell ratio among non-lung injury group, lung injury without pulmonary fibrosis group and pulmonary fibrosis group (X2 =27. 12, F = 85. 23, 78. 23, 88. 68, 323. 80, 7. 35, χ2 = 6. 71, 12. 15; all P < 0. 05). COX regression analysis revealed that EBV-DNA and serum TNF-α levels were correlated with pulmonary fibrosis significantly (OR = 3. 92, P = 0. 04; OR = 5. 95, P = 0. 01). The patients with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) had higher incidence of pulmonary fibrosis compared with non-EB-HLH (70% vs. 13%, χ2 = 4. 82, P = 0. 03). Of 18 cases, 8 cases died, including 3 cases with pulmonary fibrosis. The surviving cases with pulmonary fibrosis needed longer additional oxygen. Chest HRCT imaging of 3 cases with pulmonary fibrosis was improved at 6 months and oxygen therapy was discontinued at 18 months after discharge.EB virus infection complicated with lung injury had higher incidence of respiratory failure, pulmonary fibrosis with a fatal outcome. EBV-DNA and serum TNF-α level were found to be statistically significant indicators of pulmonary fibrosis. Pulmonary fibrosis associated with severe EB virus infection may be reversible.
1449	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis: response to HLH-04 treatment protocol.	29421229	Hemophagocytic syndrome, macrophage activation syndrome, reactive histiocytosis or hemophagocytic lymphohistiocytosis (HLH) represent a group of diseases whose common thread is reactive or neoplastic mononuclear phagocytic system cells and dendritic cell proliferation.We present a case of an HLH probably associated with Epstein-Barr virus (EBV) in a 4-year-old male patient treated with HLH-04 protocol. Viral etiology in HLH is well accepted. In this case, clinical picture of HLH was assumed secondary to EBV infection because IgM serology at the time of clinical presentation was the only positive factor in the viral panel.Diagnosis of HLH is the critical first step to successful treatment. The earlier it is identified, the less the tissue damage and reduced risk of multiple organ failure, which favors treatment response.
1450	Unique Triad of 'Pregnancy, Kala Azar and Hemophagocytic Lymphohistiocytic Syndrome from a Non-Endemic Region'.	26710404	India and neighboring Nepal, Bangladesh along with Sudan and Brazil are the four largest foci of visceral leishmaniasis and account for 90% of the world's visceral leishmaniasis (VL) burden, with India being the worst affected. High degree of suspicion is usually based on patient presenting from endemic area with features of pancytopenia hepatosplenomegaly. Hemophagocytic lymphohistiocytic (HLH) syndrome also presents with similar clinical features. Visceral leishmaniasis leading to secondary HLH syndrome is in itself a rare entity while both of these presenting in pregnant patient, to the best knowledge of the authors, is yet to be described in literature.
1451	A Novel Syntaxin 11 Gene (STX11) Mutation c.650T>C, p.Leu217Pro, in a Korean Child With Familial Hemophagocytic Lymphohistiocytosis.	26709266	We report the first Far Eastern case of a Korean child with familial hemophagocytic lymphohistiocytosis (HLH) caused by a novel syntaxin 11 (STX11) mutation. A 33-month-old boy born to non-consanguineous Korean parents was admitted for intermittent fever lasting one week, pancytopenia, hepatosplenomegaly, and HLH in the bone marrow. Under the impression of HLH, genetic study revealed a novel homozygous missense mutation of STX11: c.650T>C, p.Leu217Pro. Although no large deletion or allele drop was identified, genotype analysis demonstrated that the homozygous c.650T>C may have resulted from the duplication of a maternal (unimaternal) chromosomal region and concurrent loss of the other paternal allele, likely caused by meiotic errors such as two crossover events. A cumulative study of such novel mutations and their effects on specific protein interactions may deepen the understanding of how abnormal STX1 expression results in deficient cytotoxic function.
1452	Hemophagocytic Lymphohistiocytosis in Intensive Care Unit: A 71-Case Strobe-Compliant Retrospective Study.	26705219	Hemophagocytic lymphohistiocytosis (HLH) is a critical condition that may lead to organ failure and early death. The aim of this retrospective observational study was to describe a cohort of HLH patients admitted to intensive care unit (ICU) and investigate the risk factors of early death.A positive HLH diagnosis was defined by an HScore ≥ 169. Univariate and multivariate analyses were carried out to investigate hospital and 28-day mortality risk factors. Between January 2002 and July 2014, 71 HLH cases were seen at our institution.The overall 28-day mortality (start at ICU admission) and hospital mortality were 38% and 68%, respectively. The factors associated with increased 28-day mortality were the sequential organ failure assessment score at ICU admission (P < .001) and advance in age (P = 0.03). The factors associated with increased hospital mortality were a high sequential organ failure assessment score at ICU admission (P < 0.01), advance in age (P = 0.04), and the presence of lymphoma-related HLH or HLH of unknown origin (P < 0.01).Organ failure overtops the classical early-death risk factors in adult ICU-admitted HLH patients. This failure and the subsequent early death may be prevented by timely specific cytotoxic therapies and the control of the underlying disease.
1453	Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila.	26702412	In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H) (MAPK-) (ko) and a phospho-mimetic Su(H) (MAPK-ac) isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vg (BE) -lacZ). In general, Su(H) (MAPK-) (ko) induced a stronger response than wild-type Su(H), whereas the response to Su(H) (MAPK-ac) was very weak. Notch target genes cut, wingless and vg (BE) -lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DER (act) ) or the MAPK (rl (SEM) ) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones.
1454	MESP1 Mutations in Patients with Congenital Heart Defects.	26694203	Identifying the genetic etiology of congenital heart disease (CHD) has been challenging despite being one of the most common congenital malformations in humans. We previously identified a microdeletion in a patient with a ventricular septal defect containing over 40 genes including MESP1 (mesoderm posterior basic helix-loop-helix transcription factor 1). Because of the importance of MESP1 as an early regulator of cardiac development in both in vivo and in vitro studies, we tested for MESP1 mutations in 647 patients with congenital conotruncal and related heart defects. We identified six rare, nonsynonymous variants not seen in ethnically matched controls and one likely race-specific nonsynonymous variant. Functional analyses revealed that three of these variants altered activation of transcription by MESP1. Two of the deleterious variants are located within the conserved HLH domain and thus impair the protein-protein interaction of MESP1 and E47. The third deleterious variant was a loss-of-function frameshift mutation. Our results suggest that pathologic variants in MESP1 may contribute to the development of CHD and that additional protein partners and downstream targets could likewise contribute to the wide range of causes for CHD.
1455	Progressive Diffuse Osteonecrosis in a Patient with Secondary Hemophagocytic Lymphohistiocytosis.	26693376	This is a case report with serial imaging showing progression of diffuse osteonecrosis in a patient after a diagnosis of secondary hemophagocytic lymphohistiocytosis (HLH). While bone marrow involvement in HLH has been long noted at histological evaluation and is itself one of the diagnosis criteria, to the best of our knowledge, there has been no previous publication addressing osseous image findings in a patient with HLH.
1456	Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis.	26684649	Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics.A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes.Analyses revealed a mutation detection sensitivity of 97.3%, an average coverage per gene of 98.0%, and adequate coverage over 98.6% of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38%). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals.We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization.
1457	The significance of pre-therapeutic F-18-FDG PET-CT in lymphoma-associated hemophagocytic lymphohistiocytosis when pathological evidence is unavailable.	26666755	The significance of positron emission tomography/computed tomography (PET-CT) in identifying patients with lymphoma-associated hemophagocytic lymphohistiocytosis (LAHLH) when pathological evidence is unavailable remains uncertain.In this retrospective study, 44 HLH patients who underwent PET-CT before clinical treatment were enrolled, and 18 of them were highly suspected as LAHLH by PET-CT. We compared the PET-CT parameters between confirmed LAHLH and non-LAHLH patients. The efficacy of initial therapies for highly suspected LAHLH patients was analyzed as well.We found that the SUVSp, SUVBM, SUVLN, SUVmax, SUVLN/Li, and SUVmax/Li in LAHLH group were significantly higher than those in non-LAHLH group (p = 0.003, p = 0.034, p = 0.003, p < 0.001, p = 0.039, and p = 0.035, respectively). HLH patients with an SUVmax value >5.5, an SUVLN value >3.3, and an SUVSp value >4.8 were more likely to be LAHLH (p < 0.001, p = 0.003, and p = 0.003, respectively). And the incidence of multiple lymphadenopathy with increased FDG uptake or the incidence of multiple bone lesions in LAHLH patients was significantly higher than those in non-LAHLH group (92.9 vs. 35.7 %, p = 0.004; 42.9 vs. 0 %, p = 0.016, respectively). Furthermore, by comparing the efficacy of initial therapies for highly suspected LAHLH patients (n = 18), we indicated that the CR rate was significantly higher in lymphoma-chemotherapy group than in immunosuppressive therapy group (90 and 25 %, respectively; p = 0.013). OS analysis revealed that highly suspected LAHLH patients treated with lymphoma-chemotherapy had better prognosis (264 days) than those treated with immunosuppressive therapy (15 days) (p < 0.0001).When pathological evidence is absent, PET-CT may play an important role in identifying HLH patients underlying lymphoma. Once highly suspected as LAHLH by PET-CT, lymphoma-chemotherapies that directly treat the underling lymphoma may have a relatively favorable effect and better clinical outcomes than immunosuppressive therapy.
1458	Hemophagocytic lymphohistiocytosis: An unusual complication in disseminated Mycobacterium tuberculosis.	26664166	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon, potentially fatal, hyperinflammatory syndrome that may rarely complicate the clinical course of disseminated Mycobacterium tuberculosis (MTB). The clinical course of tuberculosis-associated HLH (TB-HLH) has been reported to be unpredictable.Here we describe the clinicopathological features, laboratory parameters, management, and outcome data of a patient who satisfied the 2004 diagnostic criteria for HLH secondary to disseminated MTB; we also do a systematic review of the international literature on TB-HLH. The literature review (January 1975-March 2014) found that HLH complicated the clinical course of 63 tuberculosis patients (41 males, 22 females, mean age = 45 ± 23.5 years) with a high mortality rate of 49% (31/63 died). The mean serum ferritin level (n = 44/63) was 5963 ng/mL (range 500-38,539 ng/mL); and a higher proportion (54.2%) of patients had pancytopenia at presentation. On univariate analysis (n = 53/63), age >30 years [hazard ratio (HR): 2.79, 95% confidence interval (CI):1.03-7.56, P = 0.03], presence of comorbidities (HR 4.59, CI: 1.08-19.52, P = 0.04), marked hemophagocytosis in bone marrow (HR: 2.65, CI: 1.16-6.05, P = 0.02), and nonusage/delayed usage of antitubercular therapy (ATT) (HR: 3.44, CI: 1.51-7.87, P = 0.003) were associated with decreased survival, though none of these parameters attained statistical significance (P > 0.05) in multivariate analysis. Usage of corticosteroids and/or immunomodulator drugs (HR 1.00, CI: 0.66-3.22, P = 0.35) did not alter the outcome in these patients.HLH should be considered as a differential diagnosis in patients with tuberculosis who present with cytopenias, organomegaly, and coagulopathy. Strong clinical suspicion and early usage of ATT might be useful in reducing the morbidity and mortality. The utility of immunosuppressive/immunomodulator therapy lacks general concensus among treating physicians, and warrants further studies.
1459	Hemophagocytic Lymphohistiocytosis Associated With Bartonella henselae Infection in an HIV-Infected Patient.	266466792716178027161782	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome that often occurs in immunocompromised patients. We report the first case of HLH due to Bartonella henselae infection in a patient with human immunodeficiency virus infection. Early recognition of HLH and B. henselae through liver biopsy and serological tests led to the patient's recovery.
1460	Treatment of hemophagocytic lymphohistiocytosis in adults.	26637720	Treatment of hemophagocytic lymphohistiocytosis (HLH) has been developed primarily in pediatric centers, where familial HLH (FHL) is the leading cause of HLH in newborns and toddlers. The Histiocyte Society Study Group for HLH developed the HLH-94 and HLH-2004 treatment protocols, and these are frequently also used by centers treating HLH in adults (aHLH). These protocols contain etoposide, dexamethasone, and cyclosporine A; these agents all have strong activity against proliferation of cytotoxic T/NK-cells and macrophages, as well as inhibitory activity against the cytokine storm that induces, and maintains HLH. In children with predominantly hereditary disease, the HLH-94 protocol can be regarded as a "one size fits all" algorithm. HLH in adults is a much more heterogeneous syndrome requiring a more individualized approach depending on the underlying trigger, disease severity and course, as well as genetic background. Additionally, treatment in adults usually needs to be modified in the face of the preceding disease history and comorbidities. Interdisciplinary patient care with rheumatologists, gastroenterologists, neurologists, pediatricians, the transplant team, and pathologists is a prerequisite to successful treatment. The preferred approach should reflect a disease- and risk-adapted treatment that includes rigorous supportive care with continuous reassessment of sequential therapeutic measures. It should be recognized that the algorithm of HLH treatment in adults is based more on expert opinion than on extensive scientific evidence.
1461	The unique aspects of presentation and diagnosis of hemophagocytic lymphohistiocytosis in adults.	26637719	Hemophagocytic lymphohistiocytosis (HLH) was initially described as an inflammatory condition affecting young children but is increasingly diagnosed in adults. Presenting features such as fever, cytopenias, phagocytosis, elevated ferritin, and increased levels of soluble IL-2 receptor are common in both age groups, but the prevalence of several clinical and biochemical criteria differ between pediatric and adult patients. Specifically, an elevated ferritin level does not have the same specificity for HLH in adults, and many other inflammatory conditions need to be considered in the differential. In contrast to the high incidence of infectious triggers seen in pediatric HLH, HLH in adults is often precipitated by a hematologic malignancy. Malignancy-associated HLH has unique manifestations and a uniformly very poor prognosis. Given these differences, diagnostic scoring systems unique to adult HLH have been proposed, and additional prognostic clinical and immunologic parameters are being explored. Although a genetic predisposition is increasingly found to underlie cases of adult-onset HLH, the mutations are less likely to be bi-allelic and differ slightly from those seen in pediatric cases of familial HLH. The facilitating genetic and environmental factors governing presentation of HLH in adults remain elusive. Understanding the clinical aspects and pathophysiology specific to adults with HLH is necessary to tailor therapies derived in pediatric disease to this under-recognized population.
1462	Pathophysiology and epidemiology of hemophagocytic lymphohistiocytosis.	26637718	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by extreme immune activation, resulting in pathologic inflammation. The diagnosis includes a spectrum of inherited or acquired defects in cytotoxic lymphocyte function, often with uncontrolled infections. HLH may also arise as the result of persistent antigen stimulation due to autoimmune disease or malignancy. HLH is often described in binary terms as "primary," indicating Mendelian inheritance of gene mutations resulting in cytotoxic lymphocyte dysfunction, or "secondary" indicating an acquired reactive disorder. Increasing evidence describes HLH as more complex phenomenon, resulting from specific immune challenges in patients with a susceptible genetic background. Early recognition of HLH and evaluation of potential causes is critically important, as survival generally requires urgent treatment with immune suppression and resolution of the activating antigen. However, the diagnosis of HLH is challenged by the myriad of pathways that lead to pathologic inflammation and the clinical overlap with other conditions. Further improvements in therapy will require prospective trials to define optimal strategies for each patient based on the individual paths that lead to pathologic inflammation.
1463	[A Fatal Case of Severe Fever with Thrombocytopenia Syndrome Complicated by Hemophagocytic Lymphohistiocytosis].	26630792	Severe fever with thrombocytopenia syndrome (SFTS) is a recently identified emerging viral infectious disease in China that is caused by a novel phlebovirus in the family Bunyaviridae, SFTS virus, with an average case fatality rate of 12-30%. A cytokine storm with abnormally expressed cytokine profiles is associated with the disease severity. Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and lifethreatening syndrome associated with excessive immune activation. We report herein on a fatal case of SFTS complicated by HLH. Consecutive plasma exchange and immunomodulatory therapy was ineffective in our case. The pathognomonic histological feature was necrotizing lymphadenitis with massive hemophagocytosis of systemic lymphoid tissues with SFTS viruses and SFTS-RNA copies. No specific treatment of SFTS is available, and an effective treatment strategy for patients with rapidly progressing SFTS has not been established. Appropriate immunomodulatory therapy is necessary for SFTS patients complicated by HLH.
1464	Interacting mechanism of ID3 HLH domain towards E2A/E12 transcription factor - An Insight through molecular dynamics and docking approach.	28955822	Inhibitor of DNA binding protein 3 (ID3) has long been characterized as an oncogene that implicates its functional role through its Helix-Loop-Helix (HLH) domain upon protein-protein interaction. An insight into the dimerization brought by this domain helps in identifying the key residues that favor the mechanism behind it. Molecular dynamics (MD) simulations were performed for the HLH proteins ID3 and Transcription factor E2-alpha (E2A/E12) and their ensemble complex (ID3-E2A/E12) to gather information about the HLH domain region and its role in the interaction process. Further evaluation of the results by Principal Component Analysis (PCA) and Free Energy Landscape (FEL) helped in revealing residues of E2A/E12: Lys570, Ala595, Val598, and Ile599 and ID3: Glu53, Gln63, and Gln66 buried in their HLH motifs imparting key roles in dimerization process. Furthermore the T-pad analysis results helped in identifying the key fluctuations and conformational transitions using the intrinsic properties of the residues present in the domain region of the proteins thus specifying their crucial role towards molecular recognition. The study provides an insight into the interacting mechanism of the ID3-E2A/E12 complex and maps the structural transitions arising in the essential conformational space indicating the key structural changes within the helical regions of the motif. It thereby describes how the internal dynamics of the proteins might regulate their intrinsic structural features and its subsequent functionality.
1465	Branched-chain amino acid catabolism is a conserved regulator of physiological ageing.	26620638	Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFβ, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan.
1466	Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza.	26597256	Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza.Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells.Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS.This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection.
1467	Cytotoxic granule secretion by lymphocytes and its link to immune homeostasis.	26594351	The granule-dependent cytotoxic activity of T and natural killer lymphocytes has progressively emerged as an important effector pathway not only for host defence but also for immune regulation. The analysis of an early-onset, severe, primary immune dysregulatory syndrome known as hemophagocytic lymphohistiocytosis (HLH) has been decisive in highlighting this latter role and identifying key effectors on the basis of gene mutation analyses and mediators in the maturation and secretion of cytotoxic granules. Studies of cytotoxicity-deficient murine counterparts have helped to define primary HLH as a syndrome in which uncontrolled T-cell activation in response to lymphocytic choriomeningitis virus infection results in excessive macrophage activation and inflammation-associated cytopenia. Recent recognition of late-onset HLH, which occurs in a variety of settings, in association with hypomorphic, monoallelic mutations in genes encoding components of the granule-dependent cytotoxic pathway or even in the absence of such mutations has broadened our view about the mechanisms that underlie the perturbation of immune homeostasis. These findings have led to the development of a model in which disease occurs when a threshold is reached through the accumulation of genetic and environmental risk factors. Nevertheless, validation of this model will require further investigations.
1468	Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span.	2658618927383456	Stabilization of the hypoxia-inducible factor 1 (HIF-1) increases life span and health span in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in Caenorhabditis elegans through a cell nonautonomous signal to the intestine, which results in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This prolongevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and is necessary for DR-mediated life-span extension, which suggests that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple life span-extending interventions in mice, which suggests that these enzymes may play a critical role in promoting health and longevity across phyla.
1469	Hemophagocytic Lymphohistiocytosis After Initiation of Chemotherapy for Bilateral Adrenal Neuroblastoma.	26583611	Hemophagocytic lymphohistiocytosis (HLH) is a rare and aggressive syndrome characterized by overactivation of the immune system. Although secondary HLH has been frequently associated with malignancies, this entity is rarely triggered by solid tumors, such as neuroblastomas. Herein, we describe a 14-month-old girl with a late diagnosis of bilateral adrenal neuroblastoma who developed HLH 6 days after the initiation of chemotherapy. On the basis of the large tumoral mass and the time of onset of her symptoms suggestive of HLH, we hypothesize that tumor cell destruction induced by chemotherapy drugs was the trigger to the development of hematophagocytic lymphohistiocytosis syndrome.
1470	A de novo whole gene deletion of XIAP detected by exome sequencing analysis in very early onset inflammatory bowel disease: a case report.	2658148726683620	Children with very early-onset inflammatory bowel disease (VEO-IBD), those diagnosed at less than 5 years of age, are a unique population. A subset of these patients present with a distinct phenotype and more severe disease than older children and adults. Host genetics is thought to play a more prominent role in this young population, and monogenic defects in genes related to primary immunodeficiencies are responsible for the disease in a small subset of patients with VEO-IBD.We report a child who presented at 3 weeks of life with very early-onset inflammatory bowel disease (VEO-IBD). He had a complicated disease course and remained unresponsive to medical and surgical therapy. The refractory nature of his disease, together with his young age of presentation, prompted utilization of whole exome sequencing (WES) to detect an underlying monogenic primary immunodeficiency and potentially target therapy to the identified defect. Copy number variation analysis (CNV) was performed using the eXome-Hidden Markov Model. Whole exome sequencing revealed 1,380 nonsense and missense variants in the patient. Plausible candidate variants were not detected following analysis of filtered variants, therefore, we performed CNV analysis of the WES data, which led us to identify a de novo whole gene deletion in XIAP.This is the first reported whole gene deletion in XIAP, the causal gene responsible for XLP2 (X-linked lymphoproliferative Disease 2). XLP2 is a syndrome resulting in VEO-IBD and can increase susceptibility to hemophagocytic lymphohistocytosis (HLH). This identification allowed the patient to be referred for bone marrow transplantation, potentially curative for his disease and critical to prevent the catastrophic sequela of HLH. This illustrates the unique etiology of VEO-IBD, and the subsequent effects on therapeutic options. This cohort requires careful and thorough evaluation for monogenic defects and primary immunodeficiencies.
1471	Efficacy of intravenous cyclosporine in a case of cytophagic histiocytic panniculitis complicated by haemophagocytic syndrome after visceral leishmania infection.	26575530	Cytophagic histiocytic panniculitis (CHP) is a rare panniculitis characterized by systemic features, due to histiocytic infiltration along with haemophagocytosis, which may also appear in bone marrow, spleen, lymph nodes, and liver. Haemophagocytic lymphohistiocytosis (HLH) is a group of autoinflammatory disorders, which include macrophage activation syndrome, sometimes observed in the course of systemic autoimmune diseases, such as juvenile chronic polyarthritis, systemic lupus erythematosus or vasculitis, and infection-associated haemophagocytic syndrome; if not promptly recognised and treated, HLH can be fatal. Visceral leishmaniasis (VL) is a systemic disease caused by different forms of Leishmania spp., an intracellular protozoa. VL is endemic in tropical countries such as in the Middle East and the Mediterranean. The typical clinical and laboratory features are fever, hepato-splenomegaly, hypergammaglobulinaemia and pancytopenia. The features of VL may mimic some haematologic diseases. We report a case of cytophagic histiocytic panniculitis and HLH, triggered by a previous visceral leishmania infection. Cyclosporine was quickly effective in this case, after failure of high-dose glucocorticoids, anakinra and etoposide.
1472	Screening assays for primary haemophagocytic lymphohistiocytosis in children presenting with suspected macrophage activation syndrome.	26572973	Primary haemophagocytic lymphohistiocytosis (HLH) screening assays are increasingly being performed in patients presenting with macrophage activation syndrome (MAS). The objective of this study was to describe their diagnostic and prognostic relevance in children who had presented to paediatric rheumatology and had undergone investigative work up for MAS.Data was obtained retrospectively from an existing protein screening assay database and patient records. Assays included: intracellular expression of perforin in CD56+ Natural Killer (NK) cells; CD107a Granule Release Assay (GRA) in response to PHA in NK cells, or anti-CD3 stimulation of CD8 lymphocytes; in males Signal Lymphocyte Activating Molecule Associated Protein (SAP), and X-linked Inhibitor of Apoptosis Protein (XIAP) expression. All assays, requested by paediatric rheumatology, of children who had undergone investigative work up for MAS over a 5-year period (2007-2011) were included.Twenty-one patients (15 female), median age 6.5 years (range 0.6-16) with follow-up of 16 months (range 1-51), were retrospectively identified. At presentation, 3/21 (14 %) fulfilled HLH-2004 diagnostic criteria. At least one screening test result was available for all 21 patients; 7/21 (33 %) had at least one persistent screening test abnormality. Of this group 4/7 (57 %) died or required haematopoietic stem cell transplantation (HSCT), compared to 1/14 (7 %) with no screening test abnormality (p = 0.025). 3/21 (14 %) ultimately had a diagnosis of primary HLH (two confirmed genetically; XIAP, familial HLH type 3, and one confirmed clinically). Of the six patients with abnormal GRA 5/6 had negative routine genetic results.Screening for primary HLH is warranted for children whose first rheumatological presentation is with MAS, since overall 14 % had an eventual diagnosis of primary HLH. A persistently abnormal GRA in patients presenting with MAS defines a high-risk group with poor outcome (mortality or HSCT), possibly due to as yet unidentified genetic cause.
1473	Hemophagocytic Lymphohistiocytosis Secondary to Human Immunodeficiency Virus-Associated Histoplasmosis.	26566535	Hemophagocytic lymphohistiocytosis (HLH) in immunocompromised hosts is a fulminant syndrome of immune activation with high rates of mortality that may be triggered by infections or immunodeficiency. Rapid diagnosis and treatment of the underlying disorder is necessary to prevent progression to multiorgan failure and death. We report a case of HLH in a patient with human immunodeficiency virus, disseminated histoplasmosis, Mycobacterium avium complex, and Escherichia coli bacteremia. We discuss management of acutely ill patients with HLH and treatment of the underlying infection versus initiation of HLH-specific chemotherapy.
1474	Antithrombotic Treatment for Recurrent Miscarriage: Bayesian Network Meta-Analysis and Systematic Review.	26559249	Combined use of heparin and aspirin is frequently prescribed for treatment of recurrent miscarriage (RM) in patients with antiphospholipid syndrome (APS), or in those without apparent cause of RM other than thrombophilia; however, this strategy is largely based on expert opinion and has not been well studied. The option for the use of different antithrombotic therapies to improve live birth remains unclear. In this network meta-analysis, we incorporated direct and indirect evidence to evaluate effects of different antithrombotic treatments on prevention of pregnancy losses.We searched PubMed and Embase for randomized clinical trials comparing effects of at least 2 antithrombotic treatments on live birth in RM patients published from 1965 through the early of May 2015. Potential risk bias of eligible trials was evaluated according to the Cochrane Collaboration guidelines. Bayesian network meta-analysis was used to estimate relative effects on live birth.A total of 19 trials involving 2391 RM patients with or without thrombophilia and 543 with APS were included. No beneficial effect of antithrombotic treatment was observed either in RM patients with or without thrombophilia or in patients with APS; however, for patients with or without thrombophilia, low molecular weight heparin therapy had the greatest probability (61.48%) of being the best option in terms of live birth; for patients with APS, unfractionated heparin plus aspirin was the superior treatment for RM with the highest possibility (75.15%) of being top 2 places for reducing pregnancy losses. Aspirin was inferior in both groups.Our results do not support the use of combined low molecular weight heparin and aspirin for RM treatment, and suggested aspirin may have negative effects for lowering the risk of pregnancy loss.
1475	Dengue fever as a cause of hemophagocytic lymphohistiocytosis.	26558245	Dengue is endemic in more than 100 countries in Southeast Asia, the Americas, the western Pacific, Africa and the eastern Mediterranean regions. The virus is transmitted by Aedes mosquitoes. Dengue disease is the most prevalent arthropod-borne viral disease in humans and is a global and national public health concern in several countries. A seasonal pattern of dengue disease is consistently observed. The highest incidences usually correspond to the period of highest rainfall and humidity, providing suitable conditions for Aedes aegypti breeding and survival. In Brazil for instance it is from January to June. Dengue may cause marked changes in bone marrow that result in hypocellularity and, consequently, thrombocytopenia and leucopenia, along with an increase in hematocrit, which is secondary to capillary leakage. However, those abnormalities are usually self-limited, and do not warrant further investigations, such as a marrow biopsy or a myelogram. The occurrence of persistent reactive hemophagocytosis is uncommon and usually leads to serious adverse outcomes. The authors report the case of an 8-year old girl complaining of high-grade fever, malaise, headache, abdominal pain and a cutaneous rash. Laboratory examination revealed atypical lymphocytosis on peripheral blood count, hyperbilirrunemia, abnormal liver enzymes and clotting tests. Serology was positive for dengue. Because of the persistence of fever and laboratory examinations were consistent with hemophagocytic lymphohistiocytosis (HLH) a bone marrow aspiration was performed, which confirmed the presence of hemophagocytosis. Hence we report a rare presentation of dengue accompanied by self-limited HLH that hopefully evolve to favorable outcome.
1476	Haemophagocytic lymphohistiocytosis in inflammatory bowel disease with virus infection.	26557937	Patients with inflammatory bowel disease (IBD) are at risk of developing haemophagocytic lymphohistiocytosis (HLH) because of chronic systemic inflammation as well as exposure to immunosuppressive medications. The two main causes of HLH in IBD patients are infection with cytomegalovirus and Epstein-Barr virus. Patients with Crohn's disease are more susceptible to HLH than those with ulcerative colitis. The majority of cases are seen in people receiving an immunosuppressive regimen that included thiopurines.
1477	Common variable immune deficiency associated Hodgkin's lymphoma complicated with EBV-linked hemophagocytic lymphohistiocytosis: a case report.	26550396	Hemophagocytic syndrome (HPS) is described by an increase in macrophages accountable for extensive phagocytosis of hematopoietic cells. Secondary HPS arises commonly in the presence of infections, neoplasia, autoimmune disorders and immune disorders. Here, we reported a patient with common variable immune deficiency (CVID) and Hodgkin's lymphoma (HL) who later developed EBV linked hemophagocytic lymphohistiocytosis. 42 year old men underwent check-up because of back pain in July 2012. He had known CVID disease. In physical examination he had no lymphadenopathies however his spleen was palpable 3 cm under arcus costa. He had hypogammaglobulinemia with IgG levels around 500 mg/dl. In abdominal computed tomography (CT) multiple lymphadenopathies reaching maximum 26×17 cm size were seen so, PET-CT was performed. Involvement in thorax, abdomen, and bone was detected with maximum SUV max 11.5. He had undergone tru-cut biopsy from lymph node in November 2012 which revealed HL. Bone marrow investigation favored with mix cell type. His cytogenetic analysis was reported as 46 XY. He was considered as stage 4 disease and ABVD (Adriamycin, bleomycin, vincristine and dexamethasone). He was given six cycles of chemotherapy in May 2013 and complete remission was observed in control CT screening in July 2013. However pancytopenia evolved in August 2013. Bone marrow investigation revealed suspicious lymphohistiocytic infiltration. Treatment was planned to apply autologous stem cell transplantation (SCT) after salvage chemotherapy. Control bone marrow investigation again revealed the lymphohistiocytic aggregates with hemophagocytosis. Our patient showed 5 criteria of hemophagocytic syndrome. He had ferritin elevation (>5000 μg/dl), splenomegaly (13 cm) cytopenia, triglyceride elevation and hemophagocytosis. He had unrelated SCT transplantation however he died from transplant related toxicity. The primary and secondary immune deficiency caused by chemotherapy are the major causes for our patient inability to control his EBV infection which eventually lead to hemophagocytic lymphohistiocytosis. To conclude, rare simultaneous manifestation of primary immune deficiencies (PID), Hodgkin's lymphoma and EBV-HLH occurred in our patient which have concordant immunological mechanism that eventually lead poor prognosis in our patient.
1478	First prospective clinical trial in adult HLH.	2654224926289641	NaN
1479	Haemophagocytic lymphohistiocytosis in adults: a multicentre case series over 7 years.	26537747	Haemophagocytic lymphohistiocytosis (HLH) is a syndrome of uncontrolled immune activation that has gained increasing attention over the past decade. Although classically known as a familial disorder of children caused by mutations that affect cytotoxic T-cell function, an acquired form of HLH in adults is now widely recognized. This is often seen in the setting of malignancy, infection or rheumatological disorders. We performed a retrospective review across 3 tertiary care centres and identified 68 adults with HLH. The average age was 53 years (range 18-77 years) and 43 were male (63%). Underlying disorders included malignancy in 33 patients (49%), infection in 22 (33%), autoimmune disease in 19 (28%) and idiopathic HLH in 15 (22%). Patients were treated with disease-specific therapy and immunomodulatory agents. After a median follow-up of 32·2 months, 46 patients had died (69%). The median overall survival was 4 months (95% CI: 0·0-10·2 months). Patients with malignancy had a worse prognosis compared to those without (median survival 2·8 months versus 10·7 months, P = 0·007). HLH is a devastating disorder with a high mortality. Further research is needed to improve treatment and outcomes.
1480	[Acquired hemophagocytic syndrome treated with HLH 94-04 chemotherapy protocol: Report of four cases].	26530200	Hemophagocytic syndrome is a severe condition of excessive immune activation that has a high mortality in the absence of treatment. The syndrome is classified as primary if associated with congenital or hereditary problems, or secondary/acquired if associated with infectious, autoimmune or oncology diseases. We report four adult cases of the syndrome, one with viral, two with autoimmune and one with idiopathic causes who were successfully treated with HLH 94-04 chemotherapy protocol. Our experience shows that a high index of suspicion, early diagnosis and an opportune therapy are essential in the treatment of this disease.
1481	Secondary hemophagocytic lymphohistiocytosis in children with brucellosis: report of three cases.	26517495	Brucellosis is a systemic zoonotic infectious disease that may cause fever, fatigue, sweating, arthritis, hepatosplenomegaly, cytopenia, and lymphadenopathy. It continues to be an important health problem worldwide. Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever, hepatosplenomegaly, cytopenias, high serum levels of ferritin and triglycerides, low serum fibrinogen levels, and hemophagocytosis in bone marrow, lymph nodes, spleen, or liver. Hemophagocytic lymphohistiocytosis associated with brucellosis is a very rare condition in the pediatric age group. Here, three pediatric cases of secondary HLH associated with brucellosis are reported. Hemophagocytic lymphohistiocytosis should be considered in patients with brucellosis having cytopenias. Hemophagocytosis in brucellosis seems to be cured with appropriate antibiotics and intravenous immunoglobulin.
1482	Hemophagocytic lymphohistiocytosis diagnosed by brain biopsy.	26512263	Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever, splenomegaly, jaundice, and pathologic findings of hemophagocytosis in bone marrow or other tissues such as the lymph nodes and liver. Pleocytosis, or the presence of elevated protein levels in cerebrospinal fluid, could be helpful in diagnosing HLH. However, the pathologic diagnosis of the brain is not included in the diagnostic criteria for this condition. In the present report, we describe the case of a patient diagnosed with HLH, in whom the brain pathology, but not the bone marrow pathology, showed hemophagocytosis. As the diagnosis of HLH is difficult in many cases, a high level of suspicion is required. Moreover, the pathologic diagnosis of organs other than the bone marrow, liver, and lymph nodes may be a useful alternative.
1483	Preceding Annular Skin Lesions in a Patient with Hemophagocytic Lymphohistiocytosis.	26512177	The cutaneous manifestations of hemophagocytic lymphohistiocytosis (HLH) are variable and nonspecific. A 42-year-old man presented with multiple annular, erythematous patches on the trunk for 3 months. Two months later, he presented with bullae along with high fever. The laboratory examination showed pancytopenia, hypertriglyceridemia, and hypofibrinogenemia. The bone marrow biopsy specimen showed an active hemophagocytosis. On the basis of these findings, a diagnosis of HLH was concluded. After five cycles of chemotherapy, his skin lesion completely resolved. Taking the results together, we suggest that annular skin lesion can be added to the list of cutaneous manifestations of HLH.
1484	The challenging diagnosis of haemophagocytic lymphohistiocytosis in an HIV-infected patient.	26511991	The differential diagnosis of febrile pancytopenia in the setting of HIV infection can be challenging. The authors report a case of a 34-year-old man with advanced HIV infection (TCD4=8 cells/mm(3)) and a 2-month history of fever, weight loss and asthaenia. On observation, he was emaciated, hyperthermic and pale, with a haemorrhagic oropharyngeal lesion, penile violaceous lesions and palpable hepatosplenomegaly. Blood tests showed pancytopenia, hyperferritinaemia and hypertriglyceridaemia; imaging confirmed hepatosplenomegaly and a bone marrow biopsy revealed HIV-associated dyshematopoiesis. Biopsy of the aforementioned lesions displayed Kaposi sarcoma; extensive investigation was negative for other opportunistic infections or lymphoproliferative disease. Despite only a posteriori histological documentation, haemophagocytic lymphohistiocytosis (HLH) was considered; chemotherapy and antiretrovirals were started, with excellent response. There was, however, disease relapse requiring an intensification course, with sustained remission at 18-month follow-up. HLH is a rare disease, with non-specific presentation, requiring a high index of suspicion since treatment delay can be fatal.
1485	Allogeneic hematopoietic stem cell transplantation for Chediak-Higashi syndrome.	26511512	The clinical outcome of allogeneic HSCT was retrospectively analyzed in eight patients with CHS. In total, six of these eight patients are alive. Four of five patients transplanted with MAC achieved prompt engraftment, and three of the four patients, including two patients with AP before transplant, are alive without disease. In contrast, three patients transplanted with RIC without active AP disease achieved prompt engraftment and survive long term. RIC-HSCT might be an alternative treatment for CHS similar to other types of HLH, at least for patients without active AP disease.
1486	Hemophagocytic Lymphohistiocytosis Complicating Dengue and Plasmodium vivax Coinfection.	26504465	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder. Dysfunction of cytotoxic T and natural killer (NK) cells causes uncontrolled activity of lymphocytes and histiocytes which leads to HLH. Infections, malignancies, and autoimmune disorders are associated with development of HLH. Dengue and Plasmodium vivax are rare causes of HLH. We report the first ever case of a young man who developed fatal HLH that complicated Dengue Hemorrhagic Fever (DHF) and Plasmodium vivax infection.
1487	Successful Treatment of Corticosteroid with Antiviral Therapy for a Neonatal Liver Failure with Disseminated Herpes Simplex Virus Infection.	26495160	Background Herpes simplex virus (HSV) infection carries one of the poorest outcomes of neonatal liver failure (NLF). Neonates with disseminated HSV infection can develop hemophagocytic lymphohistiocytosis (HLH), and occasionally need orthotopic liver transplantation. Early interventions may be critical for the cure of NLF. Case Report We describe herewith a 6-day-old neonate with fulminant hepatic failure due to disseminated HSV-1 infection, who successfully responded to high-dose corticosteroid therapy 72 hours after the onset of disease. Preceding acyclovir, gamma globulin, and exchange blood transfusion therapies failed to control the disease. Methylprednisolone pulse therapy led to a drastic improvement of liver function and cytokine storms, and prevented the disease progression to HLH. Sustained levels of plasma and cerebrospinal fluid HSV DNA declined after prolonged acyclovir therapy. Bilateral lesions of the periventricular white matter areas, assessed by magnetic resonance imaging, disappeared at 3 months of age. The infant showed normal growth and development at 4 years of age. Conclusion Early anti-hypercytokinemia therapy using corticosteroid, and prolonged antiviral therapy might only provide the transplantation-free cure of NLF with HSV dissemination.
1488	Alemtuzumab, Fludarabine, Low-Dose TBI, and Double Umbilical Cord Transplant for Primary Graft Failure in a Patient with Recurrent HLH.	26488531	Graft failure occurs at relatively low frequency, but commonly in hemophagocytic lymphohistiocytosis (HLH), especially with umbilical cord blood transplant (UCBT). No standard approaches to management of graft failure exist. We present a challenging case of relapsed HLH following first UCBT with primary graft failure following second UCBT. We report a novel reduced intensity conditioning regimen of alemtuzumab, 4 Gy total body irradiation and fludarabine for salvage of primary graft failure followed by double UCBT. The reported patient successfully engrafted with 100% donor chimerism following salvage UCBT with no occurrence of acute or chronic graft-versus-host disease.
1489	Idiopathic Hemophagocytic Lymphohistiocytosis During Pregnancy Treated with Steroids.	26487936	Hemophagocytic lymphohistiocytosis (HLH) is a rare and severe clinical syndrome characterized by a dysregulated hyperinflammatory immune response. The diagnosis of HLH during pregnancy is especially challenging due to the rarity of this condition. The highly variable clinical presentation, laboratory findings, and associated diagnoses accompanying this syndrome further complicate the problem. A pronounced hyperferritinemia in the setting of systemic signs and symptoms along with a negative infectious and rheumatological workup should raise suspicions for HLH. While treatment ideally consists of immunosuppressive chemotherapy and hematopoietic stem cell transplant, the potential toxicity to both the pregnant woman and the fetus poses a challenging decision. We report the first case of idiopathic HLH presenting as fever of unknown origin in a pregnant woman successfully treated with steroids.
1490	Secondary Hemophagocytic Lymphohistiocytosis in a Patient With Favorable Histology Wilms Tumor.	26479988	Secondary hemophagocytic lymphohistiocytosis (HLH) is most commonly associated with malignancy, infection, or an underlying autoimmune disorder. Malignancy-associated hemophagocytic syndrome is responsible for most secondary HLH cases, but it has not been well described in children. We present a case of a 4-year-old female with favorable histology of Wilms tumor who developed secondary HLH after unsuccessful resection of the tumor and initiation of chemotherapy.
1491	Adult-onset central nervous system hemophagocytic lymphohistiocytosis: a case report.	26467435	Hemophagocytic lymphohistiocytosis (HLH) is a clinical syndrome with both genetic and acquired causes characterized by elevated cytokine levels, hyperinflammation, and overactivation of lymphocytes and macrophages. It is typically a systemic disease with variable degrees of CNS involvement. Cases with predominantly central nervous system (CNS) involvement are very rare, with the vast majority of these occurring in infants and young children. This report documents a case of adult-onset CNS-HLH involving a middle-aged man.A 55 year-old man developed progressive left hemiparesis and aphasia over the course of several months. Brain MRI showed multifocal, mass-like enhancing lesions with increased susceptibility consistent with blood products. An extensive workup for infectious, autoimmune, and neoplastic etiologies was significant only for a markedly elevated serum ferritin at 1456 ng/mL. Two brain biopsies showed a non-specific inflammatory process. The patient was treated empirically with steroids and plasmapheresis, but he continued to suffer a progressive neurological decline and died one year after onset of neurological symptoms. Autopsy revealed profound histiocytic infiltration, perivascular lymphocytosis, and emperipolesis, compatible with CNS-HLH.This case report describes an exceedingly rare presentation of an adult patient with CNS predominant HLH. This diagnosis should be considered in the differential diagnosis of adults presenting with progressive brain lesions, even in the absence of typical systemic signs of HLH.
1492	The Role of Hematopoietic Stem Cell Transplantation in Treatment of Hemophagocytic Lymphohistiocytosis.	26461153	The role of reduced-intensity allogeneic hematopoietic stem cell transplantation (HSCT) from a variety of donor sources in improving survival for children with familial hemophagocytic lymphohistiocytosis (HLH) is well-documented. The heterogeneity of adult-onset HLH has complicated evaluation of initial therapy and of HSCT as definitive treatment. Therapy for adults with HLH is often individualized, but institutions are now generating algorithms that include HSCT based on growing experience. Consolidation of these data is needed to optimize management of the growing number of adults recognized to have HLH and to achieve dramatic improvements in survival.
1493	Hemophagocytic Lymphohistiocytosis in Adults.	26461151	Hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal syndrome of pathologic immune dysregulation characterized by clinical signs and symptoms of extreme inflammation. HLH can occur as a genetic or sporadic disorder and, though seen as an inherited condition affecting primarily a pediatric population, can occur at any age and can be encountered in association with a variety of underlying diseases. Clinically the syndrome, whether genetic or acquired, is characterized by fever, hepatosplenomegaly, cytopenias, and activated macrophages in hematopoietic organs. Therapy centers on suppression of this hyperinflammatory state with cytotoxic, immunosuppressive therapy and treatment of any existing HLH triggers.
1494	Familial Hemophagocytic Lymphohistiocytosis.	26461150	Familial hemophagocytic lymphohistiocytosis (FHL) is a rare heritable disorder of immune regulation that is typically characterized by sudden onset of severe systemic illness. Functional impairment or absence of 1 or more of several proteins that participate in lymphocyte cytotoxicity underlies the disease. Although FHL usually presents in infancy, age of onset is variable and dependent on genetic and environmental factors. Initial treatment consists of immune suppression, whereas definitive treatment requires hematopoietic cell transplantation.
1495	Pathogenesis of Hemophagocytic Lymphohistiocytosis.	26461149	Hemophagocytic Lymphohistiocytosis (HLH), an inherited life-threatening inflammatory disorder, has gained growing recognition not only in children but also increasingly in adults over the past 2 decades. HLH involves inborn defects in lymphocytes, which normally mediate control of infectious and inflammatory conditions within the immune system and in other tissues. In the context of inherited defects in cytotoxic cells and other immune cells, the disorder is classified as familial or primary HLH. Secondary HLH occurs in the settings of infections or underlying rheumatologic disorders. Secondary HLH also accompanies some lymphoid malignancies.
1496	[Heterogeneity of HLH pathophysiology and treatment strategies].	26458466	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome caused by immune dysregulation and hyperinflammation and is histologically characterized by the presence of benign hemophagocytic macrophages. HLH is classified as primary or secondary depending on the underlying etiology and includes conditions with various pathophysiological features. The pathogenic mechanisms underlying the different HLH subtypes remain incompletely understood and therapeutic regimens for HLH are devised on empirical bases. Herein, recent advances in the understanding of HLH pathogenesis and potential strategies for subtype-specific HLH treatment are reviewed.
1497	Hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis: recent advances and controversies.	26457279	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory clinical syndrome of uncontrolled immune response which results in hypercytokinemia due to underlying primary or secondary immune defect. A number of genetic defects in transport, processing and function of cytotoxic granules which result in defective granule exocytosis and cytotoxicity of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have been well identified at the cellular and molecular level. Important advances have been made during the last 20 years in the diagnosis and treatment of HLH. The Histiocyte Society has proposed diagnostic guideline using both clinical and laboratory findings in HLH-2004 protocol, and this has been modified partly in 2009. HLH used to be a fatal disease, but the survival of HLH patients has improved to more than 60% with the use of chemoimmunotherapy combined with hematopoietic cell transplantation (HCT) over the past 2 decades. However, HCT is still the only curative option of treatment for primary HLH and refractory/relapsed HLH after proper chemoimmunotherapy. The outcome of HCT for HLH patients was also improved steadily during last decades, but HCT for HLH still carries significant mortality and morbidity. Moreover, there remain ongoing controversies in various aspects of HCT including indication of HCT, donor selection, timing of HCT, conditioning regimen, and mixed chimerism after HCT. This review summarized the important practical issues which were proven by previous studies on HCT for HLH, and tried to delineate the controversies among them.
1498	Clinical significance of bone marrow hemophagocytosis in adult patients with malignancy and non-malignancy-induced hemophagocytic lymphohistiocytosis.	26453074	Bone marrow hemophagocytosis is a frequently observed but not mandatory finding for the diagnosis of hemophagocytic lymphohistiocytosis (HLH). However, the impact of bone marrow hemophagocytosis on the diagnosis of HLH is still not clear in adult patients. Thus, we retrospectively analyzed adult patients with bone marrow hemophagocytosis between 2000 and 2014 to determine its clinical significance. Among 264 patients with bone marrow hemophagocytosis, malignant disorders were the predominant underlying cause (n = 170, 64 %), especially T/NK-cell (n = 88) and B-cell (n = 45) lymphomas compared to infectious disease (48/264, 18 %). The data for HLH-2004 diagnostic criteria was available in 182 patients, and only 29 % (77/264) of patients with ≥ five positive criteria could be diagnosed with HLH. Among the criteria for the diagnosis of HLH, increased serum ferritin (89 %) was more common than hypofibrinogenemia, hypertriglyceridemia, and bicytopenia (<40 %). The median overall survival was worse in patients with malignancy (9.0 months, 95 % confidence interval [CI] 5.6-12.5) than in those with non-malignant disorders (71.8 months, 95 % CI 56.5-87.1, P < 0.001). In patients with malignancy, the overall survival of patients fulfilling the HLH-2004 criteria was significantly worse than patients who did not (P < 0.001). In conclusion, our results suggest that bone marrow hemophagocytosis might be an important finding in the diagnosis of HLH in adult patients. Considering the high incidence of malignancy as a predisposing disorder for HLH, immediate evaluation should be performed in adult patients with bone marrow hemophagocytosis.
1499	Prevalence of type 5 familial hemophagocytic lymphohistiocytosis in Korea and novel mutations in STXBP2.	26451869	Familial hemophagocytic lymphohistiocytosis (F-HLH or FHL) is a potentially fatal immune dysregulation syndrome with a heterogeneous genetic background. Most recently, STXBP2 has been identified as the causative gene of type 5 FHL (FHL5) with a worldwide distribution. In this study, we investigated the prevalence of FHL5 in Korea. About 50 Korean pediatric patients with HLH who lacked pathogenic mutations in PRF1, UNC13D, or in STX11 from the previous series of 72 patients with HLH were analyzed for STXBP2 mutations by conventional sequencing analyses. As a result, we found one patient with two novel mutations of STXBP2: c.184A>G and c.577A>C. c.184A>G (p.Asn62Asp) was located within a highly conserved region of the STXBP2 protein and predicted to be deleterious. c.577A>C in exon 7 resulted in incomplete splicing mutation with exon 7 skipping concurrent with exon 7-retained transcript with p.Lys193Gln substitution. The frequency of FHL5 was ~1% (1/72) in Korean pediatric patients with HLH. This is the first study on FHL5 in Korea, and the data from a nationwide patient cohort provide another piece of genetic profiles of FHL.
1500	Clonal CD8+ T Lymphocytic Proliferation and Karyotypical Abnormalities in an EBV Associated Hemophagocytic Lymphohistiocytosis.	26451265	EBV associated hemophagocytic lymphohistiocytosis and EBV-positive T cell lymphoproliferative disease of childhood share many histologic and clinical features, which sometimes makes it very difficult to render a definitive diagnosis. In this report, we present a 16-year-old male who developed symptoms clinically consistent with EBV associated hematophagocytic lymphohistiocytosis including fulfilling most of HLH diagnostic criteria and responding promptly to HLH targeted therapy. However, histologic and cytogenetics features of this case are very concerning for EBV-positive T cell lymphoproliferative disease of childhood. This case demonstrates an ambiguous boundary of these two disease entities and emphasizes the importance of comprehensive evaluation and clinical correlation with cases suspicious of EBV driven hemophagocytic or lymphoproliferative process.
1501	Coagulation Disorders and Bleedings in Critically Ill Patients With Hemophagocytic Lymphohistiocytosis.	26448017	Reactive hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition related to a cytokine storm leading to multiorgan dysfunction. A better understanding of coagulation disorders, frequently reported in HLH patients, may improve outcomes. Critically ill HLH patients managed in a multidisciplinary national reference center were retrospectively included. Relationships between coagulation disorders, severe bleedings, and outcomes were assessed. One hundred and seventeen patients fulfilled the HLH 2004 criteria. The most common HLH etiology was hematologic conditions (73%), followed by infectious diseases (20%), systemic rheumatic diseases (5%), and undetermined HLH etiology (3%). All patients exerted thrombocytopenia. Coagulation disorders were diagnosed in 79 (68%) patients (61 had hypofibrinogenemia < 1.5 g/L, 51 had prothrombin time [PT]  <  0%). The worst median value throughout ICU stay was 52% (38-65) for PT with a factor V level of 35% (27-43), 1.59 (1.30-2.09) for the activated partial thromboplastin time (APTT) ratio, and 2.33 g/L (1.13-3.86) for the fibrinogen level. Disseminated intravascular coagulation (DIC) was found in 50% of patients. Coagulation disorders were more frequent in immunocompromised patients, those with histological/cytological feature of hemophagocytosis, those with the highest ferritin concentrations, and in patients with HLH not related to infection. These patients were more prone to receive mechanical ventilation, vasopressors, or renal replacement therapy. Twenty-six (22%) patients presented severe bleeding complications, including 5 patients dying from hemorrhagic shock. Strikingly, the only coagulation parameter significantly associated with severe bleeding was low fibrinogen with a cutoff value of 2 g/L (P = 0.03). Overall, 33 (28%) patients died in the ICU and hospital mortality was 44%. Coagulation disorders were associated with higher mortality, especially fibrinogen < 2 g/L (P = 0.04) and PT value (P = 0.03). The occurrence of bleeding complications was not associated with higher risk of hospital death. Risk factors associated with mortality by multivariate analysis were fibrinogen level < 2 g/L (OR 2.42 [1.08-5.41]), SOFA score > 6 (OR 3.04 [1.32-6.98]), and age > 46 years (OR 2.26 [1.02-5.04]). Up to two-third of critically ill HLH patients present with coagulation disorders. Hypofibrinogenemia or DIC was found in half of the patients and low PT in 40%. These patients require more life support and have a higher mortality rate. Fibrinogen < 2 g/L is associated with the occurrence of severe bleeding and mortality.
1502	Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor's Extracellular Hinge Region.	26441830	The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) - secreted by the placenta, and lutropin (LH) - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.
1503	HES-Mediated Repression of Pten in Caenorhabditis elegans.	26438299	The hairy/enhancer-of-split (HES) group of transcription factors controls embryonic development, often by acting downstream of the Notch signaling pathway; however, little is known about postembryonic roles of these proteins. In Caenorhabditis elegans, the six proteins that make up the REF-1 family are considered to be HES orthologs that act in both Notch-dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate postembryonic cellular events, we performed a functional characterization of the REF-1 family member, HLH-25. We show that, after embryogenesis, hlh-25 expression persists throughout every developmental stage, including dauer, into adulthood. Like animals that carry loss-of-function alleles in genes required for normal cell-cycle progression, the phenotypes of hlh-25 animals include reduced brood size, unfertilized oocytes, and abnormal gonad morphology. Using gene expression microarray, we show that the HLH-25 transcriptional network correlates with the phenotypes of hlh-25 animals and that the C. elegans Pten ortholog, daf-18, is one major hub in the network. Finally, we show that HLH-25 regulates C. elegans lifespan and dauer recovery, which correlates with a role in the transcriptional repression of daf-18 activity. Collectively, these data provide the first genetic evidence that HLH-25 may be a functional ortholog of mammalian HES1, which represses PTEN activity in mice and human cells.
1504	Lysosomal Trafficking Regulator (LYST).	26427484	Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections.
1505	Profiling of EBV-Encoded microRNAs in EBV-Associated Hemophagocytic Lymphohistiocytosis.	26423217	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening complication of EBV infection. MicroRNAs (miRNAs) were small non-coding RNA, and EBV could encode miRNAs that are involved in the progression of infection. However, the profiles of EBV-miRNAs in EBV-HLH were unknown. Here, we aimed to profile the expression of EBV-miRNAs in children with EBV-HLH by analyzing 44 known EBV-miRNAs, encoded within the BamHI fragment H rightward open reading frame 1 (BHRF1) and the BamHI-A region rightward transcript (BART), in plasma and cellular targets by real-time quantitative PCR. The study included 15 children with EBV-HLH, 15 children with infectious mononucleosis (IM), and 15 healthy controls. CD8(+) T cells were found to be the cellular target of EBV infection in EBV-HLH, while CD19(+) B cells were infected with EBV in IM. We also found the greater levels of several miRNAs encoded by BART in EBV-HLH, compared to those in IM and healthy controls, whereas the levels of BHRF1 miRNAs were lower than those in IM. The profile and pattern of EBV-miRNAs in EBV-HLH indicated that EBV could display type II latency in EBV-HLH. Importantly, the level of plasma miR-BART16-1 continued decreasing during the whole chemotherapy, suggesting that plasma miR-BART16-1 could be a potential biomarker for monitoring EBV-HLH progression. The pathogenesis of EBV-HLH might be attributed to the abundance of EBV-miRNAs in EBV-HLH. These findings help elucidate the roles of EBV miRNAs in EBV-HLH, enabling the understanding of the basis of this disease and providing clues for its treatment.
1506	Estimation of surface-level PM concentration from satellite observation taking into account the aerosol vertical profiles and hygroscopicity.	26421659	Surface-level PM10 distribution was estimated from the satellite aerosol optical depth (AOD) products, taking the account of vertical profiles and hygroscopicity of aerosols over Jeju, Korea during March 2008 and October 2009. In this study, MODIS AOD data from the Terra and Aqua satellites were corrected with aerosol extinction profiles and relative humidity data. PBLH (Planetary Boundary Layer Height) was determined from MPLNET lidar-derived aerosol extinction coefficient profiles. Through statistical analysis, better agreement in correlation (R = 0.82) between the hourly PM10 concentration and hourly average Sunphotometer AOD was the obtained when vertical fraction method (VFM) considering Haze Layer Height (HLH) and hygroscopic growth factor f(RH) was used. The validity of the derived relationship between satellite AOD and surface PM10 concentration clearly demonstrates that satellite AOD data can be utilized for remote sensing of spatial distribution of regional PM10 concentration.
1507	Macrophage Activation Syndrome.	26400031	Macrophage activation syndrome (MAS) is a potentially life-threatening complication of rheumatic diseases such as systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus. It is often considered a type of secondary hemophagocytic lymphohistiocytosis (HLH) and results from over-activation of T lymphocytes and macrophages leading to a "cytokine storm". Characteristic features are persistent fever, lymphadenopathy, hepatosplenomegaly, cytopenias (anemia, leucopenia, thrombocytopenia), raised C-reactive protein, falling erythrocyte sedimentation rate, hypofibrinogenemia, transaminitis, hypertriglyceridemia and extreme hyperferritinemia often associated with multi-organ impairment. Key to its management is early recognition of MAS which may be difficult due to similarity to systemic sepsis or flares of the underlying rheumatic disease. To aid with this process, criteria for the diagnosis of MAS in patients with sJIA derived by international consensus have been published. Although bone marrow biopsy showing hemophagocytosis is strongly supportive it is not essential for diagnosis. Together with appropriate supportive care, first-line treatment is high-dose intravenous corticosteroids with cyclosporin or intravenous immunoglobulin (IVIg) added if there is not initial response. Although etoposide is used by hematologists in treatment of HLH, there are concerns regarding organ toxicity and bone marrow suppression which weigh against its use in initial management of MAS. With increasing understanding of the pathogenesis of MAS, use of drugs targeting specific cytokines has been reported in case series. The relatively rapid effectiveness of anakinra, a recombinant IL-1 receptor antagonist, has been documented. Further studies of this and other biologic agents are required to identify the most effective and safest treatment option for refractory MAS.
1508	Distribution characteristics of organic phosphorus in sediments from Lake Hulun, China.	26395387	The amount of organic phosphorus (OP) and its distribution among different pools in lake sediments depend on biotic and abiotic processes driving OP fractionation. The key abiotic factors governing these transformation processes between different OP fractions in sediments were studied on the basis of distribution characteristics of OP pools in sediments from Lake Hulun (HLH). The results showed the order of the size of OP pools in the surface sediments from Lake Hulun to be: Re-OP (residual OP) ≫ FAOP (fulvic acid fraction) ≥ HCl-OP (OP extracted by HCl) > HAOP (humic acid fraction) ≫ LOP (labile OP); lower concentrations of LOP were found in lake surface sediments with high pH (pH > 9) of lake overlying water indicating a governing role of pH in LOP degradation in an aquatic environment. The pattern of total OP (TOP) spatial distribution showed an obvious decreasing trend from the west to the east lake districts in surface sediments from HLH, which was attributed to the inputs of dust and dry grass driven by the prevailing wind and the finer grain size in the west lake districts. However, the spatial distribution pattern of dissolved OP (DOP) in overlying water, which presented different trends with TOP and total organic carbon (TOC) in surface sediments, indicated that the biological factors and terrestrial inputs showed a joint influence on DOP distribution in HLH. The higher levels of Re-OP and lower levels of HCl-OP observed in HLH may reveal that calcium ions and their minerals were the key governing factors of OP fractionation in sediments from HLH. This work revealed the main abiotic process or mechanism driving OP fractionation in the aquatic environment helping to understand the geochemical information archived in OP pools in lake sediments.
1509	Pregnancy-related hemophagocytic lymphohistiocytosis associated with cytomegalovirus infection: A diagnostic and therapeutic challenge.	26384065	Hemophagocytic lymphohistiocytosis (HLH) is a disorder characterized by uncontrolled mature histiocyte proliferation, hemophagocytosis, and hypercytokinemia. We describe a previously healthy pregnant patient who presented in the third trimester of pregnancy with HLH.A 35-year-old woman presented at 38 weeks' gestation with pyrexia, jaundice, severe anemia, elevated liver enzymes, and lactate dehydrogenase suggestive of HELLP (hemolysis, elevated liver enzyme, low platelet) syndrome. Unfortunately, her condition deteriorated and she was ventilated in the intensive care unit despite delivery of the baby and administration of dexamethasone. She developed microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment suggestive of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome. However, she was refractory to plasma exchange, intravenous immunoglobulin, and broad-spectrum antibiotics. HLH was eventually diagnosed from biochemical and bone marrow findings. An extensive search for possible causes yielded negative results. She improved significantly with intravenous dexamethasone and cyclosporine A and was transferred out of the intensive care unit. Unfortunately, she developed cytomegalovirus disease 2 weeks later, which improved transiently with intravenous ganciclovir; later, however, she succumbed to multidrug-resistant nosocomial infections, rapidly progressive cytomegalovirus disease, and multiorgan failure.This case highlights the challenges and difficulties involved in the diagnosis and management of pregnancy-related HLH. Immunosuppressive treatment for HLH can precipitate life-threatening opportunistic infections, which need to be promptly diagnosed and treated.
1510	Histoplasmosis-Induced Hemophagocytic Syndrome: A Case Series and Review of the Literature.	26380347	Background.  Histoplasmosis-associated hemophagocytic lymphohistiocytosis (HLH) is a relatively rare disorder for which data are limited regarding optimal treatment and clinical outcomes in adults. We describe the clinical features, treatment, and outcomes of patients with histoplasmosis-associated HLH at our institution. Methods.  We performed a retrospective chart review of all inpatients at Parkland Hospital diagnosed with HLH associated with Histoplasma capsulatum from 2003 to 2013. Results.  Eleven cases of histoplasmosis-associated HLH over this time period were identified. Nine of eleven cases were males (82%). Nine of these patients had human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), 1 was a renal transplant patient on immunosuppressants, and the other had no documented immunocompromise. The most common HLH criteria were splenomegaly (n = 10), fever (n = 10), and ferritin >500 ng/dL (n = 9). Urine Histoplasma antigen was positive in every patient tested (n = 9 of 9), and most antibodies for Histoplasma were positive if checked (n = 4 of 5). A majority of patients received liposomal amphotericin B (n = 9) with an average treatment duration of 11 days, and 5 patients also received prednisone, intravenous immunoglobulin (IVIG), or both. Overall, 5 patients died within 30 days (45.5%), and 7 patients died within 90 days (63.6%). Of the 5 patients that received immunosuppression, 4 died (80%), whereas in the group not given additional immunosuppression (n = 5), 2 died (40%). Conclusions.  Histoplasmosis-associated HLH among adults is a lethal disease of highly immunocompromised patients, especially patients with HIV/AIDS. Clinical features such as splenomegaly, elevated ferritin, and cytopenias should prompt evaluation for HLH in this population. Further data are needed to define the role of immunosuppression, IVIG, and highly active antiretroviral therapy in treating this condition.
1511	Neurological presentation of hemophagocytic lymphohistiocytosis.	26377985	Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory disorder arising from defects in critical regulatory pathways responsible for termination of inflammatory response. We are presenting a case report of a 20-year-old male, admitted to the Department of Neurology because of left lower limb weakness and balance disturbances. After a few days of hospitalization, fever occurred. Laboratory tests revealed anemia, neutropenia, lymphopenia, and thrombocytopenia. The clinical course and laboratory tests results confirmed the diagnosis of HLH. In our opinion, the disorder in the presented case occurred due to severe chronic active Epstein-Barr virus infection syndrome. We are presenting the case of pure neurological onset of hemophagocytic lymphohistiocytosis in an adult patient. Hemophagocytic lymphohistiocytosis, initially presenting with neurological symptoms, can occur in adult patients with irrelevant family history. It is a life-threatening but potentially curable condition requiring proper diagnostic and treatment management.
1512	A Hemophagocytic Lymphohistiocytosis Case with Newly Defined UNC13D (c.175G>C; p.Ala59Pro) Mutation and a Rare Complication.	26377049	Hemophagocytic lymphohistiocytosis (HLH) represents a severe hyperinflammatory condition with cardinal symptoms of prolonged fever, cytopenias, hepatosplenomegaly, and hemophagocytosis by activated, morphologically benign macrophages with impaired function of natural killer cells and cytotoxic T lymphocytes. A 2-month-old girl, who was admitted with fever, was diagnosed with HLH and her genetic examination revealed a newly defined mutation in the UNC13D (c.175G>C; p.Ala59Pro) gene. She was treated with dexamethasone, etoposide, and intrathecal methotrexate. During the second week of treatment, after three doses of etoposide, it was noticed that there was a necrotic plaque lesion on the soft palate. Pathologic examination of debrided material in PAS and Grocott staining revealed lots of septated hyphae, which was consistent with aspergillosis infection. Etoposide was stopped and amphotericin B treatment was given for six weeks. HLH 2004 protocol was completed to eight weeks with cyclosporine A orally. There was no patient with invasive aspergillosis infection as severe as causing palate and nasal septum perforation during HLH therapy. In immunocompromised patients, fungal infections may cause nasal septum perforation and treatment could be achieved by antifungal therapy and debridement of necrotic tissue.Hemofagositik lenfohistiositoz (HLH) uzamış ateş, sitopeni, hepatosplenomegali semptomları ile seyreden, active olmuş, morfolojik olarak benign makrofaj ve doğal öldürücü hücreler ile sitotosik T lenfosit fonksiyon bozukluğu sonucu gelişen hiperenflamatuvar bir durumdur. İki aylık düşmeyen ateş yakınması ile başvuran hasta HLH tanısı aldı ve hastanın genetik incelemesinde UNC13D (c.175G>C; p.Ala59Pro) geninde yeni tanımlanan bir mutasyon saptandı. Hastaya deksamatazon, etopozit ve intratekal metotreksat tedavileri başlandı. Tedavinin 2. haftasında, üç doz etopozit aldıktan sonra, yumuşak damakta plak lezyonu fark edildi ve bu nekrotik lezyon debride edildi. Debridman materyalinin patolojik incelemesinin PAS, Grocott boyamasında aspergilloz enfeksiyonu ile uyumlu olarak çok sayıda septalı hif görüldü. Etopozid tedavisi sonlandırılarak altı hafta boyunca amphotericin B tedavisi verildi. HLH 2004 tedavi protokolü oral siklosporin ile sekiz haftaya tamamlandı. HLH tedavisi sırasında yumuşak damak perforasyonuna neden olacak kadar ağır aspergilloz enfeksiyonu geçiren bir olgu bildirilmemiştir. İmmünyetmezlikli hastada mantar enfeksiyonları nazal septum perforasyonuna neden olabilmektedir ve tedavi nekrotik dokunun debridmanı ve antifungal tedavi ile sağlanabilmektedir.
1513	Disseminated Histoplasmosis and Secondary Hemophagocytic Syndrome in a Non-HIV Patient.	26347828	Histoplasma duboisii, a variant of Histoplasma capsulatum that causes "African histoplasmosis," can be resistant to itraconazole, requiring intravenous amphotericin B treatment. Rarely, these patients do not respond to intravenous antifungal therapy, and in such cases, patients may progress to develop secondary hemophagocytic lymphohistiocytosis (HLH). We present a case of a 34-year-old male patient with sickle cell disease who presented with a 5-month history of an enlarging painless axillary mass, persistent low grade fevers, night sweats, weight loss, and anorexia. An excisional biopsy of the right axillary lymph node revealed yeast and granulomas consistent with histoplasma infection. He was started on oral itraconazole. After 4 weeks of therapy, laboratory evaluation revealed worsening anemia, thrombocytopenia, and transaminitis. Due to failure of oral therapy, he was admitted for intravenous amphotericin B treatment. During his hospital course anemia, thrombocytopenia, and transaminitis all worsened. A bone marrow biopsy was done that was consistent with HLH. His clinical status continued to deteriorate, developing multiorgan failure and disseminated intravascular coagulation. He unfortunately had a cardiorespiratory arrest after eight days of admission and passed away.
1514	Genetic predisposition to hemophagocytic lymphohistiocytosis: Report on 500 patients from the Italian registry.	26342526	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease affecting mostly children but also adults and characterized by hyperinflammatory features. A subset of patients, referred to as having familial hemophagocytic lymphohistiocytosis (FHL), have various underlying genetic abnormalities, the frequencies of which have not been systematically determined previously.This work aims to further our understanding of the pathogenic bases of this rare condition based on an analysis of our 25 years of experience.From our registry, we have analyzed a total of 500 unselected patients with HLH.Biallelic pathogenic mutations defining FHL were found in 171 (34%) patients; the proportion of FHL was much higher (64%) in patients given a diagnosis during the first year of life. Taken together, mutations of the genes PRF1 (FHL2) and UNC13D (FHL3) accounted for 70% of cases of FHL. Overall, a genetic diagnosis was possible in more than 90% of our patients with FHL. Perforin expression and the extent of degranulation have been more useful for diagnosing FHL than hemophagocytosis and the cytotoxicity assay. Of 281 (56%) patients classified as having "sporadic" HLH, 43 had monoallelic mutations in one of the FHL-defining genes. Given this gene dosage effect, FHL is not strictly recessive.We suggest that the clinical syndrome HLH generally results from the combined effects of an exogenous trigger and genetic predisposition. Within this combination, different weights of exogenous and genetic factors account for the wide disease spectrum that ranges from HLH secondary to severe infection to FHL.
1515	Modulating Intrafollicular Hormonal Milieu in Controlled Ovarian Stimulation: Insights From PPAR Expression in Human Granulosa Cells.	26332656	Controlled ovarian stimulation (COS) leading to ovulation of multiple follicles is a crucial aspect of biomedical infertility care. Nevertheless, biomarkers useful for COS management are still lacking. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors relevant to steroid metabolism in granulosa cells (GCs). We investigated whether PPARs and their steroidogenic targets were differentially expressed in GCs differentiated under different recombinant or urinary gonadotropin preparations. GCs from women subjected to COS with r-hFSH, r-hFSH/r-hLH, or hMG-HP were processed to assess expression of PPARα, PPARβ/δ, PPARγ, and steroidogenic enzymes under PPAR modulation. As an evidence of their activation, all PPAR isotypes with their coactivators, the retinoic-X-receptors (RXRs), localized in the nucleus. When GCs from r-hFSH/r-hLH group were compared with r-hFSH, a significant reduction of PPARα protein was observed. By contrast, an increase of PPARβ/δ at both protein and mRNA levels along with that of PPARγ protein were detected. The steroidogenic enzymes 17βHSD IV, 3βHSD II, and HMG-CoA red were downregulated in the r-hFSH/r-hLH group in comparison to r-hFSH unlike CYP19A1 that remained unchanged. In GCs from urinary FSH-LH stimulation (hMG-HP), PPARα was more expressed in comparison with r-hFSH/r-hLH group. Likewise, 3βHSD II and 17βHSD IV were increased suggesting that hMG-HP partially mimicked r-hFSH/r-hLH effects. In summary, transcript analysis associated to protein investigation revealed differential effects of COS protocols on PPARs and their steroidogenic targets in relation to LH and gonadotropin source. These observations candidate PPARs as new biomarkers of follicle competence opening new hypotheses on COS effects on ovarian physiology. J. Cell. Physiol. 231: 908-914, 2016. © 2015 Wiley Periodicals, Inc.
1516	Prognostic Factors and Outcomes of Adult-Onset Hemophagocytic Lymphohistiocytosis: A Retrospective Analysis of 34 Cases.	26331000	Adult-onset hemophagocytic lymphohistiocytosis (HLH) has features that are distinct from that of HLH in pediatric patients. The clinical records at the Japanese Red Cross Kumamoto Hospital were reviewed. We retrospectively analyzed 34 patients who fulfilled the diagnostic criteria of HLH-2004. The median age of patients was 60.0 (range 15-86). Underlying diseases were diagnosed in 17 patients. They consisted of malignant lymphoma (n=3), other neoplastic disease (n=3), viral infection (n=4), collagen vascular disease (n=3), Kikuchi's disease (n=3) and drug (n=1). Underlying diseases were not diagnosed in 17 patients despite examination. The treatments were steroids (n=18), dexamethasone + cyclosporine A (CSA) + etoposide (n=4), multidrug chemotherapy (n=2), steroids and CSA (n=3). Eleven patients died during observation. In a multivariate analysis, the significant predictor for death was age at onset (hazard ratio, 1.22; 95%CI, 1.02-1.44; P=0.027). Autopsy was performed in 4 cases, but the underlying disease remained unknown in 3 of those cases. Adult-onset HLH has high diversity and various outcomes. The mechanism of adult-onset HLH is not fully understood and further research is required.
1517	Typhoid Fever Complicated by Hemophagocytic Lymphohistiocytosis and Rhabdomyolysis.	26324725	Hemophagocytic lymphohistiocytosis (HLH) and rhabdomyolysis are rare complications of typhoid fever from Salmonella enterica serovar Typhi. Herein, we describe the clinical features in a 21-year-old female from India who presented to the intensive care unit with fever, severe pancytopenia, and rhabdomyolysis.
1518	Hemophagocytic Lymphohistiocytosis in Malignant Hematology: Uncommon but Should Not Be Forgotten?	26297268	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening syndrome of excessive immune activation and an inflammatory cytokine storm leading to multiple organ failure. We report our experience in a large tertiary referral center on HLH in the setting of hematologic malignancies and describe responses to therapy and outcomes. Seventeen cases of HLH were included in which the most common underlying diagnosis was aggressive lymphoma (n = 7). The median time from diagnosis of primary hematologic condition to HLH was 3.1 months. The most common presenting features were fever (n = 15), splenomegaly (n = 13), and transaminitis (n = 14). The mean serum ferritin level was 21,000 ng/mL. Fourteen patients demonstrated evidence of hemophagocytosis in bone marrow or other organs. Among all patients, 12 received etoposide, 14 received dexamethasone, and 3 received cyclosporine. Intrathecal chemotherapy was administered to 3 patients. Overall, 7 patients (41%) responded to treatment with clinical and laboratory improvement. The median overall survival (OS) from the time of HLH diagnosis and the primary hematologic diagnosis was 8.4 months and 29.5 months, respectively. The OS was better among patients with HLH with aggressive lymphomas (12 months). Response to treatment was associated with better OS. Recognition of manifestations and prompt diagnosis of HLH are crucial to initiate prompt therapy and improve outcome.
1519	Dimer of arfaptin 2 regulates NF-κB signaling by interacting with IKKβ/NEMO and inhibiting IKKβ kinase activity.	26296658	IκB kinases (IKKs) are a therapeutic target due to their crucial roles in various biological processes, including the immune response, the stress response, and tumor development. IKKs integrate various upstream signals that activate NF-κB by phosphorylating IκB and also regulate many proteins related to cell growth and metabolism. Although they function as a heteromeric complex comprised of kinase subunits and an adaptor, these kinases produce distinct cellular responses by phosphorylating different target molecules, suggesting that they may also be regulated in a subtype-specific manner. In this study, arfaptin 2 was identified as an IKKβ-specific binding partner. Interestingly, arfaptin 2 also interacted with NEMO. Domain mapping studies revealed that the C-terminal region, including the IKKβ HLH domain and the first coiled-coil NEMO region were respectively required for interactions with the arfaptin 2 N-terminal flexible region. Overexpression of arfaptin 2 inhibited tumor necrosis factor (TNF)-α-stimulated nuclear factor-κB (NF-κB) signaling, whereas downregulation of arfaptin 2 by small interfering RNA enhanced NF-κB activity. Dimerization of arfaptin 2 through the Bin-Amphiphysin-Rvs domain may be essential to inhibit activation of NF-κB through multimodal interactions with IKKβs or IKKβ/NEMO, as ectopic expression of the arfaptin 2 fragment responsible for IKK interactions did not change TNFα-stimulated NF-κB activation. These data indicate that arfaptin 2 is the first molecule to regulate NF-κB signaling by interacting with the functional IKK complex but not by direct inhibiting IKKβ phosphorylation.
1520	Multicenter study of combination DEP regimen as a salvage therapy for adult refractory hemophagocytic lymphohistiocytosis.	2628964126542249	Hemophagocytic lymphohistiocytosis (HLH) is a refractory immune disorder with a significant risk of death. Although standard therapy has dramatically improved survival in HLH patients, approximately 30%, especially adults, show no response to current treatment strategies. This prospective study aimed to investigate the efficacy of liposomal doxorubicin treatment combined with etoposide and methylprednisolone (doxorubicin-etoposide-methylprednisolone; DEP) as a salvage therapy for adult refractory HLH. Adult patients who did not achieve at least partial response 2 weeks after initial standard HLH therapy were enrolled in this study between June 2013 and June 2014. Response to salvage therapy was assessed at 2 and 4 weeks after initiation of DEP therapy and patients were followed until death or until November 2014. Sixty-three refractory HLH patients were enrolled, including 29 cases of lymphoma-associated HLH, 22 cases of Epstein-Barr virus-associated HLH, and 4 cases of familial HLH. There were 8 cases with unknown underlying diseases. Seventeen cases (27.0%) achieved complete response and 31 cases (49.2%) achieved partial response. The overall response was 76.2% (48/63). Patients who showed no response to DEP died within 4 weeks after salvage therapy. Twenty-nine of the 48 patients who achieved partial or complete response survived to subsequent chemotherapy, allogenic hematopoietic stem cell transplantation, or splenectomy. Our study suggests that DEP regimen is an effective salvage regimen for adult refractory HLH, which can prolong patient survival as we continue to understand the responsible mechanisms and bridge the gap between HLH and its underlying diseases. This study was registered in the Chinese Clinical Trials Registry Platform (http://www.chictr.org.cn/) as ChiCTR-IPC-14005514.
1521	Infection is the major trigger of hemophagocytic syndrome in adult patients treated with biological therapies.	26277577	Hemophagocytic syndromes (hemophagocytic lymphohistiocytosis, HLH) are characterized by a wide range of etiologies, symptoms, and outcomes, but have a common etiopathogenic pathway leading to organ damage: an excessive inflammatory response. Biological therapies have been proposed as a therapeutic option for refractory HLH, but have also been related to the development of HLH in severe immunosuppressed patients.The purpose of this study was to analyze the clinical characteristics and outcomes of adult patients who developed HLH after receiving biological therapies.We identified 30 patients (29 from the PubMed search and one unpublished case), including 19 women and 11 men, with a mean age of 46.5 years. Underlying diseases consisted of rheumatologic/autoimmune diseases in 24 patients and hematological neoplasia in the remaining 6. Biological agents received before the development of HLH were mainly anti-TNF agents (n = 19). Search for microorganisms confirmed systemic infection in 20 (67%) patients, including Mycobacterium tuberculosis (n = 5), cytomegalovirus (CMV) (n = 4), Epstein-Barr virus (EBV) (n = 3), Histoplasma capsulatum (n = 3), Escherichia coli (n = 2), Staphylococcus aureus, Leishmania amastigotes and Brucella melitensis (n = 1, respectively); viral infections were mainly reported in inflammatory bowel disease (IBD) patients. Patients with infections had more frequently received previous immunosuppressive therapies (p = 0.036) and had lower leukocyte counts (p = 0.020) in comparison with patients without associated infections. The outcome was described in 29 patients. After a mean follow-up of 6.3 months, 8 patients died (28%) and 6 had received anti-TNF agents. There was a high mortality rate in patients aged >65 years and those with tuberculosis (62% and 60%, respectively).In patients receiving biological therapies who develop HLH, searching for a concomitant infectious process is mandatory, and specific surveillance for EBV/CMV infections (in patients with IBD) and for bacteria, including mycobacteria (in elderly patients receiving anti-TNF therapy), is recommended.
1522	Chlamydia pneumoniae-related hemophagocytic lymphohistiocytosis in an adult patient with clonal karyotype abnormality.	26275276	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease characterized by a severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages. Herein, we report a 58-year-old male who had Chlamydia pneumoniae-related pneumonia, followed by aggressive HLH. An abnormal cytogenetic profile was also detected. To our knowledge, this is the first report of an adult patient with C. pneumonia-associated HLH.
1523	Central nervous system imaging findings of hemophagocytic syndrome.	26257114	We present a rare case of intracranial involvement in hemophagocytic lymphohistiocytosis (HLH) in an adult patient. MRI features in HLH may mimic those of other neoplastic, infectious, or inflammatory disorders. Key imaging findings correlate to central nervous system inflammation and include diffuse leptomeningeal enhancement, white matter changes with variable enhancement, hemorrhage, and restricted diffusion. Recognition of the imaging characteristic in correlation with clinical presentation, laboratory values, and biopsy findings is essential for making a correct diagnosis.
1524	Undiagnosed Systemic Lupus Erythematosus Presenting as Hemophagocytic Lymphohistiocytosis.	26236531	Hemophagocytic Lymphohistiocytosis (HLH) is rarely diagnosed in adults. Incidence is reported as one case per million persons per year. It can be triggered by conditions that affect immune homeostasis as infections, malignancies, and rheumatologic disorders. The following case demonstrates a rare instance in which undiagnosed systemic lupus erythematosus (SLE) presented as HLH. A 28-year-old male presented with progressive weakness and recurrent fevers for 2 months. Vital signs were within normal limits except for temperature of 100.3°F. His exam was unremarkable except for a left cervical scar and malar rash. His labs showed pancytopenia with neutropenia, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. Hemophagocytosis was present on bone marrow biopsy. All workup for a source of infection was negative. A tentative diagnosis of HLH was made based on clinical presentation and laboratory data. The patient was treated with an HLH protocol. Later, it was determined that his HLH was actually secondary to a primary diagnosis of SLE. The patient was treated for SLE with an immunosuppressive regimen of cyclosporine and dexamethasone, and he improved dramatically. HLH rarely presents due to a rheumatologic condition such as SLE. Physicians should consider testing for SLE in patients diagnosed with HLH.
1525	SomethiNG 2 talk about-Transcriptional regulation in embryonic and adult oligodendrocyte precursors.	26232072	Glial cells that express the chondroitin sulfate proteoglycan NG2 represent an inherently heterogeneous population. These so-called NG2-glia are present during development and in the adult CNS, where they are referred to as embryonic oligodendrocyte precursors and adult NG2-glia, respectively. They give rise to myelinating oligodendrocytes at all times of life. Over the years much has been learnt about the transcriptional network in embryonic oligodendrocyte precursors, and several transcription factors from the HLH, HMG-domain, zinc finger and homeodomain protein families have been identified as main constituents. Much less is known about the corresponding network in adult NG2-glia. Here we summarize and discuss current knowledge on functions of each of these transcription factor families in NG2-glia, and where possible compare transcriptional regulation in embryonic oligodendrocyte precursors and adult NG2-glia. This article is part of a Special Issue entitled SI:NG2-glia (Invited only).
1526	Ehrlichia-Induced Hemophagocytic Lymphohistiocytosis: A Case Series and Review of Literature.	26227842	Hemophagocytic lymphohistiocytosis (HLH) is a rare potentially fatal syndrome characterized by an uncontrolled hyperinflammatory response. The secondary form of HLH is usually triggered by a causative agent. Ehrlichia chaffeensis is a rare trigger of secondary HLH. We present a case series of five adolescents and adults diagnosed with Ehrlichia-induced HLH and we discuss their clinical and laboratory findings. We also review the literature for similar cases. Between October 2003 and June 2014, we identified 76 cases of HLH in adolescents and adults, 5 of which were induced by Ehrlichia. All 5 patients had fever, cytopenias, hypertriglyceridemia, and high ferritin. Hyperferritinemia was striking with a median admission ferritin of 47,290 μg/L (range: 2,863-85,517). In addition to the positive Ehrlichia PCR testing on peripheral blood of all patients, two patients with neurologic symptoms tested positive for E. chaffeensis in CSF specimens. Early treatment with doxycycline was effective. After a median follow up of 7.3 months, all patients were alive and none had recurrence of HLH. Clinicians should consider E. chaffeensis as a potential trigger for HLH especially in areas with tick activity. Prompt diagnosis and treatment with doxycycline are required for a better outcome.
1527	IL-10-592 A/C polymorphisms is associated with EBV-HLH in Chinese children.	26222684	The aim of this study is to investigate the relationship between cytokine gene polymorphisms and Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) in children, and to further reveal the possible mechanisms of EBV-HLH.Forty-one patients with EBV-HLH, 70 patients with infectious mononucleosis (IM), and 170 EBV-seropositive healthy children were evaluated. Gene polymorphism typing was performed by a polymerase chain reaction with a sequence-specific primer of a commercially available cytokine genotyping kit. Comparison of cytokine gene polymorphisms between EBV-HLH, IM patients, and healthy controls was analyzed statistically using Chi-square test or Fisher's exact test.The frequencies of IL-10-592 C allele or IL-10-592 CC genotype were significantly higher in patients with EBV-HLH than in IM and healthy children (P < 0.001), but no significant difference was observed between IM and healthy children.IL-10-592 locus gene polymorphism is associated with the development of EBV-HLH in Chinese children.
1528	Secondary haemophagocytic lymphohistiocytosis: Experience from the Uppsala University Hospital.	26212358	Haemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by fever, hepatosplenomegaly, cytopenia, and progressive multiple-organ failure. HLH in adults is often secondary to autoimmune diseases, cancer, or infections in contrast to familial HLH. Treatment of secondary HLH is directed against the triggering disease in addition to immunosuppressive therapy, the latter commonly according to the HLH-2004 protocol.We conducted a retrospective study to identify triggering diseases, disease-specific and immunosuppressive therapy administered, and prognosis in adult patients with secondary HLH. Patient data were collected from October 2010 to January 2015.Ten adult patients with secondary HLH were identified. Seven were men, and the median age at diagnosis was 62 years. Five cases were triggered by malignant disease and five by infection. The median patient fulfilled five of the eight HLH-2004 diagnostic criteria. All patients fulfilled the criteria fever, cytopenia, and ferritin >500 µg/L. Median time from hospital admission to HLH diagnosis was 20 days. Four patients received immunosuppressive therapy according to the HLH-2004 protocol. The prognosis was dismal, especially for the patients with malignancy-associated HLH, of whom all died.HLH should be suspected in patients who present with fever, cytopenia, and ferritin >500 µg/L. Secondary HLH has a dismal prognosis. None of the patients with HLH triggered by malignancy survived. Achieving remission of the triggering disease seems to be important for a favourable outcome as, in all surviving patients, the haemophagocytic syndrome resolved after remission of the underlying infection.
1529	Familial Hemophagocytic Lymphohistiocytosis Presenting as Hydrops Fetalis.	26199792	Background Familial hemophagocytic lymphohistiocytosis (FLH) is an autosomal recessive disorder of immune regulation that leads to a hyperinflammatory syndrome. Fetal onset FHL is extremely rare and is considered to be the most severe form of FHL. Case We report a preterm case of FHL that presented as hydrops fetalis. The infant was treated with a chemotherapy regimen based on the HLH-2004 protocol from the third day of life. However, he had persistent cytopenia and died on the 18th day of life due to bacteremia. The detection of defective perforin expression in the patient's natural killer cells and mutations in the PRF1 gene resulted in a molecular diagnosis of FHL. Conclusion We suggest that early diagnosis and the development of an appropriate immunosuppressive strategy that can induce and maintain remission until hematopoietic stem cell transplantation can be performed are required to improve the outcomes of fetal onset FHL.
1530	Hemophagocytic Lymphohistiocytosis and Gastrointestinal Bleeding: What a Surgeon Should Know.	26199785	This paper presents to the surgical community an unusual and often ignored cause of gastrointestinal bleeding. Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH) is a rare medical entity characterized by phagocytosis of red blood cells, leucocytes, platelets, and their precursors in the bone marrow by activated macrophages. When intestinal bleeding is present, the management is very challenging with extremely high mortality rates. Early diagnosis and treatment seem to be the most important factors for a successful outcome. We present two cases and review another 18 from the literature.
1531	Neurological involvement and characterization in acquired hemophagocytic lymphohistiocytosis in adulthood.	26198020	To describe the neurological and neuroradiological features of acquired hemophagocytic lymphohistiocytosis (HLH) in adulthood by reporting a series of cases.Ten consecutive patients who were diagnosed with HLH at Medstar Georgetown University Hospital and Walter Reed National Military Medical Center were evaluated for neurological involvement. All underwent clinical neurological evaluation, and when indicated CSF analysis and MR imaging of the brain. Data were gathered and analyzed retrospectively.Seven of the ten patients with HLH had neurological involvement. Mean age at onset was 50 (range: 21 to 73). Four patients were males. Prominent clinical features included mild to severe encephalopathy and seizures. Other findings included hemiparesis and spastic tetraparesis. Neuroimaging revealed a wide spectrum of abnormalities including cortical and subcortical edema, gadolinium enhancement, hemorrhage, and diffusion restriction. Basal ganglia involvement was present in four out of seven patients. Three patients died due to multisystem organ failure, and the other patients displayed varying degrees of recovery.The neurological features of acquired HLH in adults have not been previously reported. These seven patients demonstrate the spectrum of neurological involvement that can occur. The diagnosis of HLH should be considered in patients who are systemically ill with unexplained fevers and hyperferritinemia who have evidence of inflammation in the CNS.
1532	Prompt diagnosis and management of Epstein-Barr virus-associated post-transplant lymphoproliferative disorder and hemophagocytosis: A dreaded complication in a post-liver transplant child.	26184957	EBV-associated PTLD is increasingly recognized as an important cause of morbidity and mortality in both solid organ and hematopoietic stem cell transplant recipients. Mortality rates due to PTLD and virus-induced HLH are reported to be quite high. We report a case of EBV-associated PTLD and HLH in a child after liver transplantation who was successfully managed due to timely intervention. This case highlights that measurement of EBV load by quantitative polymerase chain reaction assays is an important aid in the surveillance and diagnosis of PTLD and early detection of EBV-induced PTLD, and aggressive treatment with rituximab is a key to survival in patients who have undergone liver transplantation.
1533	Spectrum of Atypical Clinical Presentations in Patients with Biallelic PRF1 Missense Mutations.	26184781	Perforin, encoded by PRF1, is a pore-forming protein crucial for lymphocyte cytotoxicity. Biallelic PRF1 nonsense mutations invariably result in early-onset hemophagocytic lymphohistiocytosis (HLH), termed familial HLH type 2 (FHL2). In contrast, biallelic PRF1 missense mutations may give rise to later-onset disease and more variable manifestations.We retrospectively searched our database for patients from families with siblings carrying biallelic PRF1 missense mutations where at least one sibling did not develop HLH, and for patients with biallelic PRF1 missense mutations and an atypical presentation of disease. We reviewed their clinical, genetic, and immunological characteristics.In all, we identified 10 such patients, including three sibling pairs with discordant manifestations. Interestingly, in two families, siblings of late-onset HLH patients developed Hodgkin lymphoma but no HLH. In a third family, one sibling presented with recurrent HLH episodes, whereas the other remains healthy. Of note, the affected sibling also suffered from systemic lupus erythematosus. Additional unrelated patients with biallelic PRF1 missense mutations were affected by neurological disease without classical signs of HLH, gastrointestinal inflammation as initial presentation of disease, as well as a hematological malignancy. Compared to early-onset FHL2 patients, the patients with an atypical presentation displayed a partial recovery of NK cell cytotoxicity upon IL-2 stimulation in vitro.Our findings substantiate and expand the spectrum of clinical presentations of perforin deficiency, linking PRF1 missense mutations to lymphoma susceptibility and highlighting clinical variability within families. PRF1 mutations should, therefore, be considered as a cause of several diseases disparate to HLH.
1534	[Association between gene polymorphisms of Perforin 1 and hemophagocytic lymphohistiocytosis].	26182270	To investigate frequency distribution of gene polymorphisms of PRF1 gene in children with hemophagocytic lymphohistiocytosis (HLH), and to explore whether the possible gene polymorphisms of PRF1 gene confer an increased risk of susceptibility to HLH.Forty-eight children who were diagnosed with HLH between January 2009 and December 2013 (HLH group) and 100 healthy children (control group) were enrolled in this study. The gene polymorphisms in the coding region of PRF1 gene, which consists of three exons and two introns, were genotyped by PCR, followed by direct sequencing.Three single nucleotide polymorphisms (SNPs) were revealed in the coding sequence of PRF1 in the 48 children with HLH. Seven SNPs were detected in the noncoding sequence. Other two SNPs in the noncoding sequence including rs10999426 and rs10999427 were detected only in 5 healthy children (5%). There was no significant difference in allelic frequencies of all the SNPs above between the HLH and control groups (P>0.05). Haplotype analysis showed there was a pair-wise linkage disequilibrium between rs10999426 and rs10999427 (D=1, r2=1), but there was no significant difference in the distribution of A-T haplotype between the HLH and control groups (P>0.05).There is no association between gene polymorphisms of PRF1 gene and the susceptibility to HLH. There is a pair-wise linkage disequilibrium between rs10999426 and rs10999427, but a low detection rate of A-T haplotype in healthy children indicates that it might not play a protective role in the development of HLH.
1535	[Plasmapheresis in haematology].	26181153	Plasmapheresis also known as a therapeutic plasma exchange (TPE) is a procedure of plasma removal with it's ineligible plasma's component. Usually it is a supportive measure used simultaneously with the treatment, but in a few diseases, e.g. in trombotictrombocytopenic purpura (TTP), it is a first-choice treatment. During the plasmapheresis plasma is mostly replaced by 20% solution of albumin or combination of 20% solution of albumin and 0.9% solution of NaCl, however in some diseases fresh frozen plasma (FFP) is used. Plasmaphereses have found a wide application in different branches of medicine: hematology, neurology, nephrology, reumatology. Plasmapheresis is an invasive procedure, but when performed by qualified staff it is rather safe and serious complications are very rare.The most common complications of plasmapheresis are mild, usually caused by electrolyte disturbances (hypokalemia, hypokalcemia) or anticoagulation. More serious complication can be associated with FFP transfusion, extracorporeal circulation or presence of intravenous catheter. The latter one is usually necessary to perform the plasmapheresis. In haematology the most common indication for plasmapheresis is the supportive treatment of multiple myeloma. The procedure is performed in patients with high protein levels endangered with hyperviscosity syndrome. Less frequent indications to plasmapheresis in haematology are: Waldenström's macroblobulynaemia, idiopathic thrombocytopenic purpura (ITP), pure red cell aplasia (PRCA), polyneuropaties connected with haematological disorders. Supportive treatment of haemofagocytic syndrome (HLH--hemophagocytic lymphohistiocytosis) is one of the new indications. Plasmaphereses are used in treatment of about 150 different diseases and more and more new needs for this method are identified.
1536	Epstein-Barr virus infection-Related hemophagocytic lymphohistiocytosis.	26180435	We report a case of 27-year-old female diagnosed with hemophagocytic lymphohistiocytosis (HLH) following a recent Epstein-Barr virus (EBV) infection. A known case of relapsing remitting multiple sclerosis on corticosteroids for last 6 months presented to the critical care unit with fever, maculopapular rash and difficulty in breathing. A rapid and correct diagnosis with the precise treatment led to complete recovery of this patient. The HLH is a rare complication of primary EBV infection.
1537	[Brucellosis as a cause of hemophagocytic syndrome].	26167830	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of excessive inflammation and tissue destruction due to abnormal immune activation and inflammation. HLH can occur primarily due to genetic etiology, or secondarily associated with malignancies, autoimmmune diseases or infections. There are a number of reports that revealed the relationship of hemophagocytosis with brucellosis. In this report, we described a brucellosis-related HLH case. A 73-year-old male who work as farmer was admitted to our hospital with the complaints of fever continuing for 10 days, loss of appetite and back pain. Physical examination revealed right upper quadrant tenderness and hepatomegaly. Since the patient exhibited five of the diagnostic criteria for HLH (fever, hepatosplenomegaly, bicytopenia, hypertriglyceridemia and high ferritin level), he was diagnosed as secondary HLH. PCR, microscopic agglutination and indirect fluorescent antibody tests gave negative results for the diagnosis of Crimean-Congo hemorrhagic fever, leptospirosis and Q fever, respectively. On the other hand, Rose Bengal test for brucellosis was positive, while standard tube agglutination test (STA) was negative. The patient's serum yielded a very high positive (1/1280) result when Coombs' test was performed in terms of the possibility of blocking antibodies or prozone phenomenon. Additionally, B.melitensis was isolated from his blood culture on the sixth day. The patient was treated with doxycycline and rifampicin, and on the 10th day of antibiotic therapy the patient was discharged and recommended to complete his treatment up to 6 weeks. In conclusion, in patients with secondary HLH symptoms especially in the endemic areas, brucellosis should be considered as a predisposing infection.
1538	LIF is a new p53 negative regulator.	26161442	Leukemia inhibitory factor (LIF), a cytokine that belongs to the interleukin-6 family, regulates multiple important biological functions. Recently, we found that LIF is an important negative regulator of p53 in human colorectal cancer cells. LIF negatively regulates p53 protein levels and functions by activation of the Stat3 signaling pathway, which in turn induces the expression of ID1, the helix-loop-helix (HLH) protein inhibitor of differentiation and DNA binding. ID1 increases MDM2 expression at both mRNA and protein levels to accelerate p53 protein degradation. Overexpression of LIF increases chemoresistance of cultured colorectal cancer cells and colorectal xenograft tumors in a largely p53-dependent manner. Furthermore, LIF is overexpressed in a large percentage of human colorectal cancer specimens and LIF overexpression is associated with a poor prognosis in colorectal cancer patients. Our study revealed a new role of LIF in tumorigenesis through regulation of the p53 signaling pathway.
1539	Dimerization-driven degradation of C. elegans and human E proteins.	26159995	E proteins are conserved regulators of growth and development. We show that the Caenorhabditis elegans E-protein helix-loop-helix-2 (HLH-2) functions as a homodimer in directing development and function of the anchor cell (AC) of the gonad, the critical organizer of uterine and vulval development. Our structure-function analysis of HLH-2 indicates that dimerization drives its degradation in other uterine cells (ventral uterine precursor cells [VUs]) that initially have potential to be the AC. We also provide evidence that this mode of dimerization-driven down-regulation can target other basic HLH (bHLH) dimers as well. Remarkably, human E proteins can functionally substitute for C. elegans HLH-2 in regulating AC development and also display dimerization-dependent degradation in VUs. Our results suggest that dimerization-driven regulation of bHLH protein stability may be a conserved mechanism for differential regulation in specific cell contexts.
1540	G1/S Inhibitors and the SWI/SNF Complex Control Cell-Cycle Exit during Muscle Differentiation.	2614431826186182	The transition from proliferating precursor cells to post-mitotic differentiated cells is crucial for development, tissue homeostasis, and tumor suppression. To study cell-cycle exit during differentiation in vivo, we developed a conditional knockout and lineage-tracing system for Caenorhabditis elegans. Combined lineage-specific gene inactivation and genetic screening revealed extensive redundancies between previously identified cell-cycle inhibitors and the SWI/SNF chromatin-remodeling complex. Muscle precursor cells missing either SWI/SNF or G1/S inhibitor function could still arrest cell division, while simultaneous inactivation of these regulators caused continued proliferation and a C. elegans tumor phenotype. Further genetic analyses support that SWI/SNF acts in concert with hlh-1 MyoD, antagonizes Polycomb-mediated transcriptional repression, and suppresses cye-1 Cyclin E transcription to arrest cell division of muscle precursors. Thus, SWI/SNF and G1/S inhibitors provide alternative mechanisms to arrest cell-cycle progression during terminal differentiation, which offers insight into the frequent mutation of SWI/SNF genes in human cancers.
1541	Efficacy and Safety of Pergoveris in Assisted Reproductive Technology--ESPART: rationale and design of a randomised controlled trial in poor ovarian responders undergoing IVF/ICSI treatment.	26141305	The results of a recent meta-analysis showed that adding recombinant human luteinising hormone (r-hLH) to recombinant human follicle-stimulating hormone (r-hFSH) for ovarian stimulation was beneficial in poor responders, resulting in a 30% relative increase in the clinical pregnancy rate compared with r-hFSH monotherapy. However, a limitation of the meta-analysis was that the included studies used heterogeneous definitions of poor ovarian response (POR). Furthermore, the use of r-hLH supplementation during ovarian stimulation is a topic of ongoing debate, and well-designed, adequately powered, multicentre, randomised controlled trials in this setting are warranted. Therefore, the objective of the ESPART trial is to explore the possible superiority of a fixed-dose combination of r-hFSH plus r-hLH over r-hFSH monotherapy in patients with POR, as per a definition aligned with the European Society of Human Reproduction and Embryology (ESHRE) Bologna criteria.Phase III, randomised, single-blind, parallel-group trial in women undergoing in vitro fertilisation and/or intracytoplasmic sperm injection. Approximately 946 women aged 18-<41 years from 18 countries will be randomised (1:1) to receive a fixed-dose combination of r-hFSH plus r-hLH in a 2:1 ratio (Pergoveris) or r-hFSH monotherapy (GONAL-f). The primary end point is the total number of retrieved oocytes per participant. Secondary end points include: ongoing pregnancy rate, live birth rate, implantation rate, biochemical pregnancy rate and clinical pregnancy rate. Safety end points include: incidence and severity of ovarian hyperstimulation syndrome, and of adverse events and serious adverse events.The study will be performed in accordance with ethical principles that have their origin in the Declaration of Helsinki, with the International Conference on Harmonisation-Good Clinical Practice guidelines and all applicable regulatory requirements. All participants will provide written informed consent prior to entry. The results of this study will be publically disseminated.ClinicalTrials.gov identifier: NCT02047227; EudraCT Number: 2013-003817-16; Clinical Trial Protocol Number: EMR200061-005 V.3.0, 15 April 2014.
1542	[Treatment outcomes and prognostic analysis of 61 Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis].	26134018	To investigate the outcomes, survival status, and the prognostic factors of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) patients.A retrospective study was carried out to analyze the clinical data of 61 EBV-HLH cases, from January 2008 to July 2014. Prognostic factors were analyzed through COX model (single factor and multiple factors).A total of 246 patients with HLH were diagnosed, among which 102 cases were with EBV infection (including 61 EBV-HLH, 36 lymphoma associated HLH, 5 primary HLH. Among the 61 cases, 40 were male and 21 were female, with a ration of 1.9:1. The median age was 28 years (range, 12-78). 1, 3, 6 and 12-month overall survival rates of 61 EBV-HLH were 65.6%, 47.5%, 32.4%, and 25.0%, respectively. The median follow-up time was 3 (0.5-28) months. 12 patients didn't use etoposide within 4 weeks after diagnosis, while HLH-94 protocol was used in 33 patients and HLH-2004 protocol was used in 16 patients. Response rates of theses three groups were 33.3%, 51.5%, and 43.8%, respectively (P=0.401). There was statistically difference between the group without etoposide and the HLH-94/ HLH-2004 group in the overall survival rate (P=0.033). Serum albumin level (P=0.033) and whether EBV could became negative (P=0.010) were independent predictors for EBV-HLH.EBV-HLH patients have severe clinical feature and poor prognosis. Early application of immune chemotherapy based on etoposide can improve survival. Serum albumin level and whether EBV can become negative are independent prognostic factors for survival.
1543	Pre-B-cell colony-enhancing factor is markedly elevated in childhood hemophagocytic lymphohistiocytosis.	26125724	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome involving a final common pathway of hypercytokinemia, in which tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and soluble interleukin 2-receptor-alpha (sIL-2Rα) are the key cytokines. Pre-B-cell colony-enhancing factor (PBEF) is an inflammatory cytokine involved in several inflammatory diseases. However, its role in HLH is unknown. In this study, we examined the role of PBEF in HLH. Plasma was collected from 22 children with HLH and 14 healthy children. The concentrations of plasma PBEF, TNF-α, IFN-γ, and sIL-2Rα were determined using an enzyme-linked immunosorbent assay. All clinical data were derived from medical records. In the acute phase, children with HLH had much higher PBEF, TNF-α, IFN-γ, and sIL- 2Rα levels than did healthy children (P < 0.05). After treatment, 13 HLH children improved and PBEF, TNF-α, and IFN-γ levels decreased to normal levels (P < 0.05); sIL-2Rα levels also decreased (P < 0.05), but remained above the normal level (P < 0.05). Two patients were lost to follow-up, while 7 patients showed a bad response to therapy and eventually died, showing high PBEF levels above those of the survivors (P < 0.01). PBEF level was significantly positively correlated with TNF-α, IFN-γ, sIL-2Rα, serum ferritin, and triglycerides (all P < 0.05), and was negatively correlated with fibrin (P < 0.05). PBEF appears to be involved in the inflammatory process of HLH, and elevated PBEF is related to disease activity. We are currently evaluating the role of PBEF as a marker for the diagnosis and management of patients.
1544	Hemophagocytic lymphohistiocytosis with a leukemoid reaction in an infant with scrub typhus.	26123173	A two-month-old male infant presented with a clinical picture suggestive of fever without focus. Treatment was initiated with broad spectrum intravenous antibiotics. The infant subsequently developed septic shock, hepatosplenomegaly and tachypnoea with oxygen dependency. There was laboratory evidence of anemia, thrombocytopenia and a leukemoid reaction. Infection induced hemophagocytic lymphohistiocytosis with a leukemoid reaction was suspected because of fever, splenomegaly and bicytopenia in conjunction with elevated serum triglycerides and ferritin. Empiric therapy with doxycycline caused a rapid resolution of the fever, and the diagnosis of scrub typhus was confirmed by a positive scrub IgM ELISA. HLH with a leukemoid reaction secondary to scrub typhus has not previously been reported in early infancy.
1545	Septic Arthritis of the Hip in the Setting of Hemophagocytic Lymphohistiocytosis: A Case Report.	29252856	A twenty-two-month-old boy with septic hip arthritis had persistent elevated inflammatory markers and daily fevers despite multiple antibiotic regimens and repeated surgical debridements yielding negative cultures. After exhaustive work-up for other infectious, rheumatologic, and immunologic etiologies, he met diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) and developed cultures positive for fungal hip arthritis. Following treatment for HLH and fungal hip arthritis, he improved and was discharged.No previous report in the literature specifically associates HLH with septic hip arthritis, to our knowledge. Surgeons should suspect underlying immunologic deficiencies and atypical infectious causes of septic arthritis when usual treatment modalities have failed.
1546	Patients with an extraordinarily elevated serum ferritin: think of haemophagocytic lymphohistiocytosis.	26098856	We retrospectively analysed charts of patients with blood ferritin level >5000 µg/l. The aim of the study was to look for the likelihood of haemophagocytic lymphohistiocytosis (HLH) in these patients.Forty-two patients demonstrated hyperferritinaemia and could be evaluated. The diagnosis of HLH was based on a recently published HScore and an earlier diagnostic algorithm.According to the algorithm, 20 patients fulfilled the criteria for a diagnosis of HLH. However, patients with Still's disease have macrophage activation and, in this context, a rise in ferritin without having HLH. Fourteen patients with carcinoma, haematological malignancies or infection and hyperferritinaemia remained. Signs and symptoms were: systemic inflammatory response syndrome (SIRS 100%), fever (95%), cytopenia of ≥2 lines (70%), immunosuppression (61.5%), splenomegaly (50%), elevated liver enzymes (45%), lymphadenopathy (35%), hepatomegaly (30%). These are nonspecific parameters. Therefore HLH may be overdiagnosed. Using the HScore, only 10 patients had >80% probability of having HLH. Patients demonstrating cytopenia of ≥2 cell lines had a >60% mortality rate. Time to death was 13.8 days; death was most often due to multiorgan failure.HScore reflects a higher specificity than the algorithm for diagnosing HLH. The discrepancy may indicate the difficulty that a specific marker still is missing. Hyperferritinaemia was strongly associated with HLH in patients with haematological or oncological malignancies. HLH may be underdiagnosed because the majority of these patients suffer from a severe underlying disease, which easily might suggest a flare or infection. In this population, hyperferritinaemia and SIRS should rise suspicion because mortality in HLH is high.
1547	Hemophagocytic lymphohistiocytosis: an unusual complication of leprosy.	26094649	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition of chaotic uncontrolled immune system stimulation and not fully understood pathophysiology. Most reported cases of hemophagocytic syndrome in patients with mycobacterial infections have been associated with Mycobacterium tuberculosis. As far as we could ascertain, to date, no established HLH case complicating leprosy has been published in the medical literature.We describe here a new case of Hansen's disease in a 58-year-old Tunisian man with an unusual complicated clinical course documented as hemophagocytic syndrome. Cutaneous and neurological involvements were the main clinical signs of Hansen's disease. Histological findings suggested the diagnosis of leprosy and were somewhat more characteristic of the lepromatous leprosy type. While on antileprosy treatment, he developed unexplained persistent fever, organomegaly, bicytopenia, and elevated rate of inflammatory markers with bone marrow aspirate showing large macrophages with increased phagocytosis of mature and immature blood elements, typical features of hemophagocytic syndrome.A high index of suspicion is essential for prompt diagnosis of hemophagocytic syndrome in the setting of disseminated infection such as leprosy.
1548	Clinical features and correct diagnosis of macrophage activation syndrome.	26082353	Macrophage activation syndrome (MAS) is increasingly recognized among febrile hospitalized patients. Clinically, MAS resembles multiorgan dysfunction and shock. Laboratory features include hepatobiliary dysfunction, coagulopathy, pancytopenia, hyperferritinemia and markers of immune activation. Pathologically, hemophagocytosis is commonly seen but is only present in 60% of MAS patients. MAS, or secondary hemophagocytic lymphohistiocytosis (HLH), is triggered by infectious (e.g., herpes family viruses), rheumatologic (e.g., systemic lupus erythematosus [SLE]) and oncologic (e.g., T-cell leukemia) conditions. Formal HLH criteria, while specific, are frequently insensitive for MAS diagnosis. Thus, disease-specific (e.g., SLE) and generic MAS criteria have been published. Recently, novel criteria for MAS in children with systemic juvenile idiopathic arthritis (sJIA) were developed and are a key focus of this review.
1549	Atypical Transcriptional Activation by TCF via a Zic Transcription Factor in C. elegans Neuronal Precursors.	26073017	Transcription factors of the TCF family are key mediators of the Wnt/β-catenin pathway. TCF usually activates transcription on cis-regulatory elements containing TCF binding sites when the pathway is active and represses transcription when the pathway is inactive. However, some direct targets display an opposite regulation (activated by TCF in the absence of Wnt), but the mechanism behind this atypical regulation remains poorly characterized. Here, we use the cis-regulatory region of an opposite target gene, ttx-3, to dissect the mechanism of this atypical regulation. Using a combination of genetic, molecular, and biochemical experiments, we establish that, in the absence of Wnt pathway activation, TCF activates ttx-3 expression via a Zic binding site by forming a complex with a Zic transcription factor. This mechanism is later reinforced by specific bHLH factors. This study reveals an atypical mode of action for TCF that may apply to other binary decisions mediated by Wnt signaling.
1550	Hemophagocytic lymphohistiocytosis due to acute primary herpes simplex virus 1 infection.	26071327	NaN
1551	Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus.	26068074	The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.
1552	Approach to clinical syndrome of jaundice and encephalopathy in tropics.	26041951	A large number of patients present with jaundice and encephalopathy in tropical country like India and acute liver failure is the usual cause. Clinical presentation like ALF is also a complication of many tropical infections, and these conditions may mimic ALF but may have subtle differences from ALF. Moreover, what hepatologists see as acute liver failure in tropics is different from what is commonly described in Western Textbooks. Paracetamol overdose, which is possibly the commonest cause of ALF in UK and USA, is hardly ever seen in India. Most common etiology here is viral hepatitis (hepatitis E > hepatitis B> hepatitis A). Apart from ALF, one may also come across subacute hepatic failure (SAHF) as well as acute-on-chronic liver failure (ACLF) due to viral hepatitis. Interestingly, a host of other conditions can mimic ALF because clinical presentation in these conditions can be dominated by jaundice and encephalopathy. Malarial hepatopathy is possibly the best-known condition out of these and is not an uncommon manifestation of severe malaria. A similar presentation can also be seen in other common infections in tropics such as dengue fever, typhoid fever, leptospirosis, scrub typhus, amoebic liver abscesses, tuberculosis and other bacterial and fungal infections with or without human immunodeficiency virus (HIV) related disease. In many of these conditions, liver failure may not be underlying pathophysiology. Some pregnancy related liver diseases could also present with jaundice and encephalopathy. This review summarizes the commonly seen presentations in tropical country like India, where jaundice and encephalopathy dominate the clinical picture.
1553	EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles.	26032645	The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.
1554	[Expression of CD163 in children with Epstein-Barr virus infection].	26014702	To study the clinical significance of CD163 in the diagnosis and the evaluation of severity and prognosis of childhood hemophagocytic lymphohistiocytosis (HLH).Ninety-four children were classified into Epstein-Barr virus (EBV)-positive (n=55) and EBV-negative groups (n=39; control group). The EBV-positive group was subgrouped into infectious mononucleosis (IM; n=47) and HLH (n=8). Serum levels of soluble CD163 were measured using ELISA. Expression of CD163 on mononuclear cells was detected by flow cytometry.The serum levels of soluble CD163 were>10 000 ng/mL in all eight HLH patients (>30 000 ng/mL in 3 cases). The mean serum levels of soluble CD163 in the HLH group were significantly higher than in the control and IM groups (P<0.05). The serum levels of soluble CD163 in EBV-positive children were positively correlated with EBV-DNA copies and serum levels of ferritin and LDH, but were negatively correlated with white blood cell count, neutrophil count, hemoglobin and platelet count. The follow-up after treatment for three HLH patients showed that serum levels of soluble CD163 were significantly reduced, but the soluble CD163 levels rebounded in one patient who was complicated by fungal pneumonia infection.The levels of serum soluble CD163 may be related to the severity in children with HLH. The EBV-positive children with soluble CD163 levels >10 000 ng/mL should be considered the possibility of HLH.
1555	Hemophagocytic lymphohistiocytosis following dengue hemorrhagic fever in Hb H/Hb Constant Spring patient.	26011764	Infection-associated hemophagocytic syndrome (IAHS), a secondary form of hemophagocytic lymphohistiocytosis (HLH), has been found following several types of infections and can be fatal. We report herein a case of IAHS following dengue infection in a 14-year-old patient with underlying α-thalassemia syndrome (non-deletional Hb H/Hb Constant Spring disease). He developed prolonged fever, thrombocytopenia, and progressive splenomegaly. Further investigations indicated hyperferritinemia, and increased reactive histiocytes with hemophagocytic activity in the bone marrow. He responded promptly to dexamethasone and i.v. immune globulin. Physicians should be aware of this condition, especially in countries where both dengue hemorrhagic fever and thalassemia are prevalent. The fatal outcome of IAHS can be prevented with prompt appropriate treatment.
1556	Cerebellar swelling due to familial hemophagocytic lymphohistiocytosis: An unusual presentation.	26004995	Cerebellar swelling with obstructive hydrocephalus is a rare but life threatening condition, associated with different etiologies, familial hemophagocytic lymphohistiocytosis (HLH) being rarely one of them.2-year-7-month old boy presented with irritability, cerebellar dysfunction, and somnolence. Brain MRI showed marked diffuse cerebellar swelling and obstructive hydrocephalus with mild tonsillar herniation. Laboratory testing revealed pancytopenia, elevated liver enzymes, elevated ferritin and triglycerides levels and decreased fibrinogen. The diagnosis of familial HLH was confirmed by the presence of homozygous missense mutation of Syntaxin 11 gene. The child was treated with HLH-2004 protocol of chemotherapy followed by allogenic stem cell transplantation. His neurological condition improved significantly after treating the underlying disease.Cerebellar swelling is a rare manifestation of familial HLH. High degree of clinical suspicion may allow a timely diagnosis and appropriate therapy.
1557	Evaluating the optimal serum ferritin level to identify hemophagocytic lymphohistiocytosis in the critical care setting.	25997871	Ferritin is known to be involved in numerous physiological roles, such as iron storage, as well as various pathological conditions and in generalized inflammatory states. Hyperferritinemia is also encountered in the setting of hemophagocytic lymphohistiocytosis (HLH). Current diagnostic criteria exist to define HLH based on several clinical and biochemical markers, including the serum ferritin level. In this study, we retrospectively evaluated the value of ferritin >500 ng/mL in diagnosing HLH in 344 consecutive patients admitted to the medical intensive care unit at our hospital. Nine cases of HLH were identified. Comparison of the HLH with the non-HLH group showed that their maximum median serum ferritin level was 25,652 (range 1977-100,727 ng/mL) versus 1180 (503-85,168 ng/mL) (P < 0.001), platelets were 30 (5-92 × 10(3)/μL) versus 113 (0-507 × 10(3)/μL) (P < 0.001), absolute neutrophil counts were 2.56 (0.02-23.7 × 10(3)/μL) versus 7.7 (0.01-82.7 × 10(3)/μL) (P = 0.002), and triglycerides were 255 (156-394 mg/dL) versus 127 (17-624 mg/dL) (P = 0.002), respectively. Using a receiver operating characteristic curve, the optimal maximum serum ferritin level for the diagnosis of HLH was 3951 ng/mL, exceeding the current diagnostic cutoff set forth in the HLH-2004 guidelines. These data suggest that a higher cutoff value of ferritin level may have improved utility in the diagnosis of secondary HLH in the critical care setting.
1558	Interactional role of microRNAs and bHLH-PAS proteins in cancer (Review).	25997457	MicroRNAs (miRNAs) are recognized as an emerging class of master regulators that regulate human gene expression at the post-transcriptional level and are involved in many normal and pathological cellular processes. Mammalian basic HLH (helix-loop-helix)-PER-ARNT-SIM (bHLH-PAS) proteins are heterodimeric transcriptional regulators that sense and respond to environmental signals (such as chemical pollutants) or to physiological signals (for instance hypoxia). In the normal state, bHLH-PAS proteins are responsible for multiple critical aspects of physiology to ensure the cell accurate homeostasis, but dysregulation of these proteins has been shown to contribute to carcinogenic events such as tumor initiation, promotion, and progression. Increasing epidemiological and experimental studies have shown that bHLH-PAS proteins regulate a panel of miRNAs, whereas some miRNAs also target bHLH-PAS proteins. The interaction between miRNAs and certain bHLH-PAS proteins [hypoxia-inducible factor (HIF) and aryl hydrocarbon receptor (AHR)] is relevant to many vital events associated with tumorigenesis. This review will summarize recent findings on the interesting and complicated underlying mechanisms that miRNAs interact with HIFs or AHR in tumors, hopefully to benefit the discovery of novel drug-interfering targets for cancer therapy.
1559	Exploring the role of bone marrow increased FDG uptake on PET/CT in patients with lymphoma-associated hemophagocytic lymphohistiocytosis: a reflection of bone marrow involvement or cytokine storm?	25989865	The role of 18F-2-fluoro-2-deoxy-D-glucose (FDG) uptake of bone marrow (BM) on positron emission tomography/computed tomography (PET/CT) in patients with lymphoma-associated hemophagocytic lymphohistiocytosis (LA-HLH) remains uncertain. This retrospective study included 34 LA-HLH patients who underwent both PET/CT and comprehensive BM examinations prior to treatment. Comparison between PET/CT and BM examinations for the assessment of bone marrow involvement (BMI) indicated statistical difference (p = 0.039). The specificity of PET/CT in detecting BMI was 11.1% compared to BM examinations. However, a significant correlation was found between PET parameters of BM and laboratory parameters associated with HLH, such as C-reactive protein, ferritin, fibrinogen and soluble CD25. By multivariate analysis, PET parameters of marrow were significantly associated with overall survival. The findings suggest that FDG uptake of marrow might fail to detect lymphomatous BMI, but reflected the level of cytokine storm to a certain extent and might be a prognostic factor in patients with LA-HLH.
1560	Applying Multiple Criteria Decision Analysis to Comparative Benefit-Risk Assessment: Choosing among Statins in Primary Prevention.	25986470	Decision makers in different health care settings need to weigh the benefits and harms of alternative treatment strategies. Such health care decisions include marketing authorization by regulatory agencies, practice guideline formulation by clinical groups, and treatment selection by prescribers and patients in clinical practice. Multiple criteria decision analysis (MCDA) is a family of formal methods that help make explicit the tradeoffs that decision makers accept between the benefit and risk outcomes of different treatment options. Despite the recent interest in MCDA, certain methodological aspects are poorly understood. This paper presents 7 guidelines for applying MCDA in benefit-risk assessment and illustrates their use in the selection of a statin drug for the primary prevention of cardiovascular disease. We provide guidance on the key methodological issues of how to define the decision problem, how to select a set of nonoverlapping evaluation criteria, how to synthesize and summarize the evidence, how to translate relative measures to absolute ones that permit comparisons between the criteria, how to define suitable scale ranges, how to elicit partial preference information from the decision makers, and how to incorporate uncertainty in the analysis. Our example on statins indicates that fluvastatin is likely to be the most preferred drug by our decision maker and that this result is insensitive to the amount of preference information incorporated in the analysis.
1561	Transcription factor hlh-2/E/Daughterless drives expression of α integrin ina-1 during DTC migration in C. elegans.	25982859	Integrins are involved in a vast number of cell behaviors due to their roles in adhesion and signaling. The regulation of integrin expression is of particular interest as a mechanism to drive developmental events and for the role of altered integrin expression profiles in cancer. Dynamic regulation of the expression of integrin receptors is required for the migration of the distal tip cell (DTC) during gonadogenesis in Caenorhabditis elegans. α integrin ina-1 is required for DTC motility, yet is up-regulated by an unknown mechanism. Analysis of the promoter for α integrin ina-1 identified two E-box sequences that are required for ina-1 expression in the DTC. Knockdown of transcription factor hlh-2, an established E-box binding partner and ortholog of E/Daughterless, prevented expression of a transcriptional fusion of the ina-1 promoter to RFP and blocked DTC migration. Similarly, knockdown of hlh-2 also prevented expression of a translational fusion of the genomic ina-1 gene to GFP while blocking DTC migration. Knockdown of HLH-2 binding partner MIG-24 also reduced ina-1 expression and DTC migration. Overall, these results show that the transcription factor hlh-2 is required for up-regulation of ina-1 at the onset of DTC migration.
1562	Synergistic defects of UNC13D and AP3B1 leading to adult hemophagocytic lymphohistiocytosis.	25980904	A 32-year-old man of non-consanguineous Chinese parentage, with high-grade fever, rash, joint pain, nausea, and vomiting, was diagnosed as adult-onset still's disease at his initial admission. Although prednisone had been taken, the patient presented with recurrent high-grade fever, rash, splenomegaly, hypertriglyceridemia, cryptogenic hepatitis, apparently elevated levels of serum ferritin(>20,000 μg/L), which met the proposed HLH diagnostic criteria, 2009. Sequence analysis of genomic DNA from the patient's peripheral blood demonstrated heterozygous for UNC13D mutation: c. 1232 G>A, and AP3B1 mutation: c. 1075 A>G, which were predicted to be pathogenic. Unfortunately, at the time, molecular confirmation results for HLH were obtained, and this patient had died from progressive HLH disease with multiple organ dysfunction syndrome caused by shock. FHL should be considered in the differential diagnosis of adults who present with adult-onset still's disease-like symptoms.
1563	Inhibition of Daughterless by Extramacrochaetae mediates Notch-induced cell proliferation.	25977368	During development, the rate of cell proliferation must be constantly monitored so that an individual tissue achieves its correct size. Mutations in genes that normally promote tissue growth often result in undersized, disorganized and non-functional organs. However, mutations in genes that encode growth inhibitors can trigger the onset of tumorigenesis and cancer. The developing eye of the fruit fly, Drosophila melanogaster, has become a premier model system for studies that are focused on identifying the molecular mechanisms that underpin growth control. Here, we examine the mechanism by which the Notch pathway, a major contributor to growth, promotes cell proliferation in the developing eye. Current models propose that the Notch pathway directly influences cell proliferation by regulating growth-promoting genes such as four-jointed, cyclin D1 and E2f1. Here, we show that, in addition to these mechanisms, some Notch signaling is devoted to blocking the growth-suppressing activity of the bHLH DNA-binding protein Daughterless (Da). We demonstrate that Notch signaling activates the expression of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) transcription factor. Emc, in turn, then forms a biochemical complex with Da. As Emc lacks a basic DNA-binding domain, the Emc-Da heterodimer cannot bind to and regulate genomic targets. One effect of Da sequestration is to relieve the repression on growth. Here, we present data supporting our model that Notch-induced cell proliferation in the developing eye is mediated in part by the activity of Emc.
1564	[Analyses of 77 patients with hemophagocytic syndrome].	25976050	To summarize the clinical features and laboratory data of 77 patients with hemophagocytic syndrome (HPS).A total of 77 patients of HPS were continuously collected from 2007 to 2014 at our hospital. Their underlying diseases, clinical features, laboratory data, therapy and prognosis were analyzed.Their The patients aged from 3 months to 85 years. The gender ratio was roughly equal. Primary HPS was diagnosed in only 5 cases by gene detection Another 72 cases belonged to secondary HPS. The causes were infection (n = 28), hematologic neoplasm (n = 25), autoimmune diseases (AID, n = 11) and unknown (n = 8). HPS was the initial symptom in nearly half cases of hematologic neoplasm and AID. HPS was characterized by high fever (100%), hypersplenomegaly (81.8%) and lymphadenopathy (40.3%). Laboratory data showed cytopenia (94.8%), serum ferritin elevation (93.2%), hypofibrinogenemia (61.8%), hemophagocytosis in bone marrow (78.1%) and hypertriglyceridemia (55.3%). Low NK-cell activity (95.2%) and elevation of sCD25 (100%) were specific manifestations in HPS. Pulmonary infection (36.4%) and hepatic malfunction (33.3%) were common. Approximately 70% were treated with HLH-2004. Pulse-dose corticosteroid therapy (methylprdnisolone 200-500 mg/d) was used in 8 AID patients. And 14 patients died and 10 withdrew treatment because of exacerbation. Five had complications of DIC and another 5 progressed into MODS. Neoplasm (52.0%) had the highest mortality in secondary HPS. And infection (25.0%) and AID (18.2%) followed.Sometimes HPS occurs simultaneously with autoimmune disease or neoplasm. Relevant laboratory tests for suspected patients may aid an early diagnosis. Presence of DIC or MODS in HPS is possibly correlated with a poor prognosis and a high mortality.
1565	Hemophagocytic lymphohistiocytosis in children with visceral leishmaniasis.	25970110	Acquired hemophagocytic lymphohistiocitosis (HLH) syndrome can be a complication of visceral leishmaniasis (VL). A multicenter prospective study was conducted to determine the frequency of HLH syndrome in children with VL. Twenty-four children with VL were identified, and 10 (41%) developed HLH syndrome. VL should be ruled out in all children with HLH criteria living in or coming from endemic areas.
1566	Novel Association of Killer Cell Immunoglobulin-like Receptor Genes with EBV-infectious Diseases in Children.	25966757	Killer cell immunoglobulin-like receptors (KIRs) which are mainly expressed on natural killer (NK) cells are implicated in many virus infections. However, it is unclear whether or not KIRs are associated with susceptibility to Epstein-Barr virus (EBV) infection related diseases. Therefore, the purpose of our study was to investigate possible correlation between polymorphisms of KIR genes and infectious mononucleosis (IM)/EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The polymorphisms of KIR genes were detected by polymerase chain reaction with sequence-specific primers (PCR-SSP). The results would contribute to clarify the association of KIRs with EBV induced diseases, and provide new insights into the role of NK cells and innate immune response against viral infections and/or subsequent progression.
1567	Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides.	25960929	Glycans from six highly purified hFSH preparations were released by peptide-N-glycanase digestion and analyzed by negative mode nano-ESI mass spectrometry before and after neuraminidase digestion. Pituitary glycan structures were mainly high-mannose, di-, tri-, and tetra-antennary, and their abundance largely paralleled that reported by other investigators using different approaches. For most of the FSH preparations, the differences in glycosylation appeared to be restricted to relative abundances of the major glycan families, as defined by their neutral core oligosaccharide structures. Qualitative differences between glycan populations were largely relegated to those species that were lowest in abundance. Significant qualitative differences were noted in two cases. Recombinant GH3-hFSH triantennary glycans appeared to have the third antenna exclusively on the mannose6-branch, in contrast to all pituitary and urinary hFSH triantennary glycans, in which this antenna was exclusively attached to the mannose3-branch. The hypo-glycosylated hFSH preparation isolated from purified hLH was decorated with high mannose glycans that accounted for over 40% of the total in this population. As this preparation was found to be consistently 20-fold more active than hFSH24 in FSH receptor-binding assays, it appears that both macroheterogeneity and microheterogeneity in FSH preparations need to be taken into account.
1568	An uncommon presentation of EBV-driven HLH. Primary or secondary? An ongoing dilemma.	25948855	Haemophagocytic lymphohistiocytosis (HLH) is a potentially fatal syndrome, mainly characterised by dysregulated immune activation. The syndrome is related to a genetic cause, in the classic primary form, or to identified triggers such as infections, malignancy or rheumatological processes, in the classic secondary form. Epstein-Barr virus (EBV) is the most common agent implicated in hereditary and non-hereditary conditions. We describe a 23-month-old girl who experienced severe clinical deterioration with respiratory distress due a bilateral pleural effusion within the first week of primary EBV infection. Fever, generalised oedema and hepatosplenomegaly, along with a pruritic morbilliform erythematous rash, were the first clinical signs. Respiratory impairment followed with hypoxaemia and the patient was admitted to the intensive care unit for thoracocentesis. Further investigation showed persistent bicytopaenia, hypertriglyceridaemia, hyperferritinaemia and elevated α chain of interleukin-2 receptor (sCD25). Diagnostic criteria for HLH were fulfilled. Therapy was instituted with dexamethasone, ciclosporin A and intravenous immunoglobulin 6 days after admission with progressive clinical recovery.
1569	Analysis of prognostic risk factors in children with Epstein-Barr virus-associated hemophagocytic syndrome.	25941132	This study aims to analyze the prognostic risk factors in children with Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis (EBV-HLH).Seventy-four EBV-HLH patients were divided into two groups according to the specificity: clinical remission after four-week inductive therapy group and active disease group; death group and survival group. The risk factors that affect early efficacy and prognosis were analyzed.Overall survival rate of the 74 children was 75.7%, while the recurrence rate was 13.5%. The one-year survival rate was 71.4±5.6%, and the three-year survival rate was 65.9±6.6%, with a median survival rate of 40±19.9 months. The multivariate logistic regression analysis showed that age was the primary risk factor that affected the first 4 weeks alleviation, and the severity of splenomegaly and WBC level upon hospitalization were the risk factors that affected the prognosis. Patients with spleen>4 cm had shorter survival time than those with spleen≤4 cm, and patients with WBC≥3×10(9)/L had longer survival time than those with WBC<3×10(9)/L, which exhibited significant differences.Age negatively influences the early remission of EBV-HLH. WBC<3×10(9)/L and spleen>4 cm exhibited high correlation with the prognosis of EBV-HLH.
1570	HIV infection presenting proliferation of CD8+ T lymphocyte and hemophagocytic lymphohistiocytosis.	25941054	Hemophagocytic lymphohistiocytosis is a rare hyperinflammatory disorder characterised by CD8+ T lymphocyte activation and hypercytokinemia. Autoimmune disorders including hemophagocytic lymphohistiocytosis have been described in HIV patients; however, it is a rare initial presentation of HIV infection. We present an unusual case of HIV infection presenting with hemophagocytic lymphohistiocytosis.
1571	Malignancy-associated haemophagocytic lymphohistiocytosis in children and adolescents.	25940575	Haemophagocytic lymphohistiocytosis (HLH) in the context of malignancy is mainly considered a challenge of adult haematology. While this association is also observed in children, little is known regarding inciting factors, appropriate treatment and prognosis. We retrospectively analysed 29 paediatric and adolescent patients for presenting features, type of neoplasm or preceding chemotherapy, treatment and outcome. Haemophagocytic lymphohistiocytosis was considered triggered by the malignancy (M-HLH) in 21 patients, most of whom had T- (n = 12) or B-cell neoplasms (n = 7), with Epstein-Barr virus as a co-trigger in five patients. In eight patients, HLH occurred during chemotherapy (Ch-HLH) for malignancy, mainly acute leukaemias (n = 7); an infectious trigger was found in seven. In M- and Ch-HLH, median overall survival was 1·2 and 0·9 years, and the 6 month survival rates were 67% and 63%, respectively. Seven of 11 deceased M-HLH patients exhibited active malignancy and HLH at the time of death, while only two out of five deceased Ch-HLH patients had evidence of active HLH. To overcome HLH, malignancy- and HLH-directed treatments were administered in the M-HLH cohort; however, it was not possible to determine superiority of one approach over the other. For Ch-HLH, treatment ranged from postponement of chemotherapy to the use of etoposide-containing regimens.
1572	Laboratory parameters identify familial haemophagocytic lymphohistiocytosis from other forms of paediatric haemophagocytosis.	2592144329896892	Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of immune dysregulation and is classified as primary or secondary according to the underlying aetiology. The treatment strategies recommended for these two groups differ substantially; however, it is thought to be impossible to predict the underlying causes of HLH using conventional laboratory tests. Recent studies show that serum levels of soluble interleukin-2 receptor (sIL2R) and ferritin are useful for differentiating some forms of HLH. The present study reports that combinations of common laboratory parameters, such as the percentage of total lymphocytes within the peripheral blood leucocyte population, serum levels of lactate dehydrogenase and the sIL2R/ferritin ratio, are useful for identifying patients with familial haemophagocytic lymphohistiocytosis and for differentiating the underlying aetiology of paediatric HLH during the early course of the disease. These findings suggest that the pathogenesis of HLH differs greatly in terms of innate and adaptive immunity depending on the aetiology and may provide a new approach to unravelling the complex pathophysiology underlying this syndrome.
1573	Truth Telling and Treatment Strategies in End-of-Life Care in Physician-Led Accountable Care Organizations: Discrepancies Between Patients' Preferences and Physicians' Perceptions.	25906093	Providing patient-centered care from preventive medicine to end-of-life care in order to improve care quality and reduce medical cost is important for accountable care. Physicians in the accountable care organizations (ACOs) are suitable for participating in supportive end-of-life care especially when facing issues in truth telling and treatment strategy. This study aimed to investigate patients' attitudes toward truth telling and treatment preferences in end-of-life care and compare patients' attitudes with their ACOs physicians' perceptions.This nationwide study applied snowball sampling to survey physicians in physician-led ACOs and their contracted patients by questionnaire from August 2010 to July 2011 in Taiwan. The main outcome measures were beliefs about palliative care, attitudes toward truth telling, and treatment preferences.The data of 314 patients (effective response rate = 88.7%) and 177 physicians (88.5%) were analyzed. Regarding truth telling about disease prognosis, 94.3% of patients preferred to be fully informed, whereas only 80% of their physicians had that perception (P < 0.001). Significant differences were also found in attitudes toward truth telling even when encountering terminal disease status (98.1% vs 85.3%). Regarding treatment preferences in terminal illness, nearly 90% of patients preferred supportive care, but only 15.8% of physicians reported that their patients had this preference (P < 0.001).Significant discrepancies exist between patients' preferences and physicians' perceptions toward truth telling and treatment strategies in end-of-life care. It is important to enhance physician-patient communication about end-of-life care preferences in order to achieve the goal of ACOs. Continuing education on communication about end-of-life care during physicians' professional development would be helpful in the reform strategies of establishing accountable care around the world.
1574	Acquired hemophagocytic syndrome related to parainfluenza virus infection: case report.	25886447	We present the case of a patient with acquired hemophagocytic syndrome secondary to parainfluenza virus infection, a complication that has not, to the best of our knowledge, been previously reported.A 33-year-old Chilean man with fever secondary to parainfluenza 2 virus infection developed progressive cholestasis, hepatosplenomegaly, cytopenia and an increased ferritin level (>2000 IU/L). A bone marrow analysis showed hemophagocytosis. Our patient received HLH-94 chemotherapy, and he achieved complete and sustained remission after a two-year follow-up, without the need for hematopoietic stem cell transplantation.Hemophagocytic syndrome is a severe disease with high mortality. A high index of suspicion is essential to improve survival. A viral etiology is frequent and although Epstein-Barr virus is the most frequently associated, other viruses like parainfluenza can cause this disease.
1575	Haemophagocytic lymphohistiocytosis associated with Mycobacterium tuberculosis infection.	25870214	Haemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal condition that can be primary or secondary. Secondary HLH can occur in association with infections, most commonly viral infections, but has also been reported in association with Mycobacterium tuberculosis (TB). Prompt identification of the underlying cause of HLH is important as it guides treatment decisions. Early initiation of appropriate treatment (eg, anti-TB treatment) reduces morbidity and mortality. We present a case of HLH associated with TB infection. Initial TB investigations were negative and standard combination chemoimmunotherapy for HLH resulted in a limited clinical response. On apparent relapse of HLH, further investigation revealed TB with changes on CT chest, granuloma on bone marrow and eventual positive TB culture on bronchoalveolar lavage. Subsequent treatment with quadruple anti-TB treatment resulted in rapid clinical response and disease remission. We advocate continued monitoring for TB infection in patients with HLH, and prophylaxis or full treatment for those at high risk.
1576	Hemophagocytic lymphohistiocytosis secondary to Varicella zoster infection in a child with Henoch-Schönlein purpura.	25868958	Hemophagocytic lymphohistiocytosis (HLH) is a fatal, hyper-inflammatory syndrome that is characterized by untimely activation of macrophages, and manifests as cytopenia, organ dysfunction, and coagulopathy. Secondary HLH can be associated with infection, drugs, malignancy, and transplantation, and is mostly triggered by infection. Herein, we report the case of a patient with Henoch-Schönlein purpura (HSP) who developed severe HLH secondary to Varicella zoster infection.
1577	Conditions associated with extreme hyperferritinaemia (>3000 μg/L) in adults.	25851400	The clinical significance of extreme hyperferritinaemia has come under scrutiny with the increasing recognition of haemophagocytic lymphohistiocytosis (HLH) in adults. Most studies of hyperferritinaemia have focused on serum ferritin greater than 1000 μg/L, often in ambulatory patients. The conditions associated with more extreme hyperferritinaemia are poorly understood.To examine conditions associated with extreme hyperferritinaemia greater than 3000 μg/L in acutely ill adults at a quaternary care hospital.Patients with serum ferritin greater than 3000 μg/L at Vancouver General Hospital between 1 August 2011 and 1 August 2012 were identified. Those over 18 years of age and with clinical data available were included in the study.Eighty-three subjects were identified. Twenty-one cases (25%) were due to transfusional iron overload, 16 (19%) due to liver disease and 15 (18%) due to mixed factors. Haemophagocytic lymphohistiocytosis (HLH) was diagnosed in six of 83 patients (7%) with ferritin greater than 3000 μg/L, but six of eight patients (75%) with ferritin greater than 20 000 μg/L.Extreme hyperferritinaemia greater than 3000 μg/L is uncommon in adult patients. The highest serum ferritin values are seen in HLH, but the differential diagnosis for serum ferritin greater than 3000 μg/L remains broad with iron overload and liver disease being the most common causes.
1578	Primary lymphoma of the brain in a young man whose brother died of hemophagocytic lymphohistiocytosis: case report.	25845254	We represent the unique occurrence of primary central nervous system lymphoma (PCNSL) in a patient whose brother died of genetically confirmed hemophagocytic lymphohistiocytosis (HLH).We report a case of a 25-year-old male patient with primary aggressive diffuse large B-cell lymphoma affecting the brain and PCNSL. Despite one year of medical treatment outcome was lethal. However, our patient had a relatively longer survival compared to median survival time for PCNSL. Additionally, he had two older brothers who died at the age of about 11 years. One died of fulminate malignancy, shortly after pediatric admission, before the diagnosis could be established. The other one died from genetically confirmed (perforin mutation/PRF1) HLH. Our patient was heterozygous carrier of perforin mutation representing the genetic marker for HLH. Our patient's father was the carrier of the same mutation but had no symptoms of any disease.This case points at the presence of HLH and diffuse large B-cell PCNSL in brothers. Extensive assessment of patients with probable PCNSL and familial HLH is necessary, including genetic analysis for HLH.
1579	Disseminated histoplasmosis associated with acquired hemophagocytic lymphohistiocytosis.	25838912	Hemophagocytic lymphohistiocytosis (HLH) is a potentially life-threatening clinical syndrome caused by uncontrolled activation of lymphocytes and histiocytes resulting in high levels of cytokines. Acquired HLH occurs in autoimmune, inflammatory, infectious, and immunosuppressive disorders. Prompt identification and treatment of an underlying triggering cause improves clinical outcome.
1580	Hemophagocytic lymphohistiocytosis arising in a child with Langerhans cell histiocytosis.	25818970	Langerhans cell histiocytosis (LCH) is characterized by the proliferation of clonal dendritic cells, while hemophagocytic lymphohistiocytosis (HLH) is an extreme inflammatory process sustained by the uncontrolled activation of macrophages. HLH can be primary or secondary, the latter arising in infectious, autoimmune or neoplastic disorders. We hereby present a young girl who developed secondary HLH while being treated for relapsed multisystem LCH under the LCH III Protocol. She fulfilled 5 of 8 HLH-2004 criteria (fever, splenomegaly, pancytopenia, ferritin level >500 μ/l and sIL-2R >2400 IU/ml) and was successfully treated by the HLH-2004 Protocol for secondary HLH. She remains in good health, apart from insipid diabetes she developed as a complication of LCH. Considering that the occurrence of HLH in LCH patients has been reported before, the case history presented here yields additional support for the hypothesis that the pathogenesis of the two histiocytoses--LCH and HLH--may indeed overlap to a considerable extent.
1581	Hemophagocytic lymphohistiocytosis in a female patient due to a heterozygous XIAP mutation and skewed X chromosome inactivation.	25801017	Genetic forms of hemophagocytic lymphohistiocytosis (HLH) are caused by mutations in autosomal recessive genes affecting perforin-dependent cytotoxic function and two X-linked genes affecting distinct cell signaling pathways: SH2D1A and XIAP. HLH caused by mutations in X-linked genes is typically found only in males. Here we report the occurrence of HLH in a female caused by a heterozygous mutation in XIAP. Flow cytometric studies confirmed the absence of XIAP protein expression, while an X chromosome inactivation assay revealed an extreme skewing ratio of 99:1. This finding demonstrates that females are susceptible to X-linked forms of HLH through skewed X chromosome inactivation.
1582	Abnormalities of the lymphocyte subsets and their immunophenotype, and their prognostic significance in adult patients with hemophagocytic lymphohistiocytosis.	25800135	Hemophagocytic lymphohistiocytosis (HLH) is a final common pathway resulting from diverse immune processes. Most of the current understanding of HLH is based on studies involving pediatric patients (pHLH). Adult HLH (aHLH) is distinct from pHLH, with less frequent genetic basis, higher frequency of chemo-resistance and a higher mortality. Immunological processes underlying aHLH are poorly understood. So far, no immunophenotypic abnormalities associated with aHLH have been described, and their etiopathologic and prognostic significance has not been explored. We reviewed all adult patients with bone marrow hemophagocytosis and identified 21 who fulfilled the criteria for HLH. We then analyzed abnormalities in their lymphocyte subsets and immunophenotype and tested association of these abnormalities with survival. Twenty of 21 patients showed abnormalities in either lymphocyte subsets and/or immunophenotype: 10 showed increased CD8+ cells and decreased CD4:CD8 ratio, 8 had decreased CD3+ cells, 3 each had increased CD56+ cells, increased CD7-/CD4+ cells, and increased CD3+/CD4-/CD8- (DN) cells, and one had increased CD3-/CD4+ cells. Six of 10 patients with increased numbers of CD8+ cells and decreased CD4:CD8 ratio are alive, compared to 2/11 with normal values (p = 0.0385). On the other hand, all 8 patients with decreased CD3+ cells are dead, compared to 8/13 with normal numbers (p = 0.0417). Those who survived were younger than those who did not (median, range: 29 years, 19-88, vs 62 years, 24-81; p = 0.0272). In conclusion, most aHLH patients show diverse abnormalities in either lymphocytes and/or immunophenotype. Young age, increased numbers of CD8+ cells or decreased CD4:CD8 ratio are favorably associated with survival, while a decreased number of CD3+ cells is adversely associated with survival in aHLH patients.
1583	Chronic granulomatous disease: Review of a cohort of Egyptian patients.	25796307	Chronic granulomatous disease (CGD) is an inherited disease that results from a defect in the phagocytic cells of the immune system. It is caused by defects in one of the major subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. The clinical presentations of CGD patients are heterogeneous.This is the first report from Egypt discussing clinical and laboratory data of twenty-nine patients (from 26 families) with CGD from a single tertiary referral centre.There were twenty male and nine female patients. The consanguinity rate was 76% (19/25). Their age of diagnosis ranged from 2 to 168 months with a mean of 52.8 months ± 49.6 SD. The most common manifestations were abscesses in 79.3% (deep organ abscesses in 37.9% of patients), followed by pneumonia in 75.8% and gastrointestinal symptoms in 27.5%. Rare but fatal complications were also reported among patients as one patient developed haemophagocytic lymphohistiocytosis (HLH) syndrome. Although X linked-CGD universally constitutes the most common pattern of inheritance; only 6 of our patients 6/25 (24%) belonged to this group with a Stimulation Index (SI) of 1-5, and confirmed by carrier pattern of their mothers. Mothers were not available for testing in four male children. Nineteen patients (76%) had autosomal recessive patterns; ten males and nine females patients based on having abnormal SI, positive history of consanguinity and their mothers showing normal SI.Increasing the awareness of physicians about symptoms of CGD may lead to earlier diagnosis of the disease, thus enhancing proper management and better quality of life.
1584	Inhibitor of differentiation 4 (ID4) acts as an inhibitor of ID-1, -2 and -3 and promotes basic helix loop helix (bHLH) E47 DNA binding and transcriptional activity.	25778840	The four known ID proteins (ID1-4, Inhibitor of Differentiation) share a homologous helix loop helix (HLH) domain and act as dominant negative regulators of basic-HLH transcription factors. ID proteins also interact with many non-bHLH proteins in complex networks. The expression of ID proteins is increasingly observed in many cancers. Whereas ID-1, ID-2 and ID-3, are generally considered as tumor promoters, ID4 on the contrary has emerged as a tumor suppressor. In this study we demonstrate that ID4 heterodimerizes with ID-1, -2 and -3 and promote bHLH DNA binding, essentially acting as an inhibitor of inhibitors of differentiation proteins. Interaction of ID4 was observed with ID1, ID2 and ID3 that was dependent on intact HLH domain of ID4. Interaction with bHLH protein E47 required almost 3 fold higher concentration of ID4 as compared to ID1. Furthermore, inhibition of E47 DNA binding by ID1 was restored by ID4 in an EMSA binding assay. ID4 and ID1 were also colocalized in prostate cancer cell line LNCaP. The alpha helix forming alanine stretch N-terminal, unique to HLH ID4 domain was required for optimum interaction. Ectopic expression of ID4 in DU145 prostate cancer line promoted E47 dependent expression of CDKNI p21. Thus counteracting the biological activities of ID-1, -2 and -3 by forming inactive heterodimers appears to be a novel mechanism of action of ID4. These results could have far reaching consequences in developing strategies to target ID proteins for cancer therapy and understanding biologically relevant ID-interactions.
1585	Changes in the gene expression profile of the bladder cancer cell lines after treatment with Helix lucorum and Rapana venosa hemocyanin.	25778314	The purpose of this study was to elucidate the mechanism of action of the Helix lucorum hemocyanin (HlH), b-HlH-h, and RvH2-g hemocyanins as potential agents against bladder cancer.We evaluated the viability of 647-V, T-24, and CAL-29 bladder cancer cell lines after treatment with the tested hemocyanins. The cell viability was measured at 72 hrs with MTT and WST-1 assays. Acridine orange/propidium iodide double staining was used to discriminate between apoptotic and necrotic cells. Gene expression profiling of the 168 genes from human inflammatory cytokines and signal transduction pathways were performed on the tumor cells before and after hemocyanins' treatment.The results showed decreased survival of cancer cells in the presence of HlH and two functional units: b-HlH-h and RvH2-g. Acridine orange/propidium iodide double staining revealed that the decreased viability was due to apoptosis. The gene expression data showed upregulation of genes involved in the apoptosis as well as of the immune system activation, and downregulation of the CCL2, CCL17, CCL21, CXCL1, and ABCF1 genes.The present study is the first to report gene expression in human cells under the influence of hemocyanins. The mechanism of antitumor activity of the HlH, b-HlH-h, and RvH2-g hemocyanins includes induction of apoptosis. In addition to the antiproliferative effect, downregulation of the genes with metastatic potential was observed. Together with the already known immunogenic effect, these findings support further studies on hemocyanins as potential therapeutic agents against bladder cancer.
1586	HEB associates with PRC2 and SMAD2/3 to regulate developmental fates.	25775035	In embryonic stem cells, extracellular signals are required to derepress developmental promoters to drive lineage specification, but the proteins involved in connecting extrinsic cues to relaxation of chromatin remain unknown. We demonstrate that the helix-loop-helix (HLH) protein, HEB, directly associates with the Polycomb repressive complex 2 (PRC2) at a subset of developmental promoters, including at genes involved in mesoderm and endoderm specification and at the Hox and Fox gene families. While we show that depletion of HEB does not affect mouse ESCs, it does cause premature differentiation after exposure to Activin. Further, we find that HEB deposition at developmental promoters is dependent upon PRC2 and independent of Nodal, whereas HEB association with SMAD2/3 elements is dependent of Nodal, but independent of PRC2. We suggest that HEB is a fundamental link between Nodal signalling, the derepression of a specific class of poised promoters during differentiation, and lineage specification in mouse ESCs.
1587	The Role of Id2 Protein in Neuroblatoma in Children.	25771836	Id (DNA binding and/or differentiation) proteins occur physiologically during ontogenesis and negatively regulate the activity of other helix-loop-helix (HLH) proteins. Id2 protein causes block of cells differentiation in the S phase of the cell cycle and regulates the activity of Rb protein. The role of Id2 protein in physiological cell cycle progression and in neuroblastoma (NBL) pathogenesis was proposed by Lasorella. The aim of the study was evaluation of Id2 expression and its prognostic significance in NBL cells coming from primary tumors and evaluation of its prognostic significance, and correlation of Id2 expression with known prognostic factors. Sixty patients with primary NBL treated from 1991 to 2005 were included in the analysis. We found 50 patients with high and 10 patients with low intensity of Id2 expression. The median percentage of NBL cells with Id2 expression was 88 %. We found no correlation between the number of NBL cells or the intensity of Id2 expression and OS and DFS. In patients with stage 4 NBL, almost all patients had high expression of Id2 and it was significantly more common than in other disease stages (p = 0,03). We found no correlation between Id2 expression and other known prognostic factor in NBL patients. We assume that Id2 is not prognostic factor. However, due to its abundant expression in most of NBL cells and its role in cell cycle, it may be potential therapeutic target. Exact knowledge of expression time may be helpful in explaining mechanisms of oncogenesis.
1588	Tocilizumab as an Adjuvant Therapy for Hemophagocytic Lymphohistiocytosis Associated With Visceral Leishmaniasis.	25768375	Leishmaniasis is important as a cause of hemophagocytic lymphohistiocytosis (HLH) and must be considered and excluded in patients with HLH because it can cause severe or even fatal complications. When HLH is present, there is a deficient downregulation of the immune response, leading to an uncontrolled inflammation. We report a case of visceral leishmaniasis-HLH where the therapy with tocilizumab, targeting interleukin 6, help to regulate the immune response for the infection of Leishmania.
1589	Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-γ.	25767156	To study the role of IFN-γ in the pathogenesis of systemic JIA (sJIA) and haemophagocytic lymphohistiocytosis (HLH) by searching for an IFN-γ profile, and to assess its relationship with other cytokines.Patients with inactive (n = 10) and active sJIA (n = 10), HLH [n = 5; of which 3 had sJIA-associated macrophage activation syndrome (MAS)] and healthy controls (n = 16) were enrolled in the study. Cytokines and IFN-γ-induced genes and proteins were determined in plasma, in patient peripheral blood mononuclear cells (PBMCs) and in lymph node biopsies of one patient during both sJIA and MAS episodes. IFN-γ responses were investigated in healthy donor PBMCs, primary fibroblasts and endothelial cells.Plasma IFN-γ, IL-6 and IL-18 were elevated in active sJIA and HLH. Levels of IFN-γ and IFN-γ-induced proteins (IP-10/CXCL-10, IL-18BP and indoleamine 2,3-dioxygenase) in HLH were much higher than levels in active sJIA. Free IL-18 and ratios of IL-18/IFN-γ were higher in active sJIA compared with HLH. HLH PBMCs showed hyporesponsiveness to IFN-γ in vitro when compared with control and sJIA PBMCs. Endothelial cells and fibroblasts expressed IFN-γ-induced proteins in situ in lymph node staining of a MAS patient and in vitro upon stimulation with IFN-γ.Patients with active sJIA and HLH/MAS show distinct cytokine profiles, with highly elevated plasma levels of IFN-γ and IFN-γ-induced proteins typically found in HLH/MAS. In addition to PBMCs, histiocytes, endothelial cells and fibroblasts may contribute to an IFN-γ profile in plasma. Increasing levels of IFN-γ compared with IL-18 may raise suspicion about the development of MAS in sJIA.
1590	Hemophagocytic lymphohistiocytosis secondary to infections: a tropical experience!	25766345	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyper inflammatory condition, if not recognized and treated in time. A high index of suspicion can help identify the condition early. This condition can occur in the primary or secondary form. Secondary HLH or hemophagocytic syndrome (HPS) secondary to infections is an important clinical entity especially in tropical world. In this article, we share our experience with this entity and make an attempt to explore literature about ravenous macrophages which occurs secondary to infections. It is a series of six cases of HLH secondary to infectious disease in our center in a coastal city in South India over last one year with follow up.
1591	Elevated serum adenosine deaminase levels in secondary hemophagocytic lymphohistiocytosis.	25764155	Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition caused by activated T cells and macrophages. Adenosine deaminase (ADA) plays an important role in immune regulation, especially in the proliferation, maturation, and differentiation of lymphocytes. Its role has been studied in inflammation and malignancy. Here, we present for the first time the alteration of ADA in secondary HLH (sHLH).Serum ADA levels were measured in 20 cases with lymphoma-associated hemophagocytic syndrome (LAHS), 15 with infection-associated hemophagocytic syndrome (IAHS), six with macrophage activation syndrome (MAS) as experimental group. Additionally, we enrolled 20 cases with lymphoma, 15 with infection, six with autoimmunity who were all not associated with HLH as conditional control group and 20 healthy subjects as blank group. We also demonstrated the ADA levels in 20 LAHS cases and 21 benign disease-associated HLH cases (IAHS and MAS).Serum ADA levels were significantly higher in patients with LAHS, IAHS, and MAS compared to the healthy subjects (P < 0.001) and conditional control group (P < 0.05). Serum ADA levels of patients with LAHS were significantly higher than benign disease-associated HLH cases (P < 0.05). The optimum ADA cutoff point for LAHS was 89.25 U/L, with a sensitivity and specificity of 85.0% and 76.2%, respectively.Serum ADA levels were increased in sHLH suggesting a partial role of activated T-cell response in the disease pathophysiology. Serum ADA levels were particularly higher in LAHS and probably be a potential indicator of underlying lymphoma in sHLH patients.
1592	[Association of CCR2 gene rs1799864 polymorphism with hemophagocytic lymphohistiocytosis in children].	25760842	To investigate the association between rs1799864 single nucleotide polymorphism (SNP) of the C-C chemokine receptor 2 (CCR2) gene and susceptibility of hemophagocytic lymphohistiocytosis (HLH) in children.The clinical and laboratory data of 86 children diagnosed with HLH between January 2007 and December 2013 were retrospectively reviewed. The CCR2 gene rs1799864 was genotyped by SNaPshot technique in 86 HLH children and 128 healthy controls. The genotypic and allelic frequencies in the two groups were comparatively analyzed.No significant difference either in genotypic or allelic frequencies of rs1799864 polymorphism of the CCR2 gene was observed between HLH patients and controls (P>0.05), but there were significant differences in the age of onset and the periods of temperature and platelet returning to normal after treatment (P<0.05).There is no association between CCR2 gene rs1799864 polymorphism and the risk for HLH in children. However, the genotypic differences of this polymorphism might be associated with clinical characteristics and prognosis of HLH.
1593	How I treat hemophagocytic lymphohistiocytosis in the adult patient.	25758828	Hemophagocytic lymphohistiocytosis (HLH) is a devastating disorder of uncontrolled immune activation characterized by clinical and laboratory evidence of extreme inflammation. This syndrome can be caused by genetic mutations affecting cytotoxic function (familial HLH) or be secondary to infectious, rheumatologic, malignant, or metabolic conditions (acquired HLH). Prompt recognition is paramount and, without early treatment, this disorder is frequently fatal. Although HLH is well described in the pediatric population, less is known about the appropriate work-up and treatment in adults. Here, we review the clinical characteristics, diagnosis, and treatment of HLH in adults.
1594	Clinical characteristics and prognostic factors of adult hemophagocytic syndrome patients: a retrospective study of increasing awareness of a disease from a single-center in China.	25757854	Hemophagocytic lymphohistiocytosis (HLH) is a relatively rare but life-threatening disease with confusing clinical manifestations, rapidly deteriorating health, high morbidity and mortality.To improve the recognition as well as understanding of this disorder, we analyzed clinical characteristics and prognostic factors from 85 adult patients diagnosed with HLH in our hospital from April 2005 to June 2014.Patients with HLH displayed variable clinical markers across a wide spectrum. These included fever and hyperferritinemia (100%), elevated lactate dehydrogenase (LDH) (98.8%), two or three cytopenia (92.2%), splenomegaly (72.9%), hypofibrinogenemia (69.4%), hypertriglyceridemia (64.7%), hemophagocytosis (51.7%), and hepatomegaly (24.7%). Patients with active Epstien-Barr Virus (EBV) infection had a median overall survival (OS) of 65 days. Those displaying malignancy had very poor survival (median OS: 40 days). However, patients in rheumatic and non-EBV infection groups had relatively superior prognosis (not reached). Univariate analysis showed that Fibrinogen (Fbg) <1.5 g/L, platelet number (PLT) <40 × 10(9)/L and LDH ≥1000 U/L were factors that negatively affected survival (P = 0.004, 0.000, 0.002). Multivariate analysis showed that PLT <40 × 10(9)/L was the independent adverse factor (HR = 0.350, 95% CI: 0.145-0.844, P = 0.019).HLH had very complex clinical manifestations and high death rate. Patients with active EBV infection, malignancy, Fbg <1.5 g/L, PLT <40 × 10(9)/L and LDH ≥1000 U/L had high risk of death as well as inferior survival, and these patients require systemic targeted treatments as early as possible.
1595	Epstein-barr virus-related hemophagocytic lymphohistiocytosis: hematologic emergency in the critical care setting.	25755898	Hemophagocytic lymphohistiocytosis (HLH) is a rare and potential life-threatening clinical syndrome that results from uncontrolled activation of the immune system. Secondary HLH, more commonly observed in adult patients, is seen in the context of underlying triggering conditions. Epstein-Barr virus (EBV) has been recognized as the leading infectious cause and is associated with a poor outcome. As clinical and laboratory features of HLH could overlap with septic shock syndrome in most patients, the diagnosis of HLH, especially in adults, is the most challenging aspect of the disease that results in delayed recognition and treatment of rapidly progressive multiorgan system failure. We report a case of Hemophagocytic lymphohistiocytosis in a patient who presented with signs of septic shock syndrome and we review the literature on the topic.
1596	Hemophagocytic Lymphohistiocytosis (HLH) in Children Presenting as Liver Disease.	25755554	Hemophagocytic lymphohistiocytosis (HLH) is a rare acute hyperinflammatory condition presenting with high fever, pancytopenia, splenomegaly with the pathologic finding of hemophagocytic lymphohistiocytosis in bone marrow and other tissues. Predominant hepatic manifestations at presentation are rare. We report a series of three cases which showcase the spectrum of liver disease as presentation in hemophagocytic lymphohistiocytosis.
1597	Cytology of secondary hemophagocytic lymphohistiocytosis masquerading as lymphoma in a nonimmunocompromised adult.	25745299	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune disorder which is uncommon in a nonimmunocompromised adult. A 27-year-old female who presented with fever, hematuria, generalized lymphadenopathy was clinically suspected to have lymphoma was subjected to fine needle aspiration of cervical lymph nodes. Cytology of lymph node had numerous histiocytes with phagocytosed lymphocytes, red blood cells and nuclear debris. A diagnosis of secondary HLH was made based on cytological findings, clinical manifestations, and laboratory results. She was treated with steroids and recovered completely. A high degree of clinical suspicion coupled with comprehensive cytology with fine needle aspiration cytology is fruitful in the diagnosis of HLH, a potentially fatal disease and help in the delineation of therapeutic regimen.
1598	A female patient with incomplete hemophagocytic lymphohistiocytosis caused by a heterozygous XIAP mutation associated with non-random X-chromosome inactivation skewed towards the wild-type XIAP allele.	25744037	X-linked lymphoproliferative disease (XLP) is a rare inherited immunodeficiency that often leads to hemophagocytic lymphohistiocytosis (HLH). XLP can be classified as XLP1 or XLP2, caused by mutations in SH2D1A and XIAP, respectively. In women, X-chromosome inactivation (XCI) of most X-linked genes occurs on one of the X chromosomes in each cell. The choice of which X chromosome remains activated is generally random, although genetic differences and selective advantage may cause one of the X chromosomes to be preferentially inactivated. Here we describe three patients with pancytopenia, including one female patient, in a Japanese family with a novel XIAP mutation. All three patients exhibited deficient XIAP protein expression, impaired NOD2/XIAP signaling, and augmented activation-induced cell death. In the female patient, the paternally derived X chromosome was non-randomly and exclusively inactivated in her peripheral blood and hair root cells. In contrast to asymptomatic females, this patient exhibied non-random XCI skewed towards the wild-type XIAP allele. This is the first report of a female patient with incomplete HLH resulting from a heterozygous XIAP mutation in association with non-random XCI.
1599	Evolution of the metazoan mitochondrial replicase.	25740821	The large number of complete mitochondrial DNA (mtDNA) sequences available for metazoan species makes it a good system for studying genome diversity, although little is known about the mechanisms that promote and/or are correlated with the evolution of this organellar genome. By investigating the molecular evolutionary history of the catalytic and accessory subunits of the mtDNA polymerase, pol γ, we sought to develop mechanistic insight into its function that might impact genome structure by exploring the relationships between DNA replication and animal mitochondrial genome diversity. We identified three evolutionary patterns among metazoan pol γs. First, a trend toward stabilization of both sequence and structure occurred in vertebrates, with both subunits evolving distinctly from those of other animal groups, and acquiring at least four novel structural elements, the most important of which is the HLH-3β (helix-loop-helix, 3 β-sheets) domain that allows the accessory subunit to homodimerize. Second, both subunits of arthropods and tunicates have become shorter and evolved approximately twice as rapidly as their vertebrate homologs. And third, nematodes have lost the gene for the accessory subunit, which was accompanied by the loss of its interacting domain in the catalytic subunit of pol γ, and they show the highest rate of molecular evolution among all animal taxa. These findings correlate well with the mtDNA genomic features of each group described above, and with their modes of DNA replication, although a substantive amount of biochemical work is needed to draw conclusive links regarding the latter. Describing the parallels between evolution of pol γ and metazoan mtDNA architecture may also help in understanding the processes that lead to mitochondrial dysfunction and to human disease-related phenotypes.
1600	Alternative therapy for epstein-barr virus related hemophagocytic lymphohistiocytosis.	25737788	Hemophagocytic lymphohistiocytosis (HLH) is a rapidly fatal condition characterized by excessive immune activation. HLH can occur as a familial or sporadic acquired disorder. Acquired HLH is more frequently found in adults and is commonly secondary to infections, malignancies, or autoimmune diseases. Diagnosing HLH is challenging because of the rare occurrence, variable presentation, and nonspecific findings of this disorder. Diagnosis of HLH can be based on the diagnostic criteria which were used in the HLH-2004 trial. Given the rarity of this disease, protocols for its treatment have developed slowly, and obtaining adequate short-term and long-term control of the disease continues to be a challenge. Conventional induction therapy for HLH is dexamethasone and etoposide (VP-16), followed by or with cyclosporine. Intrathecal methotrexate ± hydrocortisone is given to those with central nervous system disease. We are reporting a patient who was diagnosed with Epstein-Barr virus (EBV) related HLH. He achieved complete remission with rituximab alone. To our knowledge, this is the first case of an adult patient with EBV related HLH who went into remission with rituximab therapy alone, without using the conventional chemotherapy.
1601	X-linked inhibitor of apoptosis protein deficiency: more than an X-linked lymphoproliferative syndrome.	25737324	X-linked inhibitor of apoptosis (XIAP) deficiency (also known as X-linked lymphoproliferative syndrome type 2, XLP-2) is a rare primary immunodeficiency. Since the disease was first described in 2006, more than 70 patients suffering from XIAP-deficiency have been reported, thus extending the clinical presentations of the disease. The main clinical features of XLP-2 are (i) elevated susceptibility to hemophagocytic lymphohistiocytosis (HLH, frequently in response to infection with Epstein-Barr virus (EBV)), (ii) recurrent splenomegaly and (iii) inflammatory bowel disease (IBD) with the characteristics of Crohn's disease. XIAP deficiency is now considered to be one of the genetic causes of IBD in infancy. Although XIAP is an anti-apoptotic molecule, it is also involved in many other pathways, including the regulation of innate immunity and inflammation. XIAP is required for signaling through the Nod-like receptors NOD1 and 2, which are intracellular sensors of bacterial infection. XIAP-deficient T cells (including innate natural killer T cells and mucosal-associated invariant T cells) are overly sensitive to apoptosis. NOD2 function is impaired in XIAP-deficient monocytes. However, the physiopathological mechanisms underlying the clinical phenotypes in XIAP deficiency, notably the HLH and the EBV susceptibility, are not well understood. Here, we review the clinical aspects, molecular etiology and physiopathology of XIAP deficiency.
1602	Rotavirus-associated hemophagocytic lymphohistiocytosis (HLH) after hematopoietic stem cell transplantation for familial HLH.	25712613	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation. HLH consists of two forms: familial and acquired, the latter which occurs in association with infection, malignancy, rheumatic disease and acquired immune deficiency. Herein, we report a case of acquired HLH in a child who had received allogeneic hematopoietic stem cell transplantation for familial HLH with UNC13D mutation. Based on microbiology, only rotavirus was identified as a possible organism triggering HLH. The patient's fulminant clinical course included acute respiratory failure, a sepsis-like pattern, disseminated intravascular coagulopathy, and rhabdomyolysis, leading to multiorgan failure and death from septic shock.
1603	A novel sterol regulatory element-binding protein gene (sreA) identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51) regulation.	25699519	Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.
1604	bHLH proteins involved in Drosophila neurogenesis are mutually regulated at the level of stability.	25694512	Proneural bHLH activators are expressed in all neuroectodermal regions prefiguring events of central and peripheral neurogenesis. Drosophila Sc is a prototypical proneural activator that heterodimerizes with the E-protein Daughterless (Da) and is antagonized by, among others, the E(spl) repressors. We determined parameters that regulate Sc stability in Drosophila S2 cells. We found that Sc is a very labile phosphoprotein and its turnover takes place via at least three proteasome-dependent mechanisms. (i) When Sc is in excess of Da, its degradation is promoted via its transactivation domain (TAD). (ii) In a DNA-bound Da/Sc heterodimer, Sc degradation is promoted via an SPTSS phosphorylation motif and the AD1 TAD of Da; Da is spared in the process. (iii) When E(spl)m7 is expressed, it complexes with Sc or Da/Sc and promotes their degradation in a manner that requires the corepressor Groucho and the Sc SPTSS motif. Da/Sc reciprocally promotes E(spl)m7 degradation. Since E(spl)m7 is a direct target of Notch, the mutual destabilization of Sc and E(spl) may contribute in part to the highly conserved anti-neural activity of Notch. Sc variants lacking the SPTSS motif are dramatically stabilized and are hyperactive in transgenic flies. Our results propose a novel mechanism of regulation of neurogenesis, involving the stability of key players in the process.
1605	Id3 induces an Elk-1-caspase-8-dependent apoptotic pathway in squamous carcinoma cells.	25693514	Inhibitor of differentiation/DNA-binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Ids typically function as dominant negative HLH proteins, which bind other HLH proteins and sequester them away from DNA promoter regions. Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor. To investigate the role of Id3 in malignant squamous cell carcinoma (SCC) cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an E-twenty-six (ETS)-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis. Furthermore, we show that Id3 synergizes with 5-FU and cisplatin therapies for nonmelanoma skin cancer cells. Our studies have shown a molecular mechanism by which Id3 induces apoptosis in SCC, and this information can potentially be used to develop new treatments for SCC patients.
1606	ShcA regulates thymocyte proliferation through specific transcription factors and a c-Abl-dependent signaling axis.	25691660	Signaling via the pre-T-cell receptor (pre-TCR), along with associated signals from Notch and chemokine receptors, regulates the β-selection checkpoint that operates on CD4(-) CD8(-) doubly negative (DN) thymocytes. Since many hematopoietic malignancies arise at the immature developmental stages of lymphocytes, understanding the signal integration and how specific signaling molecules and distal transcription factors regulate cellular outcomes is of importance. Here, a series of molecular and genetic approaches revealed that the ShcA adapter protein critically influences proliferation and differentiation during β-selection. We found that ShcA functions downstream of the pre-TCR and p56(Lck) and show that ShcA is important for extracellular signal-regulated kinase (ERK)-dependent upregulation of transcription factors early growth factor 1 (Egr1) and Egr3 in immature thymocytes and, in turn, of the expression and function of the Id3 and E2A helix-loop-helix (HLH) proteins. ShcA also contributes to pre-TCR-mediated induction of c-Myc and additional cell cycle regulators. Moreover, using an unbiased Saccharomyces cerevisiae (yeast) screen, we identified c-Abl as a binding partner of phosphorylated ShcA and demonstrated the relevance of the ShcA-c-Abl interaction in immature thymocytes. Collectively, these data identify multiple modes by which ShcA can fine-tune the development of early thymocytes, including a previously unappreciated ShcA-c-Abl axis that regulates thymocyte proliferation.
1607	A fatal case of primary cutaneous gamma-delta T-cell lymphoma complicated by HLH and cardiac amyloidosis.	25678971	Gamma-delta T-cell lymphomas (GD-TCL) are rare and rapidly fatal neoplasms that are often associated with Hemophagocytic Lymphohistiocytosis (HLH), a syndrome of fevers, cytopenias, and multiorgan failure that often leads to a rapid death. We report the first case demonstrating an association between GD-TCL, HLH, and cardiac amyloidosis, presenting a novel mechanism for rapid deterioration in these patients.
1608	Cutaneous findings in hemophagocytic lymphohistiocytosis.	25677716	Cutaneous findings associated with hemophagocytic lymphohistiocytosis (HLH) remain largely undescribed in the literature, yet are substantial and correlative with disease course.To catalog the clinical findings of cutaneous eruptions associated with HLH.We performed a retrospective chart review of patients meeting the criteria for HLH at two hospitals over 5 years. All patients meeting the criteria for HLH as defined by the HLH-2004 protocol were included.Cutaneous lesions were categorized based on clinical presentations and histology. Lesions independent of immunocompromised state were observed, including pyoderma gangrenosum, panniculitis, morbilliform eruptions, Stevens-Johnson syndrome, atypical targetoid lesions and bullae. Histologic findings were non-specific.Cutaneous eruptions as a consequence of HLH are variable in presentation and identified as a diagnosis of exclusion. Findings are both primarily and secondarily induced by altered immunity. Further study is needed to allow better understanding of the immunopathogenesis involved.
1609	A case report of hemophagocytic lymphohistiocytosis (HLH).	25672045	Hemophagocytic lymphohistiocytosis (HLH), is an uncommon, life-threatening hyperinflammatory syndrome caused by severe hypercytokinemia with excessive activation of lymphocytes and macrophages due to a highly stimulated but ineffective immune process. We report a case of Hemophagocytic Lymphohistiocytosis in a 15 year old boy presenting with fever, lymphadenopathy and pancytopenia due to infection caused by Klebsiella Pneumoniae and Acinetobacter.
1610	Hemophagocytic Syndrome in the Setting of AIDS and Disseminated Histoplasmosis: Case Report and a Review of Literature.	25670709	Hemophagocytic lymphohistiocytosis (HLH) is traditionally regarded as a rapidly progressive and often fatal illness. In patients with AIDS, HLH usually occurs secondary to opportunistic infections. Although popular guidelines exist for the diagnosis and management of HLH in general, no formal study has evaluated their applicability among adult patients who develop HLH in the setting of AIDS and opportunistic infections. The study reports on a case of HLH in a patient with AIDS and disseminated histoplasmosis. Eighteen other previously reported cases of HLH in the setting of AIDS and histoplasmosis were reviewed. Majority of the cases occurred in patients with a CD4 count of less than 70 cells/mm(3). Overall mortality was 44%. Not getting antifungal treatment and having Histoplasma in blood were the 2 main risk factors for death. Among the patients who had a timely diagnosis of histoplasmosis and were initiated on antifungal therapy, the survival rates were significantly better, especially in the post-2000 ad period.
1611	XIAP deficiency syndrome in humans.	25666262	The X-linked inhibitor of apoptosis (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome type 2 (XLP-2), is a rare primary immunodeficiency. XIAP deficiency is characterized by a key triad of clinical manisfestations, which consist of a high susceptibility to develop hemophagocytic lymphohistiocytosis (HLH) frequently triggered by Epstein-Barr virus (EBV) infection, recurrent splenomegaly and inflammatory bowel disease (IBD) with the features of a Crohn's disease. XIAP deficiency can be considered as one of the genetic causes for inherited IBD. XIAP is an anti-apoptotic molecule, but it is also involved in many other pathways. Recent findings demonstrate the role of XIAP in innate immunity and in the negative regulation of inflammation. In this review, we focus on the clinical aspects, the molecular etiology and the immunopathogenesis of XIAP deficiency. We also discuss recent progress in the understanding of XIAP function in relation to the pathophysiology of XLP-2.
1612	Production, purification, and characterization of recombinant hFSH glycoforms for functional studies.	25661536	Previously, our laboratory demonstrated the existence of a β-subunit glycosylation-deficient human FSH glycoform, hFSH(21). A third variant, hFSH(18), has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH(21) abundance in individual female pituitaries progressively decreased with increasing age. Hypo-glycosylated glycoform preparations are significantly more active than fully-glycosylated hFSH preparations. The purpose of this study was to produce, purify and chemically characterize both glycoform variants expressed by a mammalian cell line. Recombinant hFSH was expressed in a stable GH3 cell line and isolated from serum-free cell culture medium by sequential, hydrophobic and immunoaffinity chromatography. FSH glycoform fractions were separated by Superdex 75 gel-filtration. Western blot analysis revealed the presence of both hFSH(18) and hFSH(21) glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH(21/18)-derived β-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHβ subunits, as both Asn(7) and Asn(24) were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH(21/18) exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH(24). Thus, the age-related reduction in hypo-glycosylated hFSH significantly reduces circulating levels of FSH biological activity that may further compromise reproductive function. Taken together, the ability to express and isolate recombinant hFSH glycoforms opens the way to study functional differences between them both in vivo and in vitro.
1613	Laboratory features and the successful transplantation of allogeneic peripheral blood stem cells in patients with lymphoma-associated hemophagocytic lymphohistiocytosis.	25651724	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease that is commonly caused by lymphoma. The authors describe laboratory features and the successful treatment of two patients with lymphoma- associated HLH.Hematologic investigations, bone marrow aspirates and biopsies, and PET/CT scans were performed. Both patients received a transplantation of allogenic peripheral blood stem cells after chemotherapy.The patients achieved complete remission and have survived for three or more years after treatment with chemotherapy and an allogenic peripheral blood stem cell transplantation.Allogeneic peripheral blood stem cell transplantation may be the most effective therapy for patients with lymphoma-associated hemophagocytic lymphohistiocytosis.
1614	Cytokine profiles in Mycoplasma pneumoniae infection-associated hemophagocytic lymphohistiocytosis.	25649486	A 3-year-old boy with Mycoplasma pneumoniae infection associated with hemophagocytic lymphohistiocytosis (MP-HLH) presented with an elevated level of serum interleukin-12 (IL-12) and lower levels of interferon-γ and IL-10 compared to patients with Epstein-Barr virus infection associated with HLH (EBV-HLH). Unlike the patients with EBV-HLH, CD8+ CD5low HLA-DR++ T cells were not detected in our pediatric patient. Thus, the pathophysiology of MP-HLH may differ from that of EBV-HLH.
1615	Notch signaling downstream target E(spl)mbeta is dispensable for adult midgut homeostasis in Drosophila.	25637572	Adult tissue homeostasis is maintained by residential stem cells through the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs), and Notch signaling plays critical roles in the proliferation and differentiation of these ISCs. However, how Notch signaling downstream targets regulate ISC proliferation and differentiation still remains unclear. Here we find that Notch signaling downstream targets E(spl)mbeta and E(spl)malpha are differentially expressed in ISCs and their progeny. Interestingly, we find that midgut homeostasis is not affected in E(spl)mbeta null mutant. No obvious defects are observed in the intestines ectopically expressing E(spl)mbeta or E(spl)malpha. Importantly, we find that the defects in ISC proliferation and differentiation observed in Notch mutant cannot be rescued by ectopic expression of E(spl)mbeta or E(spl)malpha. Together, these data indicate that the proliferation and differentiation of ISCs are not regulated by individual Notch downstream target, but by different downstream targets collectively.
1616	Incidence and clinical presentation of primary hemophagocytic lymphohistiocytosis in Sweden.	25382070	Primary hemophagocytic lymphohistiocytosis (HLH) represents a group of inherited hyperinflammatory immunodeficiencies, including familial HLH (FHL), Griscelli syndrome type 2 (GS2), and X-linked lymphoproliferative syndrome (XLP). We previously reported an annual incidence of suspected primary HLH in Sweden 1971-1986 of 0.12 per 100,000 children. Here, we determined if the incidence had increased with concomitant awareness.Children <15 years old presenting with HLH 1987-2006 in Sweden were identified through the national mortality registry as well as by nation-wide inquiries to all pediatric centers. HLH was diagnosed according to the HLH-2004 diagnostic guidelines (in case of missing data of at least three of the eight diagnostic criteria, fulfillment of four was sufficient for inclusion). We defined primary HLH as patients presenting with HLH requiring transplantation or dying of disease.Remarkably, the minimal annual incidence rate of primary HLH remained 0.12 per 100,000 children, equating to 1.8 per 100,000 live births. Notably, an increased overall survival was observed in 1997-2006, relative to the period 1987-1996. During the subsequent 5-year period, 2007-2011, the incidence of genetically and/or functionally verified primary HLH was 0.15 per 100,000 children per year, suggesting that new assays may aid the identification of patients with primary HLH.The annual incidence of primary HLH in Sweden is 0.12-0.15 per 100,000 children per year. Pediatr Blood Cancer 2015;62:346-352. © 2014 Wiley Periodicals, Inc.
1617	Forme Fruste of HLH (haemophagocytic lymphohistiocytosis): diagnostic and therapeutic challenges.	25634853	Infants and young children often present with a persistent febrile episode, sick appearance and negative infectious disease work-up. These patients present serious diagnostic and therapeutic problems to those who provide medical care, particularly since these children are clinically sick. We present a 13 month old child who presented with this clinical challenge. She was ultimately thought to have an incomplete form of HLH with underlying pathophysiology of hypercytokinemia, but also could have been a case of incomplete form of Kawasaki disease. She responded to IVIG, but this does not differentiate one diagnosis from another. Unfortunately we failed to obtain tests to exclude genetic etiologies of HLH, which would be important for predicting severity and risks of future recurrence. We wish to present this case so that one should do a thorough work up to establish a firm diagnosis of HLH and to search for genetic causes of this disorder.
1618	Reactive hemophagocytic syndrome in adult-onset Still disease: clinical features, predictive factors, and prognosis in 21 patients.	25634183	Hemophagocytic syndrome (HPS) is a potentially life-threatening complication of systemic inflammatory disorders. Adult-onset Still disease (AOSD) is one of the systemic autoimmune diseases associated with reactive hemophagocytic syndrome (RHS). This study aimed to evaluate the characteristic findings, predictive factors, and prognosis of RHS in patients with AOSD. We retrospectively evaluated 109 patients diagnosed with AOSD and reviewed their clinical data and laboratory findings, including the biopsy results of 21 AOSD patients with RHS. Moreover, data from 17 hemophagocytic lymphohistiocytosis (HLH) patients evaluated during the same period were compared with those from the RHS patients. Twenty-one patients (19.3%) developed RHS during the course of AOSD, and only 7 patients (6.4%) were confirmed by bone marrow, liver, or lymph node biopsy. AOSD patients with RHS showed significantly higher frequencies of splenomegaly, hepatomegaly, and lymphadenopathy than did those without RHS. Moreover, patients with RHS showed significantly higher relapse rates than those without RHS (61.9% vs 18.2%, P < 0.001). Possible triggering factors inducing hemophagocytosis were detected in 16 of 21 RHS patients (76.2%): disease flare in 12 patients (75%), infection in 3 patients (18.8%), and drug use in 1 patient (6.3%). AOSD patients with RHS showed higher frequencies of leukopenia, anemia, thrombocytopenia, hypoalbuminemia, hypofibrinogenemia, hypertriglyceridemia, hyperferritinemia, and elevated lactate dehydrogenase levels than did those without RHS. Multivariate logistic regression with forward selection procedure showed that low platelet count (<121,000/mm³), anemia, and hepatomegaly were independent predictors of RHS. Patients with definite RHS and those with probable RHS showed comparable results. Although RHS is a life-threatening complication of AOSD, long-term prognosis was observed to be similar in patients with and those without RHS. Compared to RHS patients, HLH patients had poor prognosis, such as higher death rates (52.9% vs 9.5%, P = 0.005). RHS can be considered when an AOSD patient shows at least 2 of the following 3 findings: low platelet count, anemia, and hepatomegaly. Diagnostic confirmation by biopsy may not be essential if typical clinical findings of RHS are present. Moreover, prognosis of RHS was better than that of HLH diagnosed by the presence of trilineage cytopenia at admission.
1619	Gonadotrophins: The future.	25624659	The role of the IVF clinician is to make the ART treatment safe, patient-friendly, cost effective and at the same time offer good and high quality treatment. IVF protocols are a burden for women and are one of the potential reasons why women don't return for subsequent cycles. Frequent injections may increase stress and also result in high error rates. Simple short treatment regimen with optimal recovery of good quality oocytes results in development of good quality embryos followed by SET in treatment and cryopreservation cycles are a less burden and result in related lesser discontinuation, side effects, treatment cycles in time and are more cost-effective. Development of FSH analogues with longer terminal t1/2 and slower absorption to peak serum levels will increase the efficiency, decrease the side effects and also is easy to administer. This makes it convenient for the patients increasing the compliance. A certain minimum LH concentration is necessary for adequate thecal cell function and subsequent oestradiol synthesis in the granulosa cells. Adjuvant r-HLH gives clinician's precise control over the dose of LH bioactivity administered to target the therapeutic window. New parenteral, transdermal, inhaled and oral fertility drugs and regimens are currently under research and development with the objective to further simplify treatment for ART.
1620	Modeling EBV infection and pathogenesis in new-generation humanized mice.	25613732	The development of highly immunodeficient mouse strains has allowed the reconstitution of functional human immune system components in mice. New-generation humanized mice generated in this manner have been extensively used for modeling viral infections that are exclusively human tropic. Epstein-Barr virus (EBV)-infected humanized mice reproduce cardinal features of EBV-associated B-cell lymphoproliferative disease and EBV-associated hemophagocytic lymphohistiocytosis (HLH). Erosive arthritis morphologically resembling rheumatoid arthritis (RA) has also been recapitulated in these mice. Low-dose EBV infection of humanized mice results in asymptomatic, persistent infection. Innate immune responses involving natural killer cells, EBV-specific adaptive T-cell responses restricted by human major histocompatibility and EBV-specific antibody responses are also elicited in humanized mice. EBV-associated T-/natural killer cell lymphoproliferative disease, by contrast, can be reproduced in a distinct mouse xenograft model. In this review, recent findings on the recapitulation of human EBV infection and pathogenesis in these mouse models, as well as their application to preclinical studies of experimental anti-EBV therapies, are described.
1621	Evaluation of lipid peroxidation and oxidative DNA damage in patients with periodontitis and hyperlipidemia.	25612631	The purpose of this study is to determine the serum levels of malondialdehyde (MDA), as a lipid peroxidation marker, and 8-hydroxydeoxyguanosine (8-OHdG), as an oxidative DNA damage marker, in patients with chronic periodontitis (CP) and hyperlipidemia.A total of 74 individuals were divided into four age- and sex-matched groups: 18 patients with hyperlipidemia and CP (HLp), 18 periodontally healthy patients with hyperlipidemia (HLh), 19 systemically healthy individuals with CP (Cp), and 19 systemically and periodontally healthy controls (Ch). Clinical periodontal parameters were measured, and serum lipids, MDA, and 8-OHdG levels were assessed in blood samples.8-OHdG, MDA, probing depth, clinical attachment level, and percentage of sites bleeding on probing (BOP) were significantly higher in the HLp group than the Cp group. In the hyperlipidemic group, BOP was significantly correlated with total cholesterol, the ratio of total cholesterol to high-density lipoprotein cholesterol, and 8-OHdG levels. A significant correlation between 8-OHdG and MDA was also observed in the hyperlipidemia group.In this study, serum MDA and 8-OHdG were found to be highest in the HLp group. The increased levels of MDA and 8-OHdG in HLp patients may be a result of a harmful oxidative status in association with hyperlipidemia and periodontitis.
1622	Pulmonary Vasoreactivity to Materno-Fetal Hyperoxygenation Testing in Fetuses with Hypoplastic Left Heart.	25607629	The aim of the study was to describe the response of fetal lung vasculature to maternal hyperoxygenation (MH) in the case of prenatally diagnosed hypoplastic left heart (HLH) with intact or restrictive (IAS/RAS) and without restriction of the atrial septum. Furthermore, the ability of MH to differentiate between newborns with HLH who do not require immediate atrial septostomy and newborns who will undergo immediate left atrial septoplasty after birth was evaluated.Cross-sectional prospective study of fetuses ≥ 26 weeks of gestation with prenatally diagnosed HLH. Lung perfusion (LP) was qualitatively assessed by color Doppler interrogation and LP was quantitatively measured using the pulsatility index for veins (PIV). Measurements were performed both with the mother breathing room air (LPRA) and after receiving 100% oxygen for 10 minutes (LPMH). The oxygen test was defined as positive if MH led to an increase in lung perfusion and as negative if MH did not lead to an increase.A total number of 22 pregnancies with hypoplasia of the left heart structures were included. 6/20 cases presented with an intact or restrictive atrial septum (IAS/RAS). All of these fetuses presented with a reduced LPRA. MH led to an increase in LP in 2/6 cases. The overall 30-day-survival rate was 83.3% (5/6). In 14/20 fetuses an open septum was detected. 11 cases had a normal LPRA, and the LPRA was reduced in 3/14 fetuses. The overall 30-day-survival rate was 92.9% (13/14).MH might be a useful adjunct in the assessment of pulmonary vasculopathy in fetuses with HLH.
1623	Identification of genes that may regulate the expression of the transcription factor production of anthocyanin pigment 1 (PAP1)/MYB75 involved in Arabidopsis anthocyanin biosynthesis.	25604992	A putative RNA-binding protein with a single RNA Recognition Motif (At3G63450) is involved in anthocyanin biosynthesis via its ability to modulate the transcript level of a major positive regulator PAP1 in Arabidopsis. The R2R3 MYB-activator production of anthocyanin pigment 1 (PAP1)/MYB75 plays a major role in anthocyanin biosynthesis in Arabidopsis in combination with one of three bHLH activators including transparent test 8 (TT8), enhancer of glabra3 (EGL3), glabra3 (GL3), and the WD-repeat transcription factor transparent testa 1 (TTG1), forming ternary MYB-basic HLH-WD40 complexes. Transcriptional activation of PAP1 expression is largely triggered by changes in light color and intensity, temperature fluctuations, nutrient status, and sugar and hormone treatments. However, the immediate upstream and downstream regulatory factors for PAP1 transcription are largely unknown. In the present study, using a T-DNA insertional mutagenesis approach, we transformed pap1-Dominant (pap1D) plants to modulate the levels of endogenous PAP1 transcripts. We employed Restriction Site Extension (RSE)-PCR analysis of 247 homogenous T3 genetic mutant lines exhibiting variations in anthocyanin accumulation compared to pap1D and identified 92 lines with T-DNA integrated in either intra- or inter-genic locations. This analysis revealed 80 novel candidate proteins, including a putative RNA-binding protein with a single RNA Recognition Motif (At3G63450), which may directly or indirectly regulate PAP1 expression at the transcriptional level.
1624	Deletion mapping in the Enhancer of split complex.	25588303	The Enhancer of split complex [E(spl)-C] comprises twelve genes of different classes. Seven genes encode proteins of with a basic-helix-loop-helix-orange (bHLH-O) domain that function as transcriptional repressors and serve as effectors of the Notch signalling pathway. They have been named E(spl)m8-, m7-, m5-, m3-, mβ-, mγ- and mδ-HLH. Four genes, E(spl)m6-, m4-, m2- and mα-BFM are intermingled and encode Notch repressor proteins of the Bearded-family (BFM). The complex is split by a single gene of unrelated function, encoding a Kazal-type protease inhibitor (Kaz-m1). All members within a family, bHLH-O or BFM, are very similar in structure and in function. In an attempt to generate specific mutants, we have mobilised P-element constructs residing next to E(spl)m7-HLH and E(spl)mγ-HLH, respectively. The resulting deletions were mapped molecularly and by cytology. Two small deletions affected only E(spl)m7-HLH and E(spl)mδ. The deficient flies were viable without apparent phenotype. Larger deletions, generated also by X-ray mutagenesis, uncover most of the E(spl)-C. The phenotypes of homozygous deficient embryos were analysed to characterize the respective loss of Notch signalling activity.
1625	Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis (FHL) patients in India.	25577959	Inherited perforin deficiency is a rare autosomal recessive disorder that causes severe form of hemophagocytic lymphohistiocytosis (FHL2). The main aim of this study was to analyze the nature of gene mutations in a cohort of Indian patients with FHL2 and to utilize this knowledge for genetic counseling and prenatal diagnosis.13 HLH patients with abnormal perforin expression on NK cells by flow cytometry were included in the study. The entire coding region and intronic splice sites of the PRF1 gene were sequenced from the genomic DNA of these patients.10 patients from the present series had an early presentation with severe clinical manifestations, while 3 had a delayed onset with unusual presenting features viz Hodgkin's lymphoma, tuberculosis and acute lymphoblastic leukemia. Sequence analysis revealed 11 different mutations (8 novel and 3 previously reported) spread over the entire coding region of PRF1 gene. Missense mutation Trp129Ser in heterozygous state was present in all the 3 patients with a delayed onset of the disease.A wide heterogeneity was observed in the nature of mutations in Indian FHL2 patients. Molecular characterization of PRF1 gene was not only used in the confirmation of diagnosis but also in genetic counseling and pre-natal diagnosis in affected families.
1626	[Macrophage activation syndrome in a patient with systemic juvenile idiopathic arthritis].	25575650	Machrophage activation syndrome (MAS) is a rare and potentially fatal disease, commonly associated with chronic rheumatic diseases, mainly juvenile idiopathic arthritis. It is included in the group of secondary forms of haemophagocytic syndrome, and other causes are lymphoproliferative diseases and infections. Its most important clinical and laboratorial manifestations are non-remitting fever, splenomegaly, bleeding, impairment of liver function, cytopenias, hypoalbuminemia, hypertriglyceridemia, hypofibrinogenemia and hyperferritinemia. The treatment needs to be started quickly, and the majority of cases have a good response with corticosteroids and cyclosporine. The Epstein-Barr virus is described as a possible trigger for many cases of MAS, especially in these patients in treatment with tumor necrosis factor (TNF) blockers. In these refractory cases, etoposide (VP16) should be administered, associated with corticosteroids and cyclosporine. Our objective is to describe a rare case of MAS probably due to EBV infection in a subject with systemic-onset juvenile idiopathic arthritis, which achieved complete remission of the disease after therapy guided by 2004-HLH protocol.
1627	Hemophagocytic Lymphohistiocytosis: an Unusual Complication of Orientia tsutsugamushi Disease (Scrub Typhus).	25574367	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon, potentially fatal, hyperinflammatory syndrome that may rarely complicate the clinical course of Orientia tsutsugamushi disease (scrub typhus).Here we describe the clinicopathological features, laboratory parameters, management, and outcome of three adult patients (1 female, 2 males) with scrub typhus associated HLH from a tertiary center. A brief and concise review of international literature on the topic was also added.All three patients satisfied the HLH-2004 diagnostic criteria; one had multi-organ dysfunction with very high ferritin level (>30,000 ng/ml), and all had a dramatic recovery following doxycyclin therapy. Literature review from January 1990 to March 2014 revealed that scrub typhus associated HLH were reported in 21 patients, mostly from the scrub endemic countries of the world. These included 11 females and 10 males with a mean age of 35 years (range; 8 months to 81 years). Fifteen of 17 patients (where data were available) had a favorable outcome following early serological diagnosis and initiation of definitive antibiotic therapy with (N=6) or without (N=9) immunosuppressive/immunomodulator therapy. Mutation analysis for primary HLH was performed in one patient only, and HLH-2004 protocol was used in two patients.We suggest that HLH should be considered in severe cases of scrub typhus especially if associated with cytopenia (s), liver dysfunction, and coagulation abnormalities. Further studies are required to understand whether an immunosuppressive and/or immunomodulator therapy could be beneficial in those patients who remain unresponsive to definitive antibiotic therapy.
1628	Marked hyperferritinemia does not predict for HLH in the adult population.	25573993	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of uncontrolled immune activation that has gained increasing attention during the last decade. The diagnosis of HLH is based on a constellation of clinical and laboratory abnormalities, including elevated serum ferritin levels. In the pediatric population, marked hyperferritinemia is specific for HLH. To determine what conditions are associated with profoundly elevated ferritin in the adult population, we performed a retrospective analysis in a large academic health care system. We identified 113 patients with serum ferritin levels higher than 50,000 µg/L. The most frequently observed conditions included renal failure, hepatocellular injury, infections, and hematologic malignancies. Our results suggest that marked hyperferritinemia can be seen in a variety of conditions and is not specific for HLH in adults.
1629	Hemophagocytic lymphohistiocytosis caused by dominant-negative mutations in STXBP2 that inhibit SNARE-mediated membrane fusion.	25564401	Familial hemophagocytic lymphohistiocytosis (F-HLH) and Griscelli syndrome type 2 (GS) are life-threatening immunodeficiencies characterized by impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell lytic activity. In the majority of cases, these disorders are caused by biallelic inactivating germline mutations in genes such as RAB27A (GS) and PRF1, UNC13D, STX11, and STXBP2 (F-HLH). Although monoallelic (ie, heterozygous) mutations have been identified in certain patients, the clinical significance and molecular mechanisms by which these mutations influence CTL and NK cell function remain poorly understood. Here, we characterize 2 novel monoallelic hemophagocytic lymphohistiocytosis (HLH)-associated mutations affecting codon 65 of STXPB2, the gene encoding Munc18-2, a member of the SEC/MUNC18 family. Unlike previously described Munc18-2 mutants, Munc18-2(R65Q) and Munc18-2(R65W) retain the ability to interact with and stabilize syntaxin 11. However, presence of Munc18-2(R65Q/W) in patient-derived lymphocytes and forced expression in control CTLs and NK cells diminishes degranulation and cytotoxic activity. Mechanistic studies reveal that mutations affecting R65 hinder membrane fusion in vitro by arresting the late steps of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complex assembly. Collectively, these results reveal a direct role for SEC/MUNC18 proteins in promoting SNARE-complex assembly in vivo and suggest that STXBP2 R65 mutations operate in a novel dominant-negative fashion to impair lytic granule fusion and contribute to HLH.
1630	Acute Liver Failure in Infants and Young Children in a Specialized Pediatric Liver Centre in India.	2555717726334862	To study the etiological spectrum of acute liver failure in infants and young children and to identify clinical and biochemical markers for metabolic liver disease (MLD).This study was conducted at Department of Pediatric Hepatology, in a tertiary care specialized centre for liver diseases. All children less than 3 y of age, with liver dysfunction and INR ≥2 were included in the study. They were managed as per the departmental protocol. Included children were divided based on the etiology into 2 groups: MLD and non MLD group. Comparison analysis (MLD vs. non MLD) of the clinical and biochemical parameters was done.There were 30 children under 3 y of age with acute liver failure (ALF) with median age of 12.5 mo. Fifteen children were less than 12 mo. MLD (33 %) and hemophagocytic lymphohistiocytosis (HLH) (17 %) together accounted for half of the cases of ALF in children below 3 y of age. The other common etiologies were drug induced liver injury and acute viral hepatitis A. Etiology remained indeterminate in 3 cases (10 %). Comparative analysis of the clinical and biochemical parameters between MLD and non MLD group showed significant difference between the two groups in the median values of age (p = 0.014), bilirubin (p = 0.017), jaundice to encephalopathy (JE) interval (p = 0.039) and blood sugar (p = 0.001). Suggestive family history (OR 3.73, 95 %CI 1.67-8.30), developmental delay (OR 4.4 95 %CI 2.03-9.51), presence of diarrhea/vomiting (OR 3.28, 95 %CI 1.32-8.13) in the history and presence of urinary non glucose reducing substance (NGRS) (OR 15.5, 95 %CI 2.26-106.87) were also significantly associated with MLD group. Only 40 % children survived with native liver.MLD and HLH account for majority of ALF in infants. About 10 % of cases remain indeterminate. Viral hepatitis is more common in young children. Apart from clinical indicators, young age, high bilirubin, synthetic dysfunction, low sugar and NGRS in urine indicate MLD as a cause. Survival with native liver is low.
1631	Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila.	25551675	How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.
1632	Infantile hemophagocytic lymphohistiocytosis in a case of chediak-higashi syndrome caused by a mutation in the LYST/CHS1 gene presenting with delayed umbilical cord detachment and diarrhea.	25551669	A 2-month-old female infant, born to consanguineous parents, presented with infections in skin and upper respiratory tract. She was notable for delayed umbilical cord detachment, partial albinism, and neurological irritability. Giant granules were present in white blood cells. The intracellular perforin content in CD8 T cells seems to correlate to the immune activation state of the patient with 82% and 8% perforin-containing CD8 T cells at active and nonactive hemophagocytic lymphohistiocytosis (HLH) disease, respectively. HLH was confirmed by hemophagocytosis in bone marrow and absent natural killer cell activity. The patient carried a homozygous G>A mutation in the 3' splice site of intron 24 of the LYST/CHS1 gene, leading to the use of an alternative YAG splice site located in exon 25, introducing a premature STOP codon (L2355fsX2370; NP_000072.2). The early-onset accelerated phase in this severe phenotype of Chediak-Higashi syndrome was probably induced by rotaviral infection. Interestingly, the intracellular perforin content in CD8 T cells seems to correlate to the immune activation state of the patient. Late separation of the umbilical cord in concordance with clinical symptoms should lead to evaluation of a possible neutrophil dysfunction including Chediak-Higashi syndrome before onset of HLH.
1633	Familial hemophagocytic lymphohistiocytosis in two Saudi siblings.	27493422	Primary familial hemophagocytic lymphohistiocytosis (HLH; or familial erythrophagocytic lymphohistiocytosis [FEL]) is a heterogeneous autosomal recessive disorder more prevalent with parental consanguinity. There is aggressive proliferation of activated macrophages and histiocytes, which phagocytose red blood cells (RBCs), white blood cells (WBCs), and platelets, leading to anemia, neutropenia and thrombocytopenia. The exaggerated response of immune system in familial HLH can occur in the absence of infection. We report on two Saudi siblings with familial hemophagocytic lymphohistiocytosis. The first case was diagnosed and started on treatment but died after ten days of treatment while the second one was referred to a higher centre for treatment but died before commencing chemotherapy treatment. This rare inherited aggressive disease needs high index of suspicion and early treatment. Anti-inflammatory therapy consisting of steroids, etoposide or antithymocyte globulin (ATG), should be instituted promptly, followed by curative hematopoietic cell transplantation to get a better outcome. Without treatment, most patients with familial hemophagocytic lymphohistiocytosis survive only a few months.
1634	Late-onset hemophagocytic lymphohistiocytosis (HLH) in an adult female with Griscelli syndrome type 2 (GS2).	25544030	NaN
1635	[Analysis of 3 cases with Mycoplasma pneumoniae-associated hemophagocytic syndrome and review of literature].	25537549	To analyze the clinical characteristics of Mycoplasma pneumoniae-associated hemophagocytic syndrome (MP-HLH).A retrospective investigation of the clinical manifestation, laboratory test, imagelogy, clinical course and outcome of 3 cases with MP-HLH seen between June 2013 and July 2013 in Shenzhen Children's Hospital, and review of relevant literature were conducted.Of the 3 cases of MP-HLH, 2 were males, one was female, the ages were 1 year, 3 years and 6 years, respectively. They had no underlying disease previously. All the 3 cases had onset of fever, cough as main symptoms. Diagnosis of refractory Mycoplasma pneumoniae pneumonia was made, which was accompanied by decreased neutrophils [(0.08-0.68)×10(9)/L], hemoglobin [(79-103) g/L], platelet [(64-157)×10(9)/L], plasma fibrinogen [(1.3-1.5) g/L], lactate dehydrogenase [(1,170-1,285) U/L] and increased serum ferritin [(936.7-39 789.0) µg/L] in the third week of course. In two cases the T lymphocytes decreased, and the NK cell activity decreased significantly in one. Bone marrow cytology showed prompted bone marrow hyperplasia, and the phenomenon of phagocytosed blood cells. CT scan was performed for all the cases and consolidation with pleural effusion were shown. Two cases were admitted to PICU, and required endotracheal intubation and mechanical ventilation. Flexible bronchoscopy and bronchial lavage were performed and bronchial cast was found in two cases. All of them were treated with macrolide combined with other antibiotics, glucocorticoids and gamma globulin combination therapy, including one case given dexamethasone [10 mg/(m2·d)], cyclosporine[6 mg/(kg·d)], etoposide [150 mg/(m2·d)] chemotherapy. Two cases were cured, and 1 case died. The authors summarized the 18 cases reported in domestic and foreign literature. Foreign children were diagnosed and treated with steroids in 1-2 weeks, and 10 cases were cured, and 2 cases died. They died of massive hemorrhage and meningoencephalitis, and domestic children were diagnosed and treated within two to 4 weeks after onset, 5 cases were cured, one case died of severe pneumonia.MP-HLH is a rare disease in children, and had acute onset, rapid progression and high mortality. Early treatment with steroids was associated with a good prognosis, the key to successful treatment is early diagnosis and treatment, avoiding the immune cascade. Too late a diagnosis or development of serious complications may lead to death.
1636	[Clinical analyses of 80 hemophagocytic lymphohistiocytosis patients with Epstein-Barr viremia].	25534103	To analyze the clinical characteristics, treatment and prognosis of hemophagocytic lymphohistiocytosis (HLH) patients with Epstein-Barr viremia (EBV).A retrospective study was conducted to analyze the clinical data of 80 HLH patients aged ≥ 14 years with EBV-DNA >1 000 copies/ml in peripheral blood from 2008 to 2013.There were EBV-HLH (n = 46), HLH-associated lymphoma (n = 30) and primary HLH (n = 4). Among the relevant laboratory parameters, inter-group statistical differences existed only in alanine transaminase (ALT) and aspartate aminotransferase (AST) (P = 0.021, 0.035). The median follow-up period was 2.0 (0.5-20.0) months. And the 1-month, 3-month, 6-month and 12-month overall survival rates were 58.8%, 37.5%, 29.7% and 19.6% respectively.HLH patients with EBV have a pernicious clinical course with a poor prognosis. And it makes little sense for distinguishing HLH-associated lymphoma from EBV-HLH through routine laboratory tests.
1637	A novel immunoregulatory role for NK-cell cytotoxicity in protection from HLH-like immunopathology in mice.	25525117	The impairment of cytotoxic activity of lymphocytes disturbs immune surveillance and leads to the development of hemophagocytic lymphohistiocytic syndrome (HLH). Although cytotoxic T lymphocyte (CTL) control of HLH development is well documented, the role for natural killer (NK)-cell effector functions in the pathogenesis of this immune disorder remains unclear. In this study, we specifically targeted a defect in cytotoxicity to either CTL or NK cells in mice so as to dissect the contribution of these lymphocyte subsets to HLH-like disease severity after lymphocytic choriomeningitis virus (LCMV) infection. We found that NK-cell cytotoxicity was sufficient to protect mice from the fatal outcome that characterizes HLH-like disease and was also sufficient to reduce HLH-like manifestations. Mechanistically, NK-cell cytotoxicity reduced tissue infiltration by inflammatory macrophages and downmodulated LCMV-specific T-cell responses by limiting hyperactivation of CTL. Interestingly, the critical protective effect of NK cells on HLH was independent of interferon-γ secretion and changes in viral load. Therefore our findings identify a crucial role of NK-cell cytotoxicity in limiting HLH-like immunopathology, highlighting the important role of NK cytotoxic activity in immune homeostasis.
1638	Inhibitor of differentiation 4 (ID4): From development to cancer.	25512197	Highly conserved Inhibitors of DNA-Binding (ID1-ID4) genes encode multi-functional proteins whose transcriptional activity is based on dominant negative inhibition of basic helix-loop-helix (bHLH) transcription factors. Initial animal models indicated a degree of compensatory overlap between ID genes such that deletion of multiple ID genes was required to generate easily recognizable phenotypes. More recently, new model systems have revealed alterations in mice harboring deletions in single ID genes suggesting complex gene and tissue specific functions for members of the ID gene family. Because ID genes are highly expressed during development and their function is associated with a primitive, proliferative cellular phenotype there has been significant interest in understanding their potential roles in neoplasia. Indeed, numerous studies indicate an oncogenic function for ID1, ID2 and ID3. In contrast, the inhibitor of differentiation 4 (ID4) presents a paradigm shift in context of well-established role of ID1, ID2 and ID3 in development and cancer. Apart from some degree of functional redundancy such as HLH dependent interactions with bHLH protein E2A, many of the functions of ID4 are distinct from ID1, ID2 and ID3: ID4 proteins a) regulate distinct developmental processes and tissue expression in the adult, b) promote stem cell survival, differentiation and/or timing of differentiation, c) epigenetic inactivation/loss of expression in several advanced stage cancers and d) increased expression in some cancers such as those arising in the breast and ovary. Thus, in spite of sharing the conserved HLH domain, ID4 defies the established model of ID protein function and expression. The underlying molecular mechanism responsible for the unique role of ID4 as compared to other ID proteins still remains largely un-explored. This review will focus on the current understanding of ID4 in context of development and cancer.
1639	Splenectomy as a treatment for adults with relapsed hemophagocytic lymphohistiocytosis of unknown cause.	25512183	Our aim was to evaluate the clinical value of splenectomy as a treatment for relapsed hemophagocytic lymphohistiocytosis (HLH) of unknown cause in adults. We retrospectively reviewed the clinical data from medical records of 19 adults with relapsed HLH of unknown cause treated with splenectomy in our institution from June 2007 to March 2014. To rule out possible underlying diseases, including infection, autoimmune disease, neoplasms, and primary HLH, the patients had undergone examinations including F18 fluoro-2-deoxyglucose positron emission tomography/computed tomography, HLH-associated gene defects, and lymph node biopsies. Twelve patients (63.2%) achieved partial responses (PR), whereas seven patients (36.8%) had no response (NR) prior to splenectomy. Infection and hemorrhage were the main complications of splenectomy. Eighteen cases were evaluable after follow-up. Seven cases with histopathologic diagnoses of lymphoma had received chemotherapy, four of whom had achieved complete responses (CR), one PR, and two NR. Maintenance treatment was ceased 2 or 3 months after splenectomy in the other 11 cases, five of whom had CR, four PR, and two NR. Eleven of 18 cases (61.1%) survived with a median follow-up of 25 months (range 3-79 months) for survivors. Twelve- and 36-month progression-free survival rates were 48 and 24%, respectively; 12- and 36-month overall survival rates were 57 and 25%, respectively. Median survival time was 22 months. Our results indicate splenectomy may be an effective means of diagnosis and treatment of relapsed HLH of unknown cause. Further study is required to establish the mechanism and value of splenectomy in this disease.
1640	Hemophagocytic lymphohistiocytosis secondary to Epstein Barr virus and Leishmania co-infection in a toddler.	255112192576635925924240	This is the report of an EBV+Leishmanial co-infection. The patient developed hemophagocytic syndrome (HLH) and was treated with the standard HLH-2004 protocol. However, PCR in bone marrow discovered this secondary cause for HLH. In endemic countries, visceral leishmaniasis should be considered in the differential diagnosis even in EBV-related HLH, as chemotherapy toxicity may be avoided.
1641	Griscelli syndrome.	25500851	We report a case of Griscelli Syndrome (GS). Our patient initially presented with a diagnosis of haemophagocytic lymphistiocytosis (HLH). Subsequent microscopic analysis of the patient's hair follicle revealed abnormal distribution of melanosomes in the shaft, which is a hallmark for GS. Analysis of RAB27A gene in this patient revealed a homozygous mutation in exon 6, c.550C>T, p.R184X . This nonsense mutation causes premature truncation of the protein resulting in a dysfunctional RAB27A. Recognition of GS allows appropriate institution of therapy namely chemotherapy for HLH and curative haemotopoeitic stem cell transplantation.
1642	Acute kidney injury in adults with hemophagocytic lymphohistiocytosis.	25480521	Acute kidney injury (AKI) in the setting of hemophagocytic lymphohistiocytosis (HLH) is poorly characterized. This study aims to describe the incidence, clinical and biological features, and outcome associated with AKI in this population.Case series.Patients with secondary HLH admitted to a single center from February 2007 through January 2013. 95 patients were included in the study.AKI.Recovery of kidney function, 6-month mortality, and complete remission of the underlying disease.AKI was defined according to the KDIGO 2012 guideline. Recovery of kidney function was defined as improvement in serum creatinine level, with return to baseline serum creatinine level ±26.5μmol/L.HLH was related to hematologic malignancy in 73 (77%), infectious disease in 21 (22%), and autoimmune disease in 9 (10%) patients and was multifactorial in 10 (11%) patients. The cause was undetermined in 2 (2%) patients. The incidence of AKI during HLH is high (62%), and 59% of the AKI population required renal replacement therapy. Main causes of AKI were acute tubular necrosis (49%), hypoperfusion (46%), tumor lysis syndrome (29%), or HLH-associated glomerulopathies (17%). At 6 months, 32% of the patients with AKI had chronic kidney disease. Two factors were associated independently with 6-month mortality by multivariable analysis: AKI stage ≥ 2 (OR, 2.61; 95% CI, 1.08-6.29; P=0.03) and an underlying hematologic malignancy (OR, 3.1; 95% CI, 1.05-9.14; P=0.04). In patients with hematologic malignancy, AKI was associated with lower 6-month complete remission (non-AKI, 25%; AKI patients, 5%; P=0.05).Retrospective study, lack of histologic data.AKI in patients with HLH is frequent and adversely affects remission and survival. Early intensive management, including administration of etoposide, nephrotoxic drug withdrawal, prevention of tumor lysis syndrome, or aggressive supportive care, might improve kidney function and survival.
1643	Clinical characteristics, prognostic factors, and outcomes of adult patients with hemophagocytic lymphohistiocytosis.	25469675	Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by the activation of the mononuclear phagocytic system. The diagnosis of HLH in adults is challenging not only because the majority of the reported data are from pediatric patients, but also because HLH occurs in many disease entities. This study reports the clinical and laboratory findings and prognostic factors of adult HLH in a large cohort managed at a single medical center from 2003 to 2014. Seventy-three patients met the HLH-2004 diagnostic criteria. The median age was 51 years (range, 18-82 years); 41 (56.2%) were male. Patients manifested fever, cytopenias, and elevated ferritin in >85% of cases. Likely causes of HLH were as follows: 30 (41.1%) infections, 21 (28.8%) malignancies, 5 (6.8%) attributed to autoimmune disorders, 1 (1.4%) primary immunodeficiency, 2 (2.7%) post solid organ transplantation, and 13 (17.8%) idiopathic. The median overall survival was 7.67 months. Patients with malignancy-associated HLH had a markedly worse survival compared with patients with non-malignancy-associated HLH (median overall survival 1.13 vs. 46.53 months, respectively; P < 0.0001). In a multivariable analysis, malignancy (hazard ratio = 12.22; 95% CI: 2.53-59.02; P = 0.002) correlated with poor survival. Ferritin >50,000 µg/L correlated with 30-day mortality. Survival after a diagnosis of HLH is dismal, especially among those with malignancy-associated HLH. The development of a registry for adults with HLH would improve our understanding of this syndrome, validate diagnostic criteria, and help develop effective treatment strategies.
1644	Hemophagocytic lymphohistiocytosis (HLH): A heterogeneous spectrum of cytokine-driven immune disorders.	25466631	Hemophagocytic lymphohistiocytosis (HLH) comprises a group of life-threatening immune disorders classified into primary or secondary HLH. The former is caused by mutations in genes involved in granule-mediated cytotoxicity, the latter occurs in a context of infections, malignancies or autoimmune/autoinflammatory disorders. Both are characterized by systemic inflammation, severe cytokine storms and immune-mediated organ damage. Despite recent advances, the pathogenesis of HLH remains incompletely understood. Animal models resembling different subtypes of HLH are therefore of great value to study this disease and to uncover novel treatment strategies. In this review, all known animal models of HLH will be discussed, highlighting findings on cell types, cytokines and signaling pathways involved in disease pathogenesis and extrapolating therapeutic implications for the human situation.
1645	Catastrophic hemophagocytic lymphohistiocytosis in a young man with nephrotic syndrome.	25451951	In its early stage, necrotizing fasciitis mimics a milder cutaneous infection, such as cellulitis, and is an uncommon but potentially fatal complication of nephrotic syndrome. It may trigger an uncontrolled and catastrophic immune response, such as hemophagocytic lymphohistiocytosis (HLH).A 19-y-old man presented with steroid-resistant nephrotic syndrome and rapidly progressing Escherichia coli monomicrobial necrotizing fasciitis with bacteremia. The conditions developed one day after steroid therapy, leading to multiple organ dysfunction syndrome. A provisional diagnosis of HLH was promptly made, based upon the patient's fever, unremitting shock, marked pancytopenia, hyperferritinemia, hypofibrinogenemia, and the typical histiocytic hemophagocytosis in pleural effusion. Despite aggressive medical treatment and organ support, the patient died 8 days after transfer to our intensive care unit. Final bone marrow examination confirmed the diagnosis of HLH.Although nephrotic syndrome associated with E. coli infection is common, this is the first reported case of E. coli monomicrobial necrotizing fasciitis with bacteremia resulting in HLH in a patient with nephrotic syndrome.
1646	Management of the critically ill child with the sepsis/hemophagocytic lymphohistiocytosis/macrophage activation syndrome overlap syndrome.	31214471	Hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) is a frequently fatal disease, which can result in end-organ damage and death. This condition shares features with sepsis and systemic inflammatory response syndrome. Making a diagnosis of HLH can be challenging since most of the clinical and laboratory features of HLH are quite nonspecific. Timely diagnosis is critical to start therapy before damage by hypercytokinemia becomes irreversible. The treatment for patients with suspected acquired HLH/sepsis/systemic inflammatory response syndrome/multi organ dysfunction syndrome/ MAS overlap syndrome should be guided primarily by the severity of signs and symptoms, age of the patient, and underlying conditions. It is critical that the risks of treatment or non-treatment be weighed according to the clinical presentation of each patient. In this article, the authors discuss the diagnostic similarities between sepsis/HLH/MAS and management of the critically ill child with sepsis/HLH/MAS overlap syndrome.
1647	Chronic granulomatous disease presenting as hemophagocytic lymphohistiocytosis: a case report.	25422023	Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by recurrent infections and a dysregulated inflammatory response. Infection-triggered hemophagocytic lymphohistiocytosis (HLH), which manifests itself as pathologic hyperactive inflammation, has been observed in subjects with CGD. However, there have been no reports of HLH as the initial presentation with subsequent diagnosis of CGD. Furthermore, the primary therapeutic strategy for HLH focuses on immunosuppressive therapies, which limits immune-mediated tissue damage. With immunodeficiency, this therapeutic strategy may worsen the outcome. This article discusses an 8-week-old Hispanic male who presented with fever of unknown origin. The initial diagnostic evaluation demonstrated pathologic hyperactive inflammation, meeting the HLH-2004 diagnostic criteria without an identified infectious etiology. Immunosuppressive therapy was initiated, with subsequent disseminated candida septic shock and sepsis-induced multisystem organ failure. Additional evaluations ultimately established the diagnosis of CGD. We transitioned to an immune-enhancing strategy with granulocyte and immunoglobulin infusions, and intensified antifungal therapies. These interventions ultimately led to the clearance of the fungal infection and the resolution of the hyperactive inflammatory state. This case represents the first reported case of HLH as the presenting finding leading to the subsequent diagnosis of CGD. It serves as a reminder that both immunodeficiency and inflammatory disorders may share features of pathologic hyperactive inflammation and highlights the conundrum that clinicians face when treating HLH in the setting of an unresolved infection. In this case report, we demonstrate that immune-enhancing therapies may aid in the control and the clearance of the infection, thus paradoxically decreasing the pathologic hyperactive inflammatory response.
1648	Haemophagocytic lymphohistiocytosis: a cause of unresponsive malaria in a 5-year-old girl.	25410687	A 5-year-old immunocompetent girl presented with fever, jaundice, hepatosplenomegaly and pancytopenia. The peripheral blood smear demonstrated mixed malaria infection (Plasmodium vivax and Plasmodium falciparum). Fever was persistent despite antimalarials in the absence of any coexisting bacterial or viral infection. Laboratory findings included cytopaenia, hyperbilirubinaemia, hyperferritinaemia, hypertriglyceridaemia, hyponatraemia, deranged partial thromboplastin time, decreasing ESR and megaloblastic changes on bone marrow aspiration. A final diagnosis of haemophagocytic lymphohistiocytosis (HLH) with megaloblastic anaemia associated with severe mixed malaria was made. There was a dramatic response to corticosteroid treatment with improvement in her clinical condition. This report endorses the use of corticosteroids in malaria-associated HLH whenever there is no clinical improvement with antimalarials alone.
1649	Importance of hyperbilirubinemia in differentiation of primary and secondary hemophagocytic lymphohistiocytosis in pediatric cases.	25408853	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyper-inflammatory disease. It is difficult to differentiate between primary and secondary HLH based on clinical findings at the onset of disease. We aimed to find parameters that can help to differentiate primary and secondary HLH at initial diagnosis especially for physicians working in developing countries.We retrospectively analyzed data of 38 HLH patients who were admitted to the Pediatric Hematology Department of Gaziantep University between January 2009 and December 2013.Of 38 patients, 20 were defined as primary, and 18 were secondary HLH. The average age of primary and secondary HLH patients was 31±9 and 81±14 months, respectively (p=0.03). We found consanguinity rates significantly higher in primary HLH patients compared to secondary HLH patients (p=0.03). We found that total and direct bilirubin levels significantly increased in primary HLH patients compared to secondary HLH patients (p=0.006, p=0.044). Also, CRP levels were found markedly increased in secondary HLH patients compared to primary ones (p=0.017).We showed that cholestasis and hyperbilirubinemia findings of HLH patients at the initial diagnosis should be considered in favor of primary HLH, and an increased level of CRP should be considered in favor of secondary HLH.
1650	Hemophagocytic lymphohistiocytosis in association with primary cutaneous anaplastic large cell lymphoma.	25405042	Hemophagocytic lymphohistiocytosis (HLH) has a well known association with lymphomas, especially of T cell origin. Prognosis of lymphoma associated HLH is very poor, especially in T cell lymphomas; and, therefore, early diagnosis might alter the outcome. Though association of HLH with systemic anaplastic large cell lymphoma (ALCL) is known, its occurrence in primary cutaneous ALCL (C-ALCL) is distinctly rare. We aim to describe a case of C-ALCL (anaplastic lymphoma kinase (ALK)-) in an elderly male who succumbed to the complication of associated HLH, which was possibly triggered by coexistent virus infection. We briefly present the literatures on lymphoma associated HLH and discuss the histopathological differentials of cutaneous CD30+ lymphoproliferative disorders. We do suggest that HLH may pose diagnostic challenges in the evaluation of an underlying lymphoma and hence warrants proper evaluation for the underlying etiologies and/or triggering factors.
1651	A Case of Hemophagocytic Syndrome due to Tuberculosis: Uncommon Manifestation of a Common Disease.	25404945	Hemophagocytic syndrome, also known as hemophagocytic lymphohistiocytosis (HLH), is the manifestation of immune dysregulation. It is associated with ineffective but exaggerated immune response and infiltration of active lymphocytes and histiocytes in various organs. This devastating clinical condition has myriad of clinical and biochemical manifestations such as fever, splenomegaly, pancytopenia, hypertrygliceridemia, and hyperferritinemia. It can be either primary or secondary. Primary HLH usually presents in childhood. Secondary HLH occurs due to infection mostly viruses but other aetiologies are also important as early detection and treatment may improve survival. Hemophagocytosis due to tuberculosis is uncommon. Only handful of cases have been reported mostly in immunocompromised patients. We report a case of hemophagocytic syndrome associated with disseminated tuberculosis in an immunocompetent women highlighting early diagnosis and treatment is a demanding need in this devastating disease.
1652	Hematopoietic stem cell transplantation in infants.	25393821	It is rare for infants, who are less than 365 days old, to receive hematopoietic stem cell transplantation (HSCT). Our objective was to review the indications, survival, and late effects of infants who received HSCT.Between April 1992 and March 2010, a total of 1,363 children underwent HSCT (775 allogeneic [allo]; 588 autologous [auto]) in the Hospital for Sick Children, Toronto. Of these, 51 (3.7%) were infants.Seventeen infants received allo HSCT for a genetic metabolic disorder. The median age at HSCT was 211 days (29-334 days). After median follow-up of 8.9 years (2.9-20.2 years), 12 patients remained alive, representing an overall survival rate of 70%. Infants with non-metabolic disorders (n = 34); 10 (three neuroblastoma [NBL], three brain tumor, two acute meylogenous leukemia [AML], one rhabdomyosarcoma, and one retinoblastoma) received auto HSCT, and 24 (eight hemophagocytic lymphohistiocytosis [HLH], four juvenile meylomonocytic leukemia [JMML], four Wiscott-Aldrych Syndrome [WAS], three acute lymphoblastic leukemia [ALL], two AML, one severe aplastic anemia [SAA], one chronic granulomatous disease [CGD], and one amegakaryocytic thrombocytopenia) received allo HSCT. Their median age at HSCT was 255 days (142-365 days). At median follow-up of 8.7 years (2.5-17.6 years), 26 infants remained alive, representing an overall survival rate of 76%. In the auto HSCT category, eight of 10 infants are long-term survivors. Late effects such as organ dysfunction, endocrinopathy, and secondary tumors were within accepted range.The survival rate of infants who receive HSCT is encouraging.
1653	Pathogenesis of macrophage activation syndrome and potential for cytokine- directed therapies.	25386930	Macrophage activation syndrome (MAS) is an acute episode of overwhelming inflammation characterized by activation and expansion of T lymphocytes and hemophagocytic macrophages. In rheumatology, it occurs most frequently in patients with systemic juvenile idiopathic arthritis (SJIA) and systemic lupus erythematosus. The main clinical manifestations include cytopenias, liver dysfunction, coagulopathy resembling disseminated intravascular coagulation, and extreme hyperferritinemia. Clinically and pathologically, MAS bears strong similarity to hemophagocytic lymphohistiocytosis (HLH), and some authors prefer the term secondary HLH to describe it. Central to its pathogenesis is a cytokine storm, with markedly increased levels of numerous proinflammatory cytokines including IL-1, IL-6, IL-18, TNFα, and IFNγ. Although there is evidence that IFNγ may play a central role in the pathogenesis of MAS, the role of other cytokines is still not clear. There are several reports of SJIA-associated MAS dramatically benefiting from anakinra, a recombinant IL-1 receptor antagonist, but the utility of other biologics in MAS is not clear. The mainstay of treatment remains corticosteroids; other medications, including cyclosporine, are used in patients who fail to respond.
1654	More than just Langerhans cell histiocytosis: a radiologic review of histiocytic disorders.	25384298	Although Langerhans cell histiocytosis (LCH) is a familiar entity to most radiologists and to pediatric radiologists in particular, it is but one of a group of disorders caused by the overproduction of histiocytes, a subtype of white blood cells. Other less familiar diseases in this category are Erdheim-Chester disease (ECD), juvenile xanthogranuloma (JXG), Rosai-Dorfman disease (RDD), and hemophagocytic lymphohistiocytosis (HLH). This review describes the classification system, clinical manifestations, and pathophysiology of each disease, with particular attention to differential radiographic findings, including typical locations of involvement and varying appearances at radiography, computed tomography, magnetic resonance imaging, ultrasonography, and nuclear medicine imaging. Although LCH has a wide variety of manifestations and appearances, classic imaging findings include vertebra plana, skull lesions with a beveled edge, the "floating tooth" sign, bizarre lung cysts, and an absent posterior pituitary bright spot with infundibular thickening. The classic imaging findings of ECD are a perirenal rind of soft tissue and patchy long bone osteosclerosis. RDD has more nonspecific imaging findings, including lymphadenopathy (most commonly cervical) and intracranial lesions. Imaging findings in HLH are broad, with the most common abnormalities being hepatosplenomegaly, cerebral volume loss, and periventricular white matter abnormalities. JXG can manifest at imaging, but radiology does not play a major role in diagnosis. Familiarity with these disorders and their associated imaging findings facilitates correct and timely diagnosis. Imaging also features prominently in the assessment of treatment response.
1655	Acute myelocytic leukemia in a patient with hemophagocytic lymphohistiocytosis: A case report.	25364441	Hemophagocytic lymphohistiocytosis (HLH), also known as hemophagocytic syndrome, is an aggressive hyperinflammatory condition characterized by prolonged fever, cytopenias and hepatosplenomegaly, as well as hemophagocytosis by activated, morphologically benign macrophages. HLH may be characterized into two forms, familial and secondary HLH. Familial HLH usually manifests in children with genetic abnormalities associated with the cytotoxic function of NK and T cells, whereas secondary HLH usually occurs in older patients in combination with an associated condition, such as infection or malignancy, without an identifiable genetic abnormality. Malignancy-associated hemophagocytic lymphohistiocytosis is mostly accompanied by lymphoid neoplasms. The present study reports a rare case of this syndrome in combination with acute myeloblastic leukemia (AML-M2), in a patient with clonal karyotypic abnormalities. The patient was successfully treated with chemotherapy comprising daunorubicin (40 mg/m2 i.v., days 1-3) and cytosine arabinoside (100 mg/m2, 1-h i.v. infusion, days 1-7). All clinical symptoms disappeared following chemotherapy.
1656	Direct interaction between Id1 and Zrf1 controls neural differentiation of embryonic stem cells.	25361733	Id proteins are dominant-negative regulators within the HLH family of proteins. In embryonic stem cells (ESCs), Id1 and Id3 maintain the pluripotent state by preventing neural differentiation. The Id1-interacting protein Zrf1 plays a crucial role as a chromatin-bound factor in specification of the neural fate from ESCs. Here, we show that Id1 blocks Zrf1 recruitment to chromatin, thus preventing the activation of neural genes in ESCs. Upon differentiation, Id1 expression decreases thus inducing Zrf1 binding to neural genes. Importantly, depletion of Zrf1 rescues the expression of Polycomb targets involved in neural specification which are up-regulated in Id1 knock-out ESCs. We therefore identified Zrf1 as transcriptional regulator of neural fate downstream of Id1 in ESCs.
1657	Effect of perfusion pressure on the splanchnic circulation after CPB: a pilot study.	2535721425479475	The impact of different blood pressure targets is unknown for post cardiac surgery patient in the intensive care unit. We, therefore, investigated the effects of a mean arterial pressure (MAP) target of 65 or 85 mmHg on splanchnic oxygenation, metabolic function, cytokine regulation and gastric tonometry after cardiopulmonary bypass.Sixteen patients were randomized to the HLH group (high-low-high) where MAP of 85-65-85 mmHg was targeted or the LHL group where MAP 65-85-65 mmHg was targeted with norepinephrine infusion.MAP targets were achieved in all patients at all timepoints (64 ± 3, 84 ± 4; 65 ± 5, LHL group; vs. 84 ± 3; 66 ± 2; 85 ± 5 mmHg, HLH group). At corresponding timepoints, hepatic venous saturation was 41 ± 15%; 58 ± 24%; 56 ± 21% in the LHL group vs. 50 ± 19%; 43 ± 20%; 41 ± 18% in the HLH group (P<0.05). No changes were observed in cardiac output, global or trans-splanchnic lactate levels and cytokine levels or in gastric tonometry CO2.Achieving a MAP target of 85 mmHg by means of norepinephrine infusion after CPB appears safe for the splanchnic circulation.
1658	Structure of the Rad50 DNA double-strand break repair protein in complex with DNA.	25349191	The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50(NBD) (nucleotide-binding domain) in complex with Mre11(HLH) (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state.
1659	A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.	25340742	Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.
1660	[Clinical study of DEP regimen as a salvage therapy for adult refractory hemophagocytic lymphohistiocytosis].	25339326	To investigate the efficacy of liposomal doxorubicin together with etoposide and high dose methylprednisolone (DEP) as a salvage therapy for adult refractory hemophagocytic lymphohistiocytosis (HLH).Total 41 patients with refractory HLH were enrolled in this study. The efficacy of treatment with DEP regimen after 2 and 4 weeks were evaluated according to the United States Midwest Cooperative HLH Group.Of 41 refractory HLH patients, 28 were males and 13 females. The median age was 31(18-62) years old. The overall response rate (ORR) was 78.1%(32/41), including 12 patients (29.3%) achieved complete remission (CR) and 20 (48.8%) achieved partial remission (PR). The underlying disease of HLH were identified in 33 patients, including 1 case of primary HLH (CR), 20 cases of lymphoma associated HLH and 12 cases of EBV associated HLH. There were still 8 cases with unknown underlying disease. The 9 patients who had no response to DEP died within 2 to 4 weeks after salvage therapy. Twenty of the 32 patients who achieved PR or CR survived to undergo subsequent chemotherapy, allogenic hematopoietic stem cell transplantation (allo-HSCT) or splenectomy.The single-arm study suggested that DEP regimen appeared to be an effective salvage protocol for adult patients with refractory HLH.
1661	Interleukin-2-inducible T-cell kinase (ITK) deficiency - clinical and molecular aspects.	25339095	In patients with underlying immunodeficiency, Epstein-Barr virus (EBV) may lead to severe immune dysregulation manifesting as fatal mononucleosis, lymphoma, lymphoproliferative disease (LPD), lymphomatoid granulomatosis, hemophagocytic lymphohistiocytosis (HLH) and dysgammaglobulinemia. Several newly discovered primary immunodeficiencies (STK4, CD27, MAGT1, CORO1A) have been described in recent years; our group and collaborators were able to reveal the pathogenicity of mutations in the Interleukin-2-inducible T-cell Kinase (ITK) in a cohort of nine patients with most patients presenting with massive EBV B-cell lymphoproliferation. This review summarizes the clinical and immunological findings in these patients. Moreover, we describe the functional consequences of the mutations and draw comparisons with the extensively investigated function of ITK in vitro and in the murine model.
1662	The ambiguous boundary between EBV-related hemophagocytic lymphohistiocytosis and systemic EBV-driven T cell lymphoproliferative disorder.	25337215	Epstein Barr virus (EBV)-related hemophagocytic lymphohistiocytosis (EBV-HLH) is a form of acquired, infection-related HLH which typically represents a fulminant presentation of an acute EBV infection of CD8+ T cells with 30-50% mortality rate. Systemic EBV-positive lymphoproliferative disease of childhood (SE-LPD) is a rare T cell lymphoproliferative disorder predominantly arising in the setting of acute EBV infection, often presenting with HLH. Since both entities have been associated with clonal T cell populations, the discrimination between these diseases is often ambiguous. We report a unique case of a 21 years old female who presented with clinical and laboratory findings of florid HLH in the setting of markedly elevated EBV titers (>1 million) and an aberrant T cell population shown to be clonal by flow cytometry, karyotype, and molecular studies. This case raises the differential of EBV-HLH versus SE-LPD. Review of the literature identified 74 cases of reported EBV-HLH and 21 cases of SE-LPD with associated HLH in 25 studies. Of those cases with available outcome data, 62 of 92 cases (67%) were fatal. Of 60 cases in which molecular clonality was demonstrated, 37 (62%) were fatal, while all 14 cases (100%) demonstrating karyotypic abnormalities were fatal. Given the karyotypic findings in this sentinel case, a diagnosis of SE-LPD was rendered. The overlapping clinical and pathologic findings suggest that EBV-HLH and SE-LPD are a biologic continuum, rather than discrete entities. The most clinically useful marker of mortality was an abnormal karyotype rather than other standards of clonality assessment.
1663	Hemophagocytic lymphohistiocytosis associated with cytomegalovirus infection in an immunocompetent infant: a diagnostic and therapeutic challenge!	25332603	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal syndrome that results from inappropriate activation of the immune system. Many viral agents are known to trigger HLH but cytomegalovirus (CMV) associated HLH is rarely described. We report a case of CMV related HLH in a 3½ month old immunocompetent male infant who presented with fever, respiratory distress and hepatosplenomegaly. He had fulminant sepsis like course in the hospital as he continued to have hectic fever spikes, progressive pneumonia, increasing hepatosplenomegaly and multiple episodes of generalized convulsions. Investigations revealed bicytopenia, biochemical hepatitis, hyperferritinemia and hypofibrinogenemia. CMV IgM serology was reactive in both infant and mother. Diagnosis of CMV-HLH was made as per HLH 2004 diagnostic protocol. Infant was successfully treated with intravenous ganciclovir along with dexamethasone and etoposide.
1664	Hepatitis a virus infection-associated hemophagocytic lymphohistiocytosis in two children.	25332588	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by high fever, maculopapular rash, neurological symptoms, abnormal liver functions and coagulopathy. Primary HLH is due to an underlying genetic abnormality. Secondary HLH are due to an underlying infection, autoimmune disease or malignancy. Secondary HLH due to viral infections are commonly due to the herpes group commonest of which is the Ebstein Barr virus (EBV). We describe two children with virus associated hemophagocytic lymphohistiocytosis (VAHLH) secondary to hepatitis A infection.
1665	Hemophagocytic lymphohistiocytosis masking the diagnosis of lymphoma in an adolescent male.	25332560	Hemophagocytic lymphohistiocytosis (HLH) association with an underlying lymphoma is an uncommon entity in paediatrics. It may precede lymphoma diagnosis by several years or may occur at the time of remission or relapse of lymphoma. Simultaneous occurrence of HLH & lymphoma is rare. We here with report a case where HLH was the initial presentation which masked the diagnosis of lymphoma, however tissue biopsy revealed the underlying non-Hodgkin's lymphoma.
1666	Hemophagocyte lymphohistiocytosis secondary to bilateral epididymal diffuse large B-cell lymphoma.	25332548	We report a rare case of hemophagocytic lymphohistiocytosis (HLH) secondary to bilateral epididymal lymphoma. The patient is a man of 46 years old. He suffered fever 5 months before the admission. Physical examination shows splenohepatomegalia without lymphadenopathy. Laboratory studies revealed pancytopenia and coagulopathy, hyper ferritin level, large foamy macrophages containing other hematopoietic elements in bone marrow. After treatment with HLH-04 protocol, the patient recovered, but relapsed 1 month later and suffered pudendal pain. A color Doppler of the scrotum shows bilateral epididymis swollen. Pathological diagnosis of biopsy is diffuse large B cell lymphoma (DLBCL). After two cycles cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab together with intrathecal injections, the tumors on the epididymides disappeared, but relapsed intracalvarium. The patient stopped treatment due to financial reasons. Hemophagocytic lymphohistiocytosis secondary lymphoma in a sanctuary site has not been reported before and should not be ignored. Transplantation is necessary for long-time survival.
1667	Reactive hemophagocytic lymphohistiocytosis after hepatitis a infection.	25332533	Hemophagocytic lymphohistiocytosis (HLH) is a clinical condition which result in cytotoxic Tcell and antigen presenting cell overproduction and also their cytokines. Hepatitis A Virus associated HLH is very rare condition among other viruses. This condition is often difficult to diagnose, so treatment is often delayed. Here we present a case of adolescent boy with secondary virus associated HLH diagnosis with Hepatitis A infection and successfull treatment by short course of intravenous immunoglobulin and steroid.
1668	Hemophagocytosis on bone marrow aspirate cytology: single center experience in north himalayan region of India.	25328776	The differentiation of hemophagocytic lymphohistiocytosis (HLH) with other causes of hemophagocytosis is essential as HLH is life-threatening condition and requires definite clinical and biochemical criteria for its diagnosis.The present study was conducted to study hemophagocytosis on bone marrow aspirates and to observe if there is any difference on bone marrow examination between HLH and non HLH cases showing hemophagocytosis.The study reviewed all the cases of bone marrow aspirate for evidence of hemophagocytosis in which at least three smears and 500 nucleated cells were observed for every case. The cases were provisionally diagnosed as HLH according to the proposed HLH diagnostic criteria, 2009.A total of 80 cases showed hemophagocytosis in the present study with infections followed by HLH being the most common cause. Pancytopenia and erythroid hyperplasis were common hematological presentation. Moderate to severe hemophagocytosis was observed in HLH cases while mild grade in non HLH cases.The study concludes that hemophagocytosis even if observed in single cell should always be documented in the bone marrow reports. It may be the only indicator of subtle infection in the marrow. Pancytopenia and higher grade of hemophagocytosis on bone marrow examination may be helpful in establishing an early differentiation of HLH and non HLH cases.
1669	Hodgkin's Lymphoma Revealed by Hemophagocytic Lymphohistiocytosis in a Child.	25328742	Hemophagocytic lymphohistiocytosis (HLH) is a severe life-threatening disorder, responsible for extensive phagocytosis of hematopoietic cells and causing a multisystem organ failure. If lymphomas are common causes of HLH, the association with Hodgkin's lymphoma is rarely described in children. We report a case of a 9-year-old boy presenting with HLH as an initial manifestation of Hodgkin's lymphoma. He has been suffering from persistent high fever, asthenia, weight loss, and hepatosplenomegaly with no lymphadenopathy. The diagnosis of HLH secondary to infectious disease was initially worn. The patient received high-dose intravenous immunoglobulin with broad-spectrum antibiotics. However, his state got worse with the onset of dry cough and pleural effusion. Histopathologic examination of pleural fluid showed the presence of Reed-Sternberg cells. The outcome was favorable after treatment by corticosteroid and chemotherapy. Hodgkin's lymphoma revealed by HLH is a source of delayed diagnosis and should be borne in mind in children.
1670	Hemoperfusion for Hodgkin lymphoma-associated hemophagocytic lymphohistiocytosis.	25318804	Hemophagocytic lymphohistiocytosis (HLH), which is associated with various underlying conditions, is characterized by hypercytokinemia. Because it is frequently lethal, immediate mitigation of the hypercytokinemia is vital to save patients, particularly when treatments for the patient's underlying condition are ineffective on HLH. We herein present a case of Hodgkin lymphoma associated with HLH in which the HLH did not improve even after chemotherapy. We attempted to save the patient using hemoperfusion with a polymyxin B-immobilized fiber column to remove cytokines; following this treatment, the patient rapidly recovered. Hemoperfusion may be a strategic method to rescue intractable HLH patients.
1671	Hemophagocytic Lymphohistiocytosis (HLH) Associated with T-Cell Lymphomas: Broadening our Differential for Fever of Unknown Origin.	25317396	Hemophagocytic lymphohistiocytosis (HLH), due to the excessive activity of histiocytes and lymphocytes, is a rare but aggressive disease that typically occurs in infancy but can be seen in all ages. If left untreated, patients with HLH may live for only a few months and die from multi-organ failure.We present two cases of HLH diagnosis. Fever, spleen, and hepatic abnormalities were noted in both cases.Early diagnosis is the key and these two cases of similar etiology highlight how fever of unknown origin should force us to broaden our differential.
1672	Hereditary and acquired hemophagocytic lymphohistiocytosis.	25310211	Hemophagocytic lymphohistiocytosis (HLH) is a rare but life-threatening hyperinflammatory/hypercytokinemia syndrome clinicopathologically manifested by fever, hepatosplenomegaly, cytopenias, liver dysfunction, and hemophagocytosis.We searched the medical literature for English-written articles and analyzed data regarding the diagnosis, pathoetiology, prognosis, and management of HLH.HLH can be subcategorized into primary/genetic (PHLH) or secondary/acquired (SHLH) according to etiology. PHLH, including familial HLH and inherited immune deficiency syndromes, typically occurs in children harboring underlying genetic defects, whereas SHLH frequently manifests in adults and is associated with infection, autoimmunity, immune suppression, or malignancy. The pathogenesis of HLH is still elusive. Its known mechanisms include somatic mutations in gene coding for proteins implicated in the cytotoxic pathways of cytotoxic T or natural killer cells. The impaired ability of these cells to kill target cells leads to an uncontrolled hypercytokinemia and hyperinflammatory process, triggering hemophagocytosis and multiorgan failure. Corticosteroids, chemotherapy, and immunotherapy are the mainstay therapeutic strategies. The consolidation with allogeneic hematopoietic stem cell transplantation is a potentially curative option for PHLH and refractory or relapsed SHLH.Understanding of the pathophysiology of HLH has improved in the last decade. The establishment of diagnostic and treatment guidelines for PHLH and SHLH has resulted in earlier diagnoses and the rapid initiation of therapy, both of which are associated with favorable outcomes.
1673	Hemophagocytic lymphohistiocytosis: An unusual presentation of tuberculosis in hemodialysis patients.	25307022	We report a series of three patients with end-stage renal disease on maintenance hemodialysis presenting with hemophagocytic lymphohistiocytosis (HLH) as an unusual manifestation of extrapulmonary tuberculosis. All three patients were middle-aged men. They presented with fever, pancytopenia, varying degrees of hepatosplenomegaly, abnormal liver function tests, coagulopathy, increased serum ferritin, and triglycerides. Tests for fever work-up were negative. Bone marrow examination revealed hemophagocytosis and caseating granuloma. Acid fast bacilli were demonstrated in two patients. The HLH-2004 diagnostic criteria suggested by the histiocytic society were followed to arrive at the diagnosis. All of them succumbed to death even before the definitive diagnosis could be made. We suggest that aggressive diagnostic work-up must be done when hemodialysis patients present with fever and pancytopenia. Priority should be toward early diagnosis and appropriate treatment to improve the prognosis.
1674	Acute brucellosis with typical hemophagocytic lymphohistiocytosis accompanying elevated tumor markers.	25305773	We reported a typical brucellosis, which was diagnosed as hemophagocytic lymphohistiocytosis (HLH). Although some tumor markers (CEA, CYFRA21-1, NSE, CA19-9) in the patient's serum were elevated, carcinomas were excluded by a variety of inspections including bone marrow aspirations, ultrasound examinations, and whole-body PET-CT scans. It was concluded that serum tumor markers are considered medically necessary as a screening test for brucellosis with HLH, however, detailed inspections were needed to make a final diagnosis. Moreover, combination of epidemiology investigations and laboratory inspections were helpful to determine the etiology of HLH and initiate the corresponding treatments.
1675	[Analysis of clinical phenotype and genetic mutations of a pedigree of familial hemophagocytic lymphohistiocytosis].	25297583	To analyze mutations in a pedigree of familial hemophagocytic lymphohistiocytosis (FHLH) from Sichuan and provide genetic counseling for the family.Clinical data of a case with FHLH diagnosed at West China Second Hospital was retrospectively analyzed. Genomic DNA was extracted from peripheral blood samples of the proband and his family members. Eight candidate genes for primary HLH were amplified with PCR and analyzed by direct sequencing.The proband was diagnosed as HLH based on clinical manifestations of recurrent fever for 2 months, hepatosplenomegaly, lymphadenopathy, pancytopenia, hyperferritinemia, and decreased fibrinogen and hemophagocytosis in bone marrow. Genetic testing for primary HLH was carried out considering the relapse of illness after hormone therapy for 8 weeks and the family history. The results of gene sequencing showed that the proband has carried compound heterozygous mutations in PRF1 gene (c.1349C> T in exon 3 and c.445G> A in exon 2). His father has carried a heterozygous mutation (c.445G> A in exon 2) and nonsense mutation (c.900C> T in exon 3), and his mother carried a heterozygous mutation (c.1349C> T in exon 3). Both c.1349C> T and c.445G> A have been previously reported as pathogenic mutations.The family has been diagnosed as familial HLH type 2 based on clinical and laboratory examinations and molecular genetic testing. Gene sequencing has indicated that is was a recessive type familial HLH.
1676	Hemophagocytic lymphohistiocytosis in a patient with Goodpasture's syndrome: a rare clinical association.	25284133	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening clinical syndrome. HLH can occur in the setting of an autoimmune disease, chronic immunosuppression, malignancy, and infection. We discuss a rare case of a young woman who was diagnosed with Goodpasture's syndrome that was most likely complicated by HLH. To the best of our knowledge, this is the first report of HLH in the setting of this rare autoimmune disease.A 31-year-old woman who was diagnosed with Goodpasture's syndrome 7 years prior presented with febrile neutropenia. She was initially receiving treatment with azathioprine and prednisone, which was subsequently switched to hydroxychloroquine. Over time, she had developed polyarthritis and was later diagnosed with MPO-ANCA-positive vasculitides. On this admission, her clinical status deteriorated from persistent pancytopenia. This was initially attributed to the immunosuppressive effect of hydroxychloroquine. A bone marrow biopsy was performed and revealed hypercellular bone marrow without any cytogenetic abnormalities. Due to a prolonged pancytopenia thought to be of autoimmune etiology, treatment with high-dose steroids was initiated. With the persistent febrile episodes, hepatosplenomegaly on examination, and laboratory workup that revealed hyperferritinemia and pancytopenia, HLH syndrome was suspected. A repeat bone marrow biopsy confirmed this diagnosis with the presence of hemophagocytosis, demonstrated by the presence of histiocytes engulfing erythroid cells. She also met 5 of 8 diagnostic criteria, which confirmed the diagnosis of HLH. The patient eventually died despite aggressive treatment with high-dose steroid therapy for her autoimmune disorder, as well intravenous antibiotics and supportive care for her underlying infections.HLH is a syndrome marked by a hyper-inflammatory state aggravated by specific triggers. To make the diagnosis of HLH, at least 5 of the 8 criteria must be met. Treatment involves suppression of the overwhelming inflammatory response by the use of immunomodulators. The mortality rate can range from 50-90% due to delayed recognition and onset of treatment. Here, we present a rare case of Goodpasture's syndrome with overlap and pauci-immune vasculitis, which may have triggered the HLH. This correlation has not been described before in the literature.
1677	A review of luteinising hormone and human chorionic gonadotropin when used in assisted reproductive technology.	25280580	Gonadotropins extracted from the urine of post-menopausal women have traditionally been used to stimulate folliculogenesis in the treatment of infertility and in assisted reproductive technology (ART). Products, such as human menopausal gonadotropin (hMG), consist not only of a mixture of the hormones, follicle-stimulating hormone (FSH), luteinising hormone (LH) and human chorionic gonadotropin (hCG), but also other biologically active contaminants, such as growth factors, binding proteins and prion proteins. The actual amount of molecular LH in hMG preparations varies considerably due to the purification process, thus hCG, mimicking LH action, is added to standardise the product. However, unlike LH, hCG plays a different role during the natural human menstrual cycle. It is secreted by the embryo and placenta, and its main role is to support implantation and pregnancy. More recently, recombinant gonadotropins (r-hFSH and r-hLH) have become available for ART therapies. Recombinant LH contains only LH molecules. In the field of reproduction there has been controversy in recent years over whether r-hLH or hCG should be used for ART. This review examines the existing evidence for molecular and functional differences between LH and hCG and assesses the clinical implications of hCG-supplemented urinary therapy compared with recombinant therapies used for ART.
1678	In vitro antiproliferative effect of Helix aspersa hemocyanin on multiple malignant cell lines.	25265853	As an extension of our studies on the antitumour properties of various hemocyanins, we sought to compare the antiproliferative effects of hemocyanins derived from two snail species: Helix lucorum (HIH) and Helix aspersa (HaH). This is the first report on the antitumour effects of HaH. We hypothesized that HaH has antitumour effects not only against bladder cancer, as previously shown with other hemocyanins, but also on other cancer cell lines. The antiproliferative properties of the mentioned hemocyanins were investigated in vitro on the following human cell lines: bladder cancer (CAL-29 and T-24), ovarian cancer (FraWü), acute monocytic leukemia (THP-1), prostate cancer (DU-145), glioma cancer (LN-18), and Burkitt's lymphoma (Daudi). The properties of HaH were compared to those of HlH, keyhole limpet hemocyanin (KLH), and two positive controls (doxorubicin and mitomycin C). An antiproliferative effect of the total molecule and one structural subunit of HaH, betac-HaH, against both bladder cancer cell lines, T-24 and CAL-29, was observed. The cytotoxic effect of HaH ranged between 15% and 60% among the other tested cell lines. The endotoxin contamination did not affect the efficacy of HaH. Therefore, HlH and HaH could be appropriate for more detailed investigations of their use as antitumour agents for the studied cancers.
1679	A hemophagocytic lymphohistiocytosis patient initiated with prominent liver dysfunction: a case report.	25264890	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and potentially fatal syndrome that results from inappropriate activation of lymphocytes and macrophages. It is characterized by fever, hepatosplenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, and pathologic findings of hemo- phagocytosis in the bone marrow or other tissues. We report an adult HLH case admitted to hepatology department.
1680	[Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Chinese children: a primary study on clinical characteristics and gene mutations].	25253006	To study the clinical features and gene mutations of EBV-HLH in Chinese children.Sixteen pediatric patients with Epstein-Barr virus-associated HLH were enrolled the study from May 2011 to September 2012, who were admitted to Capital Institute of Pediatrics. All patients' clinical features were recorded and their nucleotide sequences of all exons and their flanking intronic sequences of perforin (PRF1) gene, protein unc-13 homolog D (UNC13D) gene, two fusion protein genes of syntaxin 11 (STX11)gene, synaptic binding protein 2 (STXBP2) gene, SH2 domain 1A (SH2D1A) gene, and the X-linked inhibitor of apoptosis protein (XIAP) gene were amplified by PCR followed by direct sequencing. Based on HLH gene detection results, all patients divided into the positive and negative gene EBV-HLH subgroups. Statistical analysis was conducted using SPSS 11.5 software.Seven of sixteen pediatric patients with EBV-HLH were identified with heterozygous, hemizygous, and homozygous mutations in PRF1, UNC13D, STXBP2, and SH2D1A. No significant differences were found on the gender, age, illness duration, EBV load, the positive duration of EBV-DNA, lab results, clinical symptoms, treatment, and the outcome between the HLH gene positive and negative subgroups (P > 0.05).A considerable EBV-HLH pediatric patients have genetic defects, and HLH gene defects may not affect the clinical features and treatment of EBV-HLH pediatric patients.
1681	Expression dynamics and functions of Hes factors in development and diseases.	25248479	Hes genes, encoding basic helix-loop-helix (HLH) transcriptional repressors, are mammalian homologues of Drosophila hairy and Enhancer of split genes, both of which are required for normal neurogenesis in Drosophila. There are seven members in the human Hes family, Hes1-7, which are expressed in many tissues and play various roles mainly in development. All Hes proteins have three conserved domains: basic HLH (bHLH), Orange, and WRPW domains. The basic region binds to target DNA sequences, while the HLH region forms homo- and heterodimers with other bHLH proteins, the Orange domain is responsible for the selection of partners during heterodimer formation, and the WRPW domain recruits corepressors. Hes1, Hes5, and Hes7 are known as downstream effectors of canonical Notch signaling, which regulates cell differentiation via cell-cell interaction. Hes factors regulate many events in development by repressing the expression of target genes, many of which encode transcriptional activators that promote cell differentiation. For example, Hes1, Hes3, and Hes5 are highly expressed by neural stem cells, and inactivation of these genes results in insufficient maintenance of stem cell proliferation and prematurely promotes neuronal differentiation. Recently, it was shown that the expression dynamics of Hes1 plays crucial roles in proper developmental timings and fate-determination steps of embryonic stem cells and neural progenitor cells. Here, we discuss some key features of Hes factors in development and diseases.
1682	E(spl): genetic, developmental, and evolutionary aspects of a group of invertebrate Hes proteins with close ties to Notch signaling.	25248478	Enhancer-of-split (E(spl)) was genetically characterized in Drosophila as a dominant mutation that interacts with an allele of Notch, the receptor in a multipurpose signaling pathway throughout development. Although dominant mutations are often not informative of the normal gene function, E(spl) turned out to encode a family of seven paralogous basic helix-loop-helix proteins of utmost importance in the implementation of the Notch signal in the receiving cell. They are transcriptionally induced by Notch in almost every instance where the signal is deployed, and they participate in numerous feedback circuits, where they interface with a panoply of additional more tissue-specific Notch targets to ensure the proper signaling outcome. Besides the bHLH domain, E(spl) contain a characteristic Orange domain and are classified in the Hes (hairy and enhancer-of-split) branch of the bHLH-Orange proteins. They act as DNA-binding repressors in close collaboration with the corepressor Groucho. In this review, we will focus on the regulation of E(spl) expression and on the function of E(spl) proteins. In the latter section, we will present some of the best-studied developmental events where E(spl) function has been analysed as well as the molecular mechanism of E(spl) activity that has transpired. Finally, we will review the evolution of this protein family, which, albeit of relatively recent origin, present only in insects and crustaceans, has undergone extensive diversification, including gene loss and duplication. Importantly, many of the characteristics of E(spl) proteins are more deeply rooted in the very ancient larger bHLH-O family, which seems to have forged a connection with the Notch pathway from the very beginning of multicellular animal life.
1683	Id proteins: small molecules, mighty regulators.	25248477	The family of inhibitor of differentiation (Id) proteins is a group of evolutionarily conserved molecules, which play important regulatory roles in organisms ranging from Drosophila to humans. Id proteins are small polypeptides harboring a helix-loop-helix (HLH) motif, which are best known to mediate dimerization with other basic HLH proteins, primarily E proteins. Because Id proteins do not possess the basic amino acids adjacent to the HLH motif necessary for DNA binding, Id proteins inhibit the function of E protein homodimers, as well as heterodimers between E proteins and tissue-specific bHLH proteins. However, Id proteins have also been shown to have E protein-independent functions. The Id genes are broadly but differentially expressed in a variety of cell types. Transcription of the Id genes is controlled by transcription factors such as C/EBPβ and Egr as well as by signaling pathways triggered by different stimuli, which include bone morphogenic proteins, cytokines, and ligands of T cell receptors. In general, Id proteins are capable of inhibiting the differentiation of progenitors of different cell types, promoting cell-cycle progression, delaying cellular senescence, and facilitating cell migration. These properties of Id proteins enable them to play significant roles in stem cell maintenance, vasculogenesis, tumorigenesis and metastasis, the development of the immune system, and energy metabolism. In this review, we intend to highlight the current understanding of the function of Id proteins and discuss gaps in our knowledge about the mechanisms whereby Id proteins exert their diverse effects in multiple cellular processes.
1684	The Hand2 gene dosage effect in developmental defects and human congenital disorders.	25248475	Heart- and neural crest derivatives-expressed (Hand) proteins belong to the Twist family of the basic helix-loop-helix (bHLH) transcription factors, and play crucial roles in the development of several organs. They form heterodimers with Twist1 via their HLH domain. Disruption of the expression balance between Hand2 and Twist1 causes limb malformation, indicating that the expression level of Hand2 relative to Twist1 is essential for limb development. Mutations of the TWIST1 and TWIST2 genes are involved in human diseases. Although, the functions of the Hand proteins are indispensable for limb, heart, and craniofacial development, mutations of the Hand genes that are causative of human diseases remain elusive. Recently, comparative analyses of a human chromosomal disorder, partial trisomy distal 4q, and its mouse model, which is a spontaneously occurring mutant, clearly demonstrated that over dosage of Hand2 results in developmental defects of limbs, craniofacial, and lumbar vertebrae, and that trisomy of the Hand2 gene directly causes a human congenital disorder. In this review, we focus on gene dosage effect of Hand2 in limb, heart, and craniofacial development, and discuss its implication in human diseases.
1685	[Establishment of HLH-like mouse model with CPG-ODN and IFN-γ].	25246254	To establish a hemophagocytic lymphohistiocytosis (HLH)-like mouse model induced by CpG oligodeoxynucleotide (CpG-ODN1826) and interferon (IFN)-γ for further study on therapy.Wild type adult C57BL/6 mice were administered with PBS or CpG-ODN1826 (50 μg) by intraperitoneal injection every two day and IFN-γ subcutaneous injection every day. Parameters of HLH were evaluated on day 10.As compared to control, HLH-like symptoms in CpG group were characterized with pancytopenia accompanied by increased ratios of monocytes, alanine aminotransferase [(198.7±54.2)IU/L], triglyceride level [(12.1±0.6)g/L], and serum ferritin [(708.4±11.8)pmol/L]; decreased albumin [(217.7±4.3)g/L], fibrinogen [(17.1±1.9)g/L] (all P<0.05). Hepatosplenomegaly was obvious in CpG group. The liver in CpG group had multifocal hepatocytes necrosis and perivascular inflammations. Spleen had expanding red pulp and hyperplastic nucleated cells. Furthermore, macrophages in the liver and spleen were largely activated. Hemophagocytosis were observed in liver, spleen and bone marrow smear. The CpG group was alive during experiment, other than significant decreased activity after the first injection of CpG-ODN.These data demonstrate that repeated administration of CpG-ODN1826 and IFN-γ could induce HLH-like symptoms without fatal condition in wild type C57B/L mice. This protocol could establish a mild HLH-like mouse model, which could be useful for further study on HLH.
1686	[Clinical characteristics of 192 adult hemophagocytic lymphohistiocytosis].	25246246	To analyze the clinical manifestations, laboratory data, therapy, and prognosis in patients with hemophagocytic lymphohistiocytosis (HLH).A retrospective study was carried out in 192 adult patients with HLH between 2003 and 2013.Of the 192 cases, 70 cases were secondary to cancer and 64 cases secondary to infection. According to HLH-2004 criteria, the coincidence rate of indices were: fever (98.96%), high level of serum ferritin (94.27%), increased level of soluble interleukin- 2 receptor(sCD25) (94.79%), decreased or absent activity of NK cells (94.27%), cytopenias (80.73%), splenomegaly (80.21%), emophagocytosis in bone marrow, spleen or lymph nodes (74.48%), hypofibrinogenemia (50.52%), hypertriglyceridemia (37.50%). In addition, 94.27% of patients were presented with liver dysfunction, 96.35% with infections, and 75.52% with coagulopathy. Incidences of central nervous system symptoms and rash were 19.27% and 20.31%, respectively. Among cancer, infection and rheumatic group, there were statistically differences on white blood cells (WBC), platelet (PLT), sCD25, alanine aminotransferase, aspartate aminotransferase, total bilirubin and globulin(GLO) (P<0.05). The differences of WBC, PLT, albumin (ALB), GLO, brain natriuretic peptide, creatinine, urea nitrogen between survival group and death group had statistical significance.The secondary HLH occurs from various underlined diseases. Cancer, especially T- cell lymphoma, is the main cause, Secondly, it is EB virus infection. The diagnostic sensitive indicators are Persistent fever, higher level of serum ferritin, low or absent NK-cell activity, and increased sCD25 were the most valuable parameters for diagnosis. Cytopenias were not common in early phase of HLH secondary to rheumatic diseases. WBC, PLT, ALB, GLO could be used as the preliminary parameters for diagnosis. Cardiac insufficiency, renal insufficiency and coagulation dysfunction play important roles in prognosis.
1687	Gene therapy for haemophagocytic lymphohistiocytosis.	25245087	Haemophagocytic lymphohistiocytosis (HLH) describes a severe and often fatal immunodysregulatory disorder caused primarily by the uncontrolled activation and proliferation of T cells and macrophages. A number of genetic defects mainly involving defective granule exocytosis and effector cell cytotoxicity have been identified and well characterised at the molecular and cellular level. These conditions have limited therapeutic options and given the predominant restriction of the causative gene to the haematopoietic system, they have become attractive targets for haematopoietic cell gene therapy. Pre clinical studies in murine models of HLH due to perforin deficiency have shown correction of the disease phenotype as a result of autologous haematopoietic stem cell (HSC) gene transfer using lentiviral vectors. In a murine model of X-linked lymphoproliferative disease (XLP1), HSC gene transfer is able to correct the immunological manifestations of the disease. These encouraging murine studies have led to further work to develop clinically applicable strategies. An alternative approach is to correct defective T cells as this approach is safer than HSC gene therapy and may allow early control of the HLH through the engraftment of functional gene modified effector T cells. Both strategies are now in development and a gene therapy option for certain genetic forms of HLH may soon enter clinical trials.
1688	Ileal mass-like lesion induced by Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in a patient with aplastic anemia.	25243347	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory syndrome characterized by activated macrophages engulfing erythrocytes, leukocytes, platelets, and their precursor cells in bone marrow, liver, spleen, or lymph nodes. We report a case of Epstein-Barr virus (EBV)-associated HLH unusually presenting as an ileal mass. A 23-year-old man presented initially with persistent fever unresponsive to antibiotics and pancytopenia. A bone marrow aspiration and biopsy were used to diagnose the patient with aplastic anemia and HLH. A relatively well-defined low-density mass was radiologically noted in the terminal ileum, along with enlarged lymph nodes, and was suspected to be malignant lymphoma or an abscess. The ileocecectomy specimen revealed a transmural hemorrhagic infarction with numerous activated macrophages phagocytosing erythrocytes, plasma cells, and lymphocytes, and he was diagnosed with EBV-associated HLH. The patient received an allo-unrelated peripheral blood stem-cell transplantation and expired due to graft-versus-host disease following liver failure. The present case is very unique, in that EBV-associated HLH presented with an unusual ileal mass resulting from hemorrhagic infarction in a patient with aplastic anemia, suggesting variability in the biological behavior of EBV-associated disease.
1689	Primary Epstein-Barr virus infection and probable parvovirus B19 reactivation resulting in fulminant hepatitis and fulfilling five of eight criteria for hemophagocytic lymphohistiocytosis.	25242697	A case of primary Epstein-Barr virus (EBV) infection/parvovirus B19 reactivation fulfilling five of eight criteria for hemophagocytic lymphohistiocytosis (HLH) is presented. Despite two coinciding viral infections, massive splenomegaly, and fulminant hepatitis, the patient had a good clinical outcome, probably due to an early onset form of HLH with normal leukocyte count, normal natural killer (NK) cell function, and a lack of hemophagocytosis.
1690	Hemophagocytic lymphohistiocytosis and primary immune deficiency disorders.	25241079	Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled immune activation and is traditionally associated with inherited gene defects or acquired causes. In addition to abnormalities in cytotoxic granules and lysosomes, various primary immune deficiency disorders (PID) have been identified among patients suffering from HLH. Our purpose was twofold: to better characterize and detail the association between PID and HLH. We found that HLH occurs infrequently among patients with PID, particularly those suffering from abnormalities that impair T cell function. The prognosis of patients suffering from PID and HLH is poor, emphasizing the need for rapid clinical and genetic diagnosis of the PID as well as initiation of appropriate management of the HLH, including allogeneic hematopoietic stem cell transplantations. The association of HLH and PID implicates abnormal T cell function as an important factor in HLH development. It also suggests that the partition of HLH into genetic versus acquired forms might be misleading.
1691	Genetic features of late onset primary hemophagocytic lymphohistiocytosis in adolescence or adulthood.	25233452	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of uncontrolled immune activation leading to extreme inflammation. Primary HLH was once believed to be a disease that occurred only in infancy or young children, and was rarely diagnosed in adults. It is now understood that patients can develop primary HLH in their adolescence or adulthood. This study included 252 adolescent and adult patients with a clinical diagnosis of HLH from 35 general medical institutions across mainland China. All exons and 50 bp of flanking intronic sequence of six HLH-related genes (PRF1, UNC13D, STX11, STXBP2, SH2D1A, and BIRC4) were sequenced in these patients. We identified mutations in 18/252 (7.1%) of the patients, with changes in PRF1 being most common. Late-onset HLH often features viral infection and other predisposing factors. We conclude that late-onset primary HLH is not as rare as previously thought. Older patients should not be delayed to receive HLH-related genes testing when they are suspected with HLH.
1692	Expression of rat oligodendrocyte transcription factor 2 in COS-7 cells following its eukaryotic expression vector construction.	25219831	The basic HLH transcription factor Olig is a key regulator for differentiating the oligodendrocyte lineage cells during development. Oligodendrocyte transcription factor 2 (Olig2) plays a crucial role in differentiating the oligodendrocytes in the spinal cord. We aimed to construct and investigate the eukaryotic expression recombinant plasmid in the rat Olig2.The experiment was performed at the Laboratory of Neurobiology, Xuzhou Medical College from October 2011 to March 2012.The pEGFP-N1 vector was purchased from Invitrogen. JM101 competent cells and COS-7 cells were preserved at the Laboratory of Neurobiology, Xuzhou Medical College, China. The Olig2 cDNA fragment was cloned by RT-PCR with the total RNA from the neonatal rat spinal cord, and subsequently cloned into pGEM-T vector. The confirmed Olig2 fragment was then cloned into the pEGFP-N1 vector. The right recombinant was transfected into COS-7 cells by lipofectamine 2000. The expression of the Olig2 in COS-7 cells was detected by RT-PCR and immunoblot analysis. Enzyme digestion and sequencing of the recombinant plasmid; and expression of the Olig2 were analyzed by fluorescence microscope and western blot.The correct pEGFP-N1-Olig2 cloning was verified by restriction endonuclease digestion and sequencing. The western blot analysis indicated that the Olig2-GFP fusion protein was expressed in the COS-7/pEGFP-N1-Olig2 cells at 72 h.The pEGFP-N1-Olig2 vector was constructed successfully. The Olig2-GFP fusion protein was expressed in the COS-7/pEGFP-N1-Olig2 cells. This study lays the foundation for further research in gene therapy for central nervous system demyelinating diseases.
1693	Cytomegalovirus infections, kidney transplantation, and secondary hemophagocytic lymphohistiocytosis.	2521929824966022	NaN
1694	Neurosurgical capacity building in the developing world through focused training.	25216067	In Tanzania, there are 4 neurosurgeons for a population of 46 million. To address this critical shortage of neurosurgical care, the authors worked with local Tanzanian health care workers, neurosurgeons, the Ministry of Health and Social Welfare, and the Office of the President of Tanzania to develop a train-forward method for sustainable, self-propagating basic and emergency neurosurgery in resource-poor settings. The goal of this study was to assess the safety and effectiveness of this method over a 6-year period.The training method utilizes a hands-on bedside teaching technique and was introduced in 2006 at a remote rural hospital in northern Tanzania. Local health care workers were trained to perform basic and emergency neurosurgical procedures independently and then were taught to train others. Outcome information was retrospectively collected from hospital records for the period from 2005 (1 year before method implementation) through 2010. Analysis of de-identified data included descriptive statistics and multivariable assessment of independent predictors of complications following a patient's first neurosurgical procedure.By 2010, the initial Tanzanian trainee had trained a second Tanzanian health care worker, who in turn had trained a third. The number of neurosurgical procedures performed increased from 18 in 2005 to an average of 92 per year in the last 3 years of the study period. Additionally, the number of neurosurgical cases performed independently by Tanzanian health care providers increased significantly from 44% in 2005 to 86% in 2010 (p < 0.001), with the number of complex cases independently performed also increasing over the same time period from 34% to 83% (p < 0.001). Multivariable analysis of clinical patient outcome information to assess safety indicated that postoperative complications decreased significantly from 2005 through 2010, with patients who had been admitted as training progressed being 29% less likely to have postoperative complications (OR 0.71, 95% CI 0.52-0.96, p = 0.03).The Madaktari Africa train-forward method is a reasonable and sustainable approach to improving specialized care in a resource-poor setting.
1695	X-linked severe combined immunodeficiency due to a novel mutation complicated with hemophagocytic lymphohistiocytosis and presented with invagination: A case report.	25215194	Severe combined immunodeficiency (SCID) is an inherited disease with profoundly defective T cells, B cells, and natural killer (NK) cells. X-linked SCID (X-SCID) is its most common form. In this report, we describe a 4-month-old male with X-SCID who presented invagination and also showed hemophagocytic lymphohistiocytosis (HLH). The patient was admitted to our hospital with fever, cough, vomiting, monoliasis, and hepatosplenomegaly in postoperative period at the age of 3 months. The laboratory finding revealed no detectable T cells and hypogammaglobulinemia despite normal B-cell counts. Diagnosis of X-SCID was established by DNA analysis of the interleukin (IL)-2 receptor gamma chain gene (IL2RG); namely, we detected the novel mutation in the splice-site of exon 5 (c.595-1G>T). The patient died due to infection at the age of 4 months. Also, this case is the first report that describes the patient with X-SCID with presented invagination.
1696	Haemophagocytic lymphohistiocytosis: A fulminant syndrome associated with multiorgan failure and high mortality that frequently masquerades as sepsis and shock.	25214245	Acquired haemophagocytic lymphohistiocytosis (HLH) is a condition involving cytokine overproduction by defective cytotoxic T lymphocytes and natural killer cells, resulting in life-threatening cytopenias and multiorgan infiltration and dysfunction. Triggers for acquired HLH vary and include viruses, malignancies and autoimmune conditions. Recent reports suggest that HLH may be underdiagnosed owing to variable clinical presentations, diagnostic criteria and a low level of awareness on the part of medical personnel, thus delaying prompt treatment and contributing to high mortality rates. Five patients in whom acquired HLH was diagnosed, following bone marrow investigations, for the period of May - September 2013 are presented. All were at an advanced stage of their disease at time of diagnosis. The three patients who were HIV-positive had a coexisting malignancy at the time of HLH diagnosis, which may have triggered HLH. A definite trigger was not identified in the remaining two HIV-negative patients despite early concerns regarding autoimmune disease. Two patients received timeous diagnosis, started chemotherapy and are currently improving. The remaining three succumbed to their illness. Adult acquired HLH may be more common in the acute care setting than currently appreciated. As awareness of this condition and its treatment is currently low, it may remain undiagnosed until the disease has evolved into multiorgan failure. Fever in the absence of infectious agents, marked hyperferritinaemia, unexplained cytopenias, organomegaly or liver dysfunction should raise the suspicion of HLH. Timeous introduction of therapy will improve outcomes.
1697	Macrophage activation syndrome in Kawasaki disease: more common than we thought?	2520094527639924	To analyze the clinical characteristics, treatment, and outcomes of Kawasaki Disease (KD) patients associated with macrophage activation syndrome (MAS) and to compare two diagnostic standards (the HLH 2009 and Ravelli׳s criteria).All of the studied patients with Kawasaki Disease (KD) were treated at The Children׳s Hospital, Zhejiang University School of Medicine, during 2007-2010. Clinical and laboratory findings were analyzed.In 719 KD patients, eight patients (1.11%, 81.3 ± 49.4 months, all male) were diagnosed by Ravelli׳s criteria, but only three (0.42%) patients were diagnosed by the HLH 2009 criteria. Aspartate aminotransferase increased significantly in all cases. Alanine aminotransferase, lactate dehydrogenase, and serum ferritin increased significantly in seven cases. Cytopenia and hypertriglyceridemia (>1.5mmol/L) were found in six and five cases, respectively. Hypofibrinogenemia (<1.5g/L) was found in two cases. Three cases showed evidence of hemophagocytosis, but only one case met the HLH 2009 criteria. Ectasia of the coronary arteries occurred in two cases. Seven patients were non-responsive to IVIG. One case died after the combined application of DXM, VP16, and CSA.MAS may be a frequently under-recognized complication of KD, because the understanding of complications and diagnostic criteria are still in progress. The HLH 2009 criteria have low sensitivity and specificity for the diagnosis of MAS complicating KD. When hepatosplenomegaly is present in KD patients with abnormal laboratory findings, such as cytopenia, liver dysfunction, hyperferritinemia, elevated serum LDH, hypofibrinogenemia, and hypertriglyceridemia, the presence of MAS should be considered.
1698	Up-regulation of the Sirtuin 1 (Sirt1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) genes in white adipose tissue of Id1 protein-deficient mice: implications in the protection against diet and age-induced glucose intolerance.	25190816	Id1, a helix-loop-helix (HLH) protein that inhibits the function of basic HLH E protein transcription factors in lymphoid cells, has been implicated in diet- and age-induced obesity by unknown mechanisms. Here we show that Id1-deficient mice are resistant to a high fat diet- and age-induced obesity, as revealed by reduced weight gain and body fat, increased lipid oxidation, attenuated hepatosteatosis, lower levels of lipid droplets in brown adipose tissue, and smaller white adipocytes after a high fat diet feeding or in aged animals. Id1 deficiency improves glucose tolerance, lowers serum insulin levels, and reduces TNFα gene expression in white adipose tissue. Id1 deficiency also increased expression of Sirtuin 1 and peroxisome proliferator-activated receptor γ coactivator 1α, regulators of mitochondrial biogenesis and energy expenditure, in the white adipose tissue. This effect was accompanied by the elevation of several genes encoding proteins involved in oxidative phosphorylation and fatty acid oxidation, such as cytochrome c, medium-chain acyl-CoA dehydrogenase, and adipocyte protein 2. Moreover, the phenotype for Id1 deficiency was similar to that of mice expressing an E protein dominant-positive construct, ET2, suggesting that the balance between Id and E proteins plays a role in regulating lipid metabolism and insulin sensitivity.
1699	Treatment of rare co-occurrence of Epstein-Barr virus-driven post-transplant lymphoproliferative disorder and hemophagocytic lymphohistiocytosis after allogeneic stem cell transplantation.	25179757	In both conditions, post-transplant lymphoproliferative disorder (PTLD) and hemophagocytic lymphohistiocytosis (HLH), infection with Epstein-Barr virus (EBV) is a key mechanism: almost all PTLD in allogeneic stem cell transplantation (alloSCT) is caused by EBV-related neoplastic lymphoproliferation, and secondary HLH is most frequently triggered by EBV infection. Therefore, concomitant EBV-driven PTLD and HLH early after alloSCT require an approach to eliminate EBV and balance immune activation simultaneously. We report on a patient who developed simultaneous PTLD and signs of HLH on day 64 after alloSCT. Treatment was comprised of stopping cyclosporine, short-course dexamethasone, and 3 courses of rituximab. The patient showed full recovery and complete remission of lymphadenopathy. This result indicates that immediate reduction in EBV-carrying B cells by rituximab, suppression of general inflammation, and parallel support of reconstitution of long-term T-cell function, might be an appropriate therapeutic approach in this rare situation.
1700	Visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis in a traveler returning from a pilgrimage to the Camino de Santiago.	25145768	We report the case of a 73-year-old American traveler who presented with 3 weeks of fatigue, fevers, chills, and pancytopenia. Clinical and laboratory findings were consistent with hemophagocytic lymphohystiocytosis (HLH) and bone marrow biopsy revealed amastigotes consistent with visceral leishmaniasis. The range of endemic visceral leishmaniasis transmission now extends into northern Spain and travelers to this region should use personal protective measures against sand fly exposure.
1701	Haemophagocytic lymphohistiocytosis associated with coccidiomycosis.	25139924	Haemophagocytic lymphohistiocytosis (HLH) is a rapidly fatal disease caused by dysregulated histiocytes leading to an excessive inflammatory reaction. While genetic forms of HLH exist, the most common form is acquired, frequently associated with infection. Here we report the first case of HLH associated with a coccidiomycosis infection. This patient is a 13-year-old previously healthy boy who presented with a flu-like illness, which rapidly progressed to refractory shock, severe ARDS, multiorgan failure and death despite maximal medical therapy, including broad-spectrum antibiotics to treat well-established causes of acquired HLH. Autopsy findings revealed the diagnosis of HLH in the setting of pulmonary coccidiomycosis. Antifungal therapy should be considered in cases of acquired HLH when the underlying aetiology is not clear.
1702	[Research advances in the pathogenesis, diagnosis and treatment of hemophagocytic lymphohistiocytosis in children].	25130849	Hemophagocytic lymphohistiocytosis (HLH), or hemophagocytic syndrome (HPS), is characterized clinically by abrupt onset and progressive deterioration and even death. HLH is much more prevalent in children, and is potentially fatal if early diagnosis is not made and appropriate HLH-directed therapy not instituted. Increasing genetic defects and underlying diseases or causative factors have been identified to be closely implicated in the pathogenesis of HLH. In addition, great advances have been made in the past few years in terms of HLH diagnosis and clinical management. In the present review, the cause of disease, contemporary classification, epidemiology, genetic defects and molecular mechanisms, updated diagnostic criteria and novel treatment strategies for childhood HLH are summarized.
1703	Towards the targeted management of Chediak-Higashi syndrome.	25129365	Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive congenital immunodeficiency caused by mutations in CHS1, a gene encoding a putative lysosomal trafficking protein. In the majority of patients, this disorder is typically characterized by infantile-onset hemophagocytic lymphohistiocytosis (HLH), which is lethal unless allogeneic transplantation is performed. A small number of individuals have the attenuated form of the disease and do not benefit from transplant. Improved outcomes of transplantation have been reported when performed before the development of HLH, thus it is important to quickly differentiate patients that present with the childhood form of disease and to prematurely enroll them into a transplantation protocol. In addition, this would also preclude those that exhibit clinical phenotypes of adolescent and adult CHS from this treatment. Patients with an absence of cytotoxic T lymphocyte (CTL) function have a high risk for developing HLH, and could therefore benefit the most from early hematopoietic stem cell transplantation (HSCT). However, although normal CTL cytotoxicity or bi-allelic missense mutations do not exclude the occurrence of HLH in childhood, a more conservative approach is justified. This article summarizes recent advances in the clinical characterization of CHS patients, provides updates on promising new testing methods, and focuses on specific therapeutic approaches.
1704	Congenital hemophagocytic lymphohistiocytosis presenting as thrombocytopenia in a newborn.	25121636	Hemophagocytic lymphohistiocytosis (HLH) is a disease caused by dysregulation and hyperactivation of the immune system, and can be familial or acquired. HLH presenting in infancy can be rapidly fatal if not promptly recognized and treated. Congenital HLH can be caused by various genetic mutations or part of immunodeficiency syndromes. We present an infant with Griscelli syndrome and familial HLH with atypical genetic mutations, presenting as thrombocytopenia on the first day of life, cured with chemotherapy and unrelated cord blood transplant.
1705	Hemophagocytic lymphohistiocytosis.	25114410	Hemophagocytic lymphohistiocytosis (HLH) is an immune disorder characterized by uncontrolled inflammation due to defective immune response. It may be familial or acquired, but both share a common feature of threatening the life of a patient and may lead to death unless treated by appropriate treatment. Here in we report a case of adult HLH.
1706	A 6-year-old girl with undiagnosed hemophagocytic lymphohistiocytosis and takotsubo cardiomyopathy: a case report and review of the literature.	2509880126148109	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder of highly stimulated immune responses to antigens that leads to life-threatening inflammation and multiple organ dysfunction. At presentation, HLH may uncommonly mimic septic shock. In this case, we present a 6-year-old girl presenting to the pediatric emergency department with a febrile illness and anemia that subsequently developed decompensated shock, initially thought to be septic. She was ultimately diagnosed with HLH. During initial HLH treatment, this patient also developed takotsubo cardiomyopathy, a unique pattern of reversible left ventricular dysfunction characterized by transient apical ballooning and hypokenesis of the left ventricle that spontaneously resolves. There are very few case reports of HLH-associated takotsubo cardiomyopathy. We believe that this is the first case of takotsubo cardiomyopathy in a child with HLH.
1707	Recombinant human luteinizing hormone to trigger ovulation: randomized, controlled, dose-finding pilot study in ovulation induction.	25098025	To evaluate recombinant human luteinizing hormone (r-hLH) versus urine-derived human chorionic gonadotropin (u-hCG) to trigger ovulation in women (aged 20-40 years) with WHO Group II anovulatory infertility undergoing ovulation induction (OI) with recombinant human follicle-stimulating hormone (r-hFSH) (150 IU/day starting dose).For this Phase II, open-label, dose-finding pilot study, patients were randomized to doses of 825, 2,750, 5,500, 11,000, or 22,000 IU r-hLH or u-hCG (5,000 IU). Primary endpoints were ovulation and ratio of ruptured follicles/follicle > or = 15 mm (day of r-hLH/ u-hCG administration). Secondary endpoints included monofollicular ovulation and clinical pregnancy rates.All 67 randomized patients completed treatment. All patients in the r-hLH 2,750 (13/13), 5,500 (12/ 12), 11,000 IU (13/13), and u-hCG 5,000 IU (12/ 12) groups ovulated; 3/5 patients in the r-hLH 825 IU and 2/12 in the r-hLH 22,000 IU group failed to ovulate (p = 0.105 between evaluable groups). The mean ratio of ruptured follicles/ follicle > or = 15 mm was 1.1 (p = 0.675 between groups). The monofollicular ovulation rate was 15/60 (25%). Two cases of ovarian hyperstimulation syndrome were reported.This open-label, pilot study (conducted in 1999-2001) suggests that the minimal effective dose of r-hLH to trigger ovulation in women with WHO Group II anovulatory infertility undergoing OI with r-hFSH (150 IU starting dose) was 2,750 IU.
1708	Hemophagocytic lymphohistiocytosis secondary to Mycoplasma pneumoniae infection without pneumonia.	25088886	Mycoplasma pneumoniae typically causes respiratory tract infections, including pneumonia. We herein report the case of a 30-year-old Japanese woman with hemophagocytic lymphohistiocytosis (HLH), which is rarely caused by bacteria, secondary to Mycoplasma pneumoniae infection and ensuing hypercytokinemia without pneumonia who was successfully treated with a combination of antibiotics and corticosteroids. Therefore, Mycoplasma pneumoniae infection, which is treatable with antibiotics, should be considered a possible trigger of HLH in patients who present with a viral-like illness without pneumonia.
1709	Regulation of p21 by TWIST2 contributes to its tumor-suppressor function in human acute myeloid leukemia.	25088197	TWIST2 has a dual function in tumors. Its implication in the initiation and metastasis of various solid tumors is well established, and its tumor-suppressor role in murine osteosarcoma cells has been reported recently. However, the function of TWIST2 and its underlying mechanisms in human normal and malignant hematopoiesis remain unclear. In the present study, we found that TWIST2 directly regulated p21 in human hematopoietic cells and whose silence promoted cell proliferation and cell cycle progression. Hypermethylation of TWIST2 occurred to 23 out of the 75 adult acute myeloid leukemia (AML) patients and resulted in the impaired expression of both TWIST2 and p21. Conversely, TWIST2 overexpression inhibited the growth of AML cells partially through its direct activation of p21 with intact HLH (helix-loop-helix) domain. The microarray data and gene expression validation showed that TWIST2 was sufficient to activate known tumor-suppressor genes, whereas suppress known oncogenes, which further supported its inhibitory effect against AML cells. Taken together, our data have identified a novel TWIST2-p21 axis that modulates the cell cycle of both normal and leukemic cells and demonstrated that the direct regulation of p21 by TWIST2 has a role in its tumor-suppressor function in AML.
1710	Epstein-Barr virus associated hemophagocytic lymphohistiocytosis in a rheumatic patient receiving abatacept therapy.	25083955	Hemophagocytic lymphohistiocytosis (HLH) is a life- threatening hyperinflammatory disease that causes extensive organ damage. It is generally triggered by viral, fungal, or parasitic infections in the setting of hematologic disease-induced immune deficiency. Occurrences in rheumatologic disease are less frequent, with the syndrome developing most often in patients with systemic lupus erythematosus and adult-onset Still disease. It is believed that the immunosuppression induced by rheumatologic disease itself and exacerbation by immunomodulatory therapies predispose to infection and subsequently HLH. Abatacept is a relatively new disease-modifying agent for rheumatoid arthritis (RA) that has been associated with varicella zoster virus, cytomegalovirus, and Epstein-Barr virus (EBV) infections, but not previously in the setting of HLH. Here we report a unique case of EBV-associated HLH in a RA patient receiving abatacept therapy.
1711	[Natural killer cell cytotoxic activity in critical pediatric patients with suspected hemophagocytic syndrome].	25081072	To determine the role of natural killer (NK) cytotoxic activity in patients with suspected hemophagocytic lymphohistiocytosis syndrome (HLH).A prospective study was conducted from September 2008 to February 2014.The study was carried out in the Hematological Oncology Laboratory of Hospital Infantil Universitario Niño Jesús, Madrid (Spain).We analyzed 30 peripheral blood samples from intensive care patients with suspected HLH. There were 18 males and 12 females, with a mean age of 4.7 years (range 0.2-22). NK cell cytotoxicity was compared with healthy controls according to age and sex.In vitro NK cell cytotoxicity against the K562 cell line was determined by time-resolved fluorescence (Europium-TDA) under resting conditions, after interleukin 15 stimulation, and following block with Fas ligand antibody.NK cell cytotoxicity.A total of 20 patients showed a significant decrease of NK cell activity compared with controls (P=.001). Nine of these patients were diagnosed with primary HLH. A total of 10 patients were diagnosed with secondary HLH. Cytotoxic activity was normal in 10 subjects. None of them were diagnosed with HLH. Interleukin 15 stimulation increased NK cell cytotoxicity in secondary HLH, and blocking Fas ligand on NK cells decreased cytotoxic activity in primary HLH patients (P=.001).In our experience, NK cell cytotoxic activity measured by time-resolved fluorescence is a simple and useful clinical diagnostic test for HLH. Interleukin 15 stimulation and Fas ligand blocking on NK cells could help differentiate between primary and secondary HLH.
1712	A mutation in X-linked inhibitor of apoptosis (G466X) leads to memory inflation of Epstein-Barr virus-specific T cells.	25079909	Mutations in the X-linked inhibitor of apoptosis (XIAP) gene have been associated with XLP-like disease, including recurrent Epstein-Barr virus (EBV)-related haemophagocytic lymphohystiocytosis (HLH), but the immunopathogenic bases of EBV-related disease in XIAP deficiency is unknown. We present the first analysis of EBV-specific T cell responses in functional XIAP deficiency. In a family of patients with a novel mutation in XIAP (G466X) leading to a late-truncated protein and varying clinical features, we identified gradual hypogammaglobulinaemia and large expansions of T cell subsets, including a prominent CD4(+) CD8(+) population. Extensive ex-vivo analyses showed that the expanded T cell subsets were dominated by EBV-specific cells with conserved cytotoxic, proliferative and interferon (IFN)-γ secretion capacity. The EBV load in blood fluctuated and was occasionally very high, indicating that the XIAP(G466X) mutation could impact upon EBV latency. XIAP deficiency may unravel a new immunopathogenic mechanism in EBV-associated disease.
1713	Lymphoma presenting as secondary HLH: a review with a tale of two cases.	25065778	NaN
1714	[Analysis of clinical and laboratory features of 217 pediatric hemophagocytic lymphohistiocytosis].	25052607	To investigate the incidence, clinical symptoms, signs and laboratory features of childhood hemophagocytic lymphohistiocytosis (HLH) in China.A retrospective study was performed on 217 pediatric patients with HLH who were admitted to Children's Hospital of Chongqing Medical University from January 2006 to April 2013. All patients'medical records were reviewed and analyzed.The Male to female ratio was 1.11:1. The median onset age was 3 years and 5 months old (range of 6 months old to 16 years and 9 months old), and the age of onset peaked between 1-5 years old (61.3%). The most common causes of HLH was infection, especially Epstein-Barr virus-associated HLH (71.0%). Other causes included malignant hemophagocytic syndrome (MAHS), macrophage activation syndrome (MAS) and so on. The outstanding clinical manifestations including persistent fever (100.0%), hepatomegaly (92.6%), splenomegaly (88.4%), and more than half of cases with central nervous system involvement and pulmonary manifestations. Laboratory data indicated that the most prominent abnormality was elevated ferritin (98.0%), and the others were hemophagocytosis in bone marrow (90.7%) and coagulation abnormalities (76.5%). Abnormal lymphocytes classification is very common in HLH.HLH is a heterogeneous disease, with a variety of the etiology and clinical manifestations. HLH-2004 diagnostic protocol had theoretical basis and clinical operability. The hepatitis damages related indicators, lymphocytes classification, central nervous system involvement and pulmonary performance can be used as reference value for HLH diagnosis.
1715	Whole-exome sequencing reveals overlap between macrophage activation syndrome in systemic juvenile idiopathic arthritis and familial hemophagocytic lymphohistiocytosis.	25047945	Macrophage activation syndrome (MAS), a life-threatening complication of systemic juvenile idiopathic arthritis (JIA), resembles familial hemophagocytic lymphohistiocytosis (HLH), a constellation of autosomal-recessive immune disorders resulting from deficiency in cytolytic pathway proteins. We undertook this study to test our hypothesis that MAS predisposition in systemic JIA could be attributed to rare gene sequence variants affecting the cytotolytic pathway.Whole-exome sequencing was used in 14 patients with systemic JIA and MAS and in their parents to identify protein-altering single-nucleotide polymorphisms/indels in known HLH-associated genes. To discover new candidate genes, the entire whole-exome sequencing data were filtered to identify protein-altering, rare recessive homozygous, compound heterozygous, and de novo variants with the potential to affect the cytolytic pathway.Heterozygous protein-altering rare variants in the known genes (LYST,MUNC13-4, and STXBP2) were found in 5 of 14 patients with systemic JIA and MAS (35.7%). This was in contrast to only 4 variants in 4 of 29 patients with systemic JIA without MAS (13.8%). Homozygosity and compound heterozygosity analysis applied to the entire whole-exome sequencing data in systemic JIA/MAS revealed 3 recessive pairs in 3 genes and compound heterozygotes in 73 genes. We also identified 20 heterozygous rare protein-altering variants that occurred in at least 2 patients. Many of the identified genes encoded proteins with a role in actin and microtubule reorganization and vesicle-mediated transport. "Cellular assembly and organization" was the top cellular function category based on Ingenuity Pathways Analysis (P < 3.10 × 10(-5) ).Whole-exome sequencing performed in patients with systemic JIA and MAS identified rare protein-altering variants in known HLH-associated genes as well as in new candidate genes.
1716	Performance of current guidelines for diagnosis of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis.	25044674	To compare the capacity of the 2004 diagnostic guidelines for hemophagocytic lymphohistiocytosis (HLH-2004) with the capacity of the preliminary diagnostic guidelines for systemic juvenile idiopathic arthritis (JIA)-associated macrophage activation syndrome (MAS) to discriminate MAS complicating systemic JIA from 2 potentially confusable conditions, represented by active systemic JIA without MAS and systemic infection.International pediatric rheumatologists and hemato-oncologists were asked to retrospectively collect clinical information from patients with systemic JIA-associated MAS and confusable conditions. The ability of the guidelines to differentiate MAS from the control diseases was evaluated by calculating the sensitivity and specificity of each set of guidelines and the kappa statistics for concordance with the physician's diagnosis. Owing to the fact that not all patients were assessed for hemophagocytosis on bone marrow aspirates and given the lack of data on natural killer cell activity and soluble CD25 levels, the HLH-2004 guidelines were adapted to enable the diagnosis of MAS when 3 of 5 of the remaining items (3/5-adapted) or 4 of 5 of the remaining items (4/5-adapted) were present.The study sample included 362 patients with systemic JIA and MAS, 404 patients with active systemic JIA without MAS, and 345 patients with systemic infection. The best capacity to differentiate MAS from systemic JIA without MAS was found when the preliminary MAS guidelines were applied. The 3/5-adapted HLH-2004 guidelines performed better than the 4/5-adapted guidelines in distinguishing MAS from active systemic JIA without MAS. The 3/5-adapted HLH-2004 guidelines and the preliminary MAS guidelines with the addition of ferritin levels ≥500 ng/ml discriminated best between MAS and systemic infections.The preliminary MAS guidelines showed the strongest ability to identify MAS in systemic JIA. The addition of hyperferritinemia enhanced their capacity to differentiate MAS from systemic infections. The HLH-2004 guidelines are likely not appropriate for identification of MAS in children with systemic JIA.
1717	Dual roles for Id4 in the regulation of estrogen signaling in the mammary gland and ovary.	25038044	The HLH transcriptional regulator Id4 exerts important roles in different organs, including the neural compartment, where Id4 loss usually results in early lethality. To explore the role of this basally restricted transcription factor in the mammary gland, we generated a cre-inducible mouse model. MMTV- or K14-cre-mediated deletion of Id4 led to a delay in ductal morphogenesis, consistent with previous findings using a germ-line knockout mouse model. A striking increase in the expression of ERα (Esr1), PR and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands. Together with chromatin immunoprecipitation of Id4 on the Esr1 and Foxa1 promoter regions, these data imply that Id4 is a negative regulator of the ERα signaling axis. Unexpectedly, examination of the ovaries of targeted mice revealed significantly increased numbers of secondary and antral follicles, and reduced Id4 expression in the granulosa cells. Moreover, expression of the cascade of enzymes that are crucial for estrogen biosynthesis in the ovary was decreased in Id4-deficient females and uterine weights were considerably lower, indicating impaired estrogen production. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice. Collectively, these data identify Id4 as a novel regulator of estrogen signaling, where Id4 restrains ERα expression in the basal and luminal cellular compartments of the mammary gland and regulates estrogen biosynthesis in the ovary.
1718	Phagocytized neutrophil fragments in the bone marrow: a phenomenon most commonly associated with hodgkin lymphoma.	25031871	Bone marrow macrophages containing other cells, or large pieces of other cells, represent a distinctive feature of diseases such as Hemophagocytic Lymphohistiocytosis (HLH) and Rosai-Dorfman disease. We describe a distinct variation of phagocytic histiocyte morphology, featuring histiocytes containing predominantly fragments of neutrophil nuclei. We retrospectively reviewed initial bone marrow samples for Hodgkin lymphoma, Burkitt lymphoma, Ewing sarcoma, or evaluation for nonneoplastic conditions, scoring the presence or absence of the above-described histiocytes. We find that these histiocytes, which we term "fragmentophages," are associated with staging marrow sampling for malignancy, especially Hodgkin lymphoma (Hodgkin lymphoma: 28/34 or 82.4%, Ewing sarcoma: 11/26 or 42.3%, Burkitt lymphoma: 4/13 or 30.8%). These cells are significantly less common in marrow samples for nonneoplastic conditions (4/21 or 19.0%). Fragmentophages are significantly associated with malignancy, especially Hodgkin lymphoma, and their recognition has the potential to provide a clue to an underlying malignancy.
1719	Cloning and expression characteristics of the pig Stra8 gene.	25029539	Stra8 (Stimulated by Retinoic Acid 8) is considered a meiotic gatekeeper gene. Using reverse transcriptase PCR and rapid amplification of cDNA ends (RACE), the complete sequence of the pig Stra8 gene was cloned. Bioinformatics analyses of this sequence were performed. Using semi-quantitative methods, the expression characteristics of Stra8 in Testis, cauda epididymis, body epididymis, caput epididymis, seminal vesicles, prostate gland, Cowper's gland, heart, liver, spleen, lung, kidney, stomach, hypothalamus, pituitary gland, cerebrum, cerebellum, and hippocampus of adult Meishan boar and sow tissues were examined. The expression pattern in the testis of 2-, 30-, 60-, 90-, and 150-day old Meishan boars were analyzed using real-time PCR. We constructed a eukaryotic expression vector for the Stra8 gene and used it to transfect NIH-3T3 cells and third generation pig spermatogonial stem cells (SSCs) cultured in vitro. Testes weight and sperm count in the cauda epididymis were evaluated at various time points. The results showed that the length of the pig Stra8 gene cDNA was 1444 bp encoding 366 amino acids with one typical helix-loop-helix (HLH) domain. It is testes-specific expression. Expression was first detected in boar testis starting at day 2, and its expression significantly (p<0.05) increased with age and body weight. When NIH-3T3 cells and pig SSCs were transfected with the eukaryotic expression vector EGFP (enhanced green fluorescent protein)-N1-pStra8, it was expressed in the cytoplasm of NIH-3T3 cells. However, in SSCs, Stra8 was expressed predominantly in cytoplasm and few in nucleus. Our data suggest that perhaps Stra8 acts as a transcription factor to initiate meiosis in young boar.
1720	The comprehensive transcriptional analysis in Caenorhabditis elegans by integrating ChIP-seq and gene expression data.	25023089	The fundamental step of learning transcriptional regulation mechanism is to identify the target genes regulated by transcription factors (TFs). Despite numerous target genes identified by chromatin immunoprecipitation followed by high-throughput sequencing technology (ChIP-seq) assays, it is not possible to infer function from binding alone in vivo. This is equally true in one of the best model systems, the nematode Caenorhabditis elegans (C. elegans), where regulation often occurs through diverse TF binding features of transcriptional networks identified in modENCODE. Here, we integrated ten ChIP-seq datasets with genome-wide expression data derived from tiling arrays, involved in six TFs (HLH-1, ELT-3, PQM-1, SKN-1, CEH-14 and LIN-11) with tissue-specific and four TFs (CEH-30, LIN-13, LIN-15B and MEP-1) with broad expression patterns. In common, TF bindings within 3 kb upstream of or within its target gene for these ten studies showed significantly elevated level of expression as opposed to that of non-target controls, indicated that these sites may be more likely to be functional through up-regulating its target genes. Intriguingly, expression of the target genes out of 5 kb upstream of their transcription start site also showed high levels, which was consistent with the results of following network component analysis. Our study has identified similar transcriptional regulation mechanisms of tissue-specific or broad expression TFs in C. elegans using ChIP-seq and gene expression data. It may also provide a novel insight into the mechanism of transcriptional regulation not only for simple organisms but also for more complex species.
1721	Hemophagocytic lymphohistiocytosis and pelger-huët anomaly associated with colchicine intoxication.	25013716	Colchicine is frequently used in the treatment of familial Mediterranean fever (FMF). First symptoms of colchicine intoxication are gastrointestinal disturbances, such as abdominal cramps, diarrhea, pancytopenia and so on. Herein, we report a female FMF patient with pancytopenia and hemophagocytic lymphohitiocytosis (HLH), following colchicine intoxication for committing suicide. To our knowledge, this is the first reported case of a patient with HLH associated with colchicine intoxication.
1722	Cost-effectiveness of the "helping babies breathe" program in a missionary hospital in rural Tanzania.	25006802	The Helping Babies Breathe" (HBB) program is an evidence-based curriculum in basic neonatal care and resuscitation, utilizing simulation-based training to educate large numbers of birth attendants in low-resource countries. We analyzed its cost-effectiveness at a faith-based Haydom Lutheran Hospital (HLH) in rural Tanzania.Data about early neonatal mortality and fresh stillbirth rates were drawn from a linked observational study during one year before and one year after full implementation of the HBB program. Cost data were provided by the Tanzanian Ministry of Health and Social Welfare (MOHSW), the research department at HLH, and the manufacturer of the training material Lærdal Global Health.Costs per life saved were USD 233, while they were USD 4.21 per life year gained. Costs for maintaining the program were USD 80 per life saved and USD 1.44 per life year gained. Costs per disease adjusted life year (DALY) averted ranged from International Dollars (ID; a virtual valuta corrected for purchasing power world-wide) 12 to 23, according to how DALYs were calculated.The HBB program is a low-cost intervention. Implementation in a very rural faith-based hospital like HLH has been highly cost-effective. To facilitate further global implementation of HBB a cost-effectiveness analysis including government owned institutions, urban hospitals and district facilities is desirable for a more diverse analysis to explore cost-driving factors and predictors of enhanced cost-effectiveness.
1723	The Ser/Thr phosphatase PP2A regulatory subunit widerborst inhibits notch signaling.	25006677	Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit widerborst (wdb) in three developmental contexts; the bristle, wing and the R8 photoreceptors of the eye. wdb overexpression elicits bristle and wing defects akin to reduced Notch signaling, whereas hypomorphic mutations in this PP2A subunit elicit opposite effects. We have also evaluated wdb functions using mutations in Notch and E(spl) that affect the eye. We find that the eye and R8 defects of the well-known Nspl mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread.
1724	Diagnosing XLP1 in patients with hemophagocytic lymphohistiocytosis.	24985396	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, heterogeneous, hyperinflammmatory disorder. Prompt identification of inherited forms resulting from mutation in genes involved in cellular cytotoxicity can be crucial. X-linked lymphoproliferative disease 1 (XLP1), due to mutations in SH2D1A (Xq25) encoding signaling lymphocyte activation molecule-associated protein (SAP), may present with HLH. Defective SAP induces paradoxical inhibitory function of the 2B4 coreceptor and impaired natural killer (NK) (and T) cell response against EBV-infected cells.To characterize a cohort of patients with HLH and XLP1 for SAP expression and 2B4 function in lymphocytes, proposing a rapid diagnostic screening to direct mutation analysis.We set up rapid assays for 2B4 function (degranulation or (51)Cr-release) to be combined with intracellular SAP expression in peripheral blood NK cells. We studied 12 patients with confirmed mutation in SH2D1A and some family members.The combined phenotypic/functional assays allowed efficient and complete diagnostic evaluation of all patients with XLP1, thus directing mutation analysis and treatment. Nine cases were SAP(-), 2 expressed SAP with mean relative fluorescence intensity values below the range of healthy controls (SAP(dull)), and 1, carrying the R55L mutation, was SAP(+). NK cells from all patients showed inhibitory 2B4 function and defective killing of B-EBV cells. Carriers with SH2D1A mutations abolishing SAP expression and low percentage of SAP(+) cells showed neutral 2B4 function at the polyclonal NK cell level. Three novel SH2D1A mutations have been identified.Study of SAP expression is specific but may have insufficient sensitivity for screening XLP1 as a single tool. Combination with 2B4 functional assay allows identification of all cases.
1725	Self-recognition behavior of a helix-loop-helix domain by a fragment scan.	24981796	The inhibitors of DNA binding Id1-4 are helix-loop-helix (HLH) proteins that exert their biological function by interacting with members of the basic-HLH (bHLH) transcription-factor family. The HLH domains of the Id and bHLH proteins allow both self- and hetero-association. Due to their abnormal expression in cancer cells, the Id proteins are potential protein targets for cancer treatment. Suitable Id-protein inactivators should promote self-association and/or prevent hetero-association. In this work we evaluated the ability of the Id-protein HLH domain to recognize itself in form of short sequences extracted from the helical and loop regions. We performed a peptide scan of the Id1 HLH domain 64-106 based on three-residue overlapping octapeptides. Interaction of each octapeptide with the natively folded Id1 HLH domain was investigated by CD and fluorescence spectroscopy. The results from both techniques showed that the helix-based but not the loop-based octapeptides interacted with the Id1 HLH domain in the low-micromolar range. In contrast, a nitrotyrosine-containing analog of the Id1 HLH region, which was unable to reproduce the native-like conformation, quenched only the 2-amino-benzoyl-(Abz)-labeled loop-based octapeptides. This opposite self-recognition pattern suggests that the short helix-based and loop-based sequences should be able to distinguish different folding states of the Id1 HLH domain. This feature may be biologically relevant, as the Id proteins are predicted to behave as intrinsically disordered proteins, being in equilibrium between rapidly exchanging monomeric conformations and structurally better-defined homo-/heterodimers displaying the parallel four-helix bundle.
1726	Haemophagocytic lymphohistiocytosis (HLH): case series in tertiary referral hospital over three years.	24974508	Haemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal disease of normal but overactive histiocytes and lymphocytes that commonly appears in infancy, although it has been seen in all age groups. We are reporting a series of 5 cases of HLH in young adult and paediatric patients identified over three years.
1727	Macrophage activation syndrome and cytokine-directed therapies.	24974063	Macrophage activation syndrome (MAS) is an episode of overwhelming inflammation that occurs most commonly in children with systemic juvenile idiopathic arthritis (SJIA). It is characterized by expansion and activation of T lymphocytes and hemophagocytic macrophages and bears great similarity to hemophagocytic lymphohistiocytosis (HLH). This disorder has substantial morbidity and mortality, and there is frequently a delay in recognition and initiation of treatment. Here, we will review what is known about the pathogenesis of MAS and, in particular, its similarities to HLH. The development of MAS is characterized by a cytokine storm, with the elaboration of numerous pro-inflammatory cytokines. We will examine the evidence for various cytokines in the initiation and pathogenesis of MAS and discuss how new biologic therapies may alter the risk of MAS. Finally, we will review current treatment options for MAS and examine how cytokine-directed therapy could serve as novel treatment modalities.
1728	Spectrum of Epstein-Barr virus-associated T-cell lymphoproliferative disorder in adolescents and young adults in Taiwan.	24966953	Epstein-Barr Virus (EBV) is a herpesvirus usually infecting B-cells but may occasionally infect T- or natural killer (NK)-cells. EBV-associated T- or NK-cell lymphoproliferations represent a continuous spectrum of diseases ranging from asymptomatic infection, infectious mononucleosis (IM), to clonal and malignant lymphoproliferations including systemic EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD) of childhood and hydroa-vacciniforme-like lymphoma of the skin. The clonal diseases are more prevalent in East Asia and exhibit overlapping clinical and pathological features with chronic active EBV infection. Here we report our experience on 10 cases of EBV-associated T-cell lymphoproliferation from Taiwan including five males and five females with a median age of 18 years old (range, 15-28). The most common clinical symptoms were fever, neck mass and hepatosplenomegaly. Eight of these patients showed elevated lactate dehydrogenase level and half of the patients had cytopenia. All patients had either elevated EBV antibody titers or increased serum EBV DNA levels. Five cases were clinically IM-like with polyclonal (3 cases) or clonal (2 cases) T-cell lymphoproliferation. Two patients each had chronic active EBV infection (CAEBV) and hemophagocytic lymphohistiocytosis (HLH). One patient had both CAEBV and HLH. One of the HLH patients with marrow infiltration by intra-sinusoidal large atypical lymphocytes experienced a fulminant course. In a median follow-up time of 21.5 months, seven patients were free of disease, one was alive with disease, and two died of disease in 31 and 3 months, respectively, despite chemotherapy. We confirmed a wide clinicopathological range of EVB-associated T-cell lymphoproliferation in Taiwan. Furthermore, monomorphic LPD and the single case with fulminant course as defined by Ohshima et al (Pathol Int 2018) as categories A3 and B, respectively, died of disease despite chemotherapy. Our report, the largest series in the recent decade from Taiwan, adds to the understanding of these rare diseases with variable clinical and histopathological presentations.
1729	Hemophagocytic lymphohistiocytosis: review of etiologies and management.	24966707	Hemophagocytic lymphohistiocytosis (HLH) covers a wide array of related life-threatening conditions featuring ineffective immunity characterized by an uncontrolled hyperinflammatory response. HLH is often triggered by infection. Familial forms result from genetic defects in natural killer cells and cytotoxic T-cells, typically affecting perforin and intracellular vesicles. HLH is likely under-recognized, which contributes to its high morbidity and mortality. Early recognition is crucial for any reasonable attempt at curative therapy to be made. Current treatment regimens include immunosuppression, immune modulation, chemotherapy, and biological response modification, followed by hematopoietic stem-cell transplant (bone marrow transplant). A number of recent studies have contributed to the understanding of HLH pathophysiology, leading to alternate treatment options; however, much work remains to raise awareness and improve the high morbidity and mortality of these complex conditions.
1730	Epstein-Barr virus induced hemophagocytic lymphohistiocytosis in X-linked lymphoproliferative disease.	24966560	X-linked lymphoproliferative disease (XLP) is a rare, often fatal genetic disorder characterized by extreme vulnerability to Epstein-Barr virus (EBV). EBV-induced hemophagocytic lymphohistiocytosis (HLH) is a known presentation in XLP. In EBV-induced HLH in XLP, the brain imaging findings in the acute phase include a non specific pattern. In this report, we highlight the magnetic resonance imaging and magnetic resonance spectroscopy findings in a child with EBV induced HLH in XLP.
1731	Targeting the bHLH transcriptional networks by mutated E proteins in experimental glioma.	24965159	Glioblastomas (GB) are aggressive primary brain tumors. Helix-loop-helix (HLH, ID proteins) and basic HLH (bHLH, e.g., Olig2) proteins are transcription factors that regulate stem cell proliferation and differentiation throughout development and into adulthood. Their convergence on many oncogenic signaling pathways combined with the observation that their overexpression in GB correlates with poor clinical outcome identifies these transcription factors as promising therapeutic targets. Important dimerization partners of HLH/bHLH proteins are E proteins that are necessary for nuclear translocation and DNA binding. Here, we overexpressed a wild type or a dominant negative form of E47 (dnE47) that lacks its nuclear localization signal thus preventing nuclear translocation of bHLH proteins in long-term glioma cell lines and in glioma-initiating cell lines and analyzed the effects in vitro and in vivo. While overexpression of E47 was sufficient to induce apoptosis in absence of bHLH proteins, dnE47 was necessary to prevent nuclear translocation of Olig2 and to achieve similar proapoptotic responses. Transcriptional analyses revealed downregulation of the antiapoptotic gene BCL2L1 and the proproliferative gene CDC25A as underlying mechanisms. Overexpression of dnE47 in glioma-initiating cell lines with high HLH and bHLH protein levels reduced sphere formation capacities and expression levels of Nestin, BCL2L1, and CDC25A. Finally, the in vivo induction of dnE47 expression in established xenografts prolonged survival. In conclusion, our data introduce a novel approach to jointly neutralize HLH and bHLH transcriptional networks activities, and identify these transcription factors as potential targets in glioma.
1732	Hemophagocytic lymphohistiocytosis secondary to T-cell/histiocyte-rich large B-cell lymphoma.	24955327	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening clinical syndrome characterized by dysregulation of the immune system. Impaired function of cytotoxic T cells and natural killer cells is often seen, and T-cell malignancies represent most cases of lymphoma-associated HLH. HLH associated with B-cell lymphoma is rare. We describe a case of a 30-year-old man who presented with fever, splenomegaly, and hyperferritinemia. Bone marrow biopsy revealed T-cell/histiocyte-rich large B-cell lymphoma, a rare, aggressive B-cell malignancy. This case highlights the interplay between a pro-inflammatory cytokine microenvironment and tumor-mediated immune suppression, and addresses the importance of accurately diagnosing these entities for appropriate clinical management.
1733	Of worms and men: HLH-30 and TFEB regulate tolerance to infection.	2495020624882217	In this issue of Immunity, Visvikis et al. (2014) use the model host Caenorhabditis elegans to discover a role in innate immunity for the basic helix-loop-helix transcription factor, HLH-30. The finding inspires study of the mammalian ortholog TFEB, in which a similar role in immune response is ascertained.
1734	Complications of Reduced Intensity Conditioning HSCT for XIAP Deficiency (Alloimmune Cytopenias and HLH) Successfully Managed With Donor Lymphocyte Infusion.	24942029	X-linked inhibitor of apoptosis protein deficiency is a rare illness and although stem cell transplant is curative, full intensity conditioning is associated with high mortality rates. We describe a child with unusual complications associated with residual host lymphocytes following reduced intensity stem cell transplant. Recipient derived, donor directed, antigranulocyte antibodies led to life-threatening and prolonged neutropenia and residual recipient lymphocytes reestablished hemophagocytic lymphohistiocytosis after withdrawal of immune suppression despite high levels of whole blood chimerism. Hemophagocytic lymphohistiocytosis was abolished following specific improvement in donor T-cell chimerism after donor lymphocyte infusions, and alloimmune cytopenias were no longer evident.
1735	Mixed chimerism and graft loss in pediatric recipients of an alemtuzumab-based reduced-intensity conditioning regimen for non-malignant disease.	24939325	Reduced-intensity conditioning (RIC) regimens can mitigate the toxicity of hematopoietic cell transplantation (HCT) in children with non-malignant diseases, but are associated with increased risk for post-transplant mixed donor/recipient chimerism (MC) and/or graft loss (GL). Intervention with donor lymphocytes or stem cell boosts (DLI/boost) may be necessary, but there is limited information about timing and results of intervention.We retrospectively evaluated 31 consecutive pediatric recipients of an alemtuzumab-based RIC HCT at the Children's Hospital of Philadelphia from May 2007 to December 2012 to determine the incidence of MC, GL, and use of DLI/boost. All patients received alemtuzumab with either fludarabine (150 mg/m(2) )/melphalan (140 mg/m(2) ) (n = 30) or fludarabine/busulfan (n = 1), and unmanipulated marrow grafts from related (48%) or matched unrelated (52%) donors.Of surviving patients, 67% and 44% displayed MC and MC with ≤80% donor contribution (MC ≤ 80%), respectively. Rates of MC, MC ≤ 80%, DLI/boost, and GL were significantly higher in recipients of proximal/intermediate (100%, 73%, 46%, and 46%, respectively) compared to distal alemtuzumab (44%, 25%, 6%, and 6%, respectively). Event-free and overall survival was significantly lower in HLH compared with non-HLH patients. Twenty percent of patients required DLI/boost, and DLI/boost did not affect the incidence of GL.RIC with proximal/intermediate alemtuzumab is associated with high rates of MC, need for DLI/boost, and GL.
1736	Clinical features, genetics, and outcome of pediatric patients with hemophagocytic lymphohistiocytosis in Korea: report of a nationwide survey from Korea Histiocytosis Working Party.	24935083	We analyzed a nationwide registry of pediatric patients with hemophagocytic lymphohistiocytosis (HLH) in Korea to assess the clinical and genetic features and treatment outcomes in pediatric HLH.The Korea Histiocytosis Working Party retrospectively analyzed data on 251 pediatric patients diagnosed with HLH between 1996 and 2011.In the study cohort, 25 cases were categorized with familial HLH, 64 with presumed secondary HLH, and 162 with unspecified HLH. Of 217 evaluable patients, 91 (42%) had concomitant Epstein-Barr virus infection. Of 238 evaluable patients, central nervous system (CNS) involvement, which was more frequent in the familial group, was evident in 81 cases (34%). Genetic tests revealed a predominant UNC13D mutation with a high incidence of two recurrent splicing mutations (c.118-308C>T and c.754-1G>C). The 5-yr overall survival rate was 68% (38% in the familial group and 81% in the presumed secondary group). The 5-yr overall survival rate among 32 patients who underwent allogeneic hematopoietic stem cell transplantation was 64%. In multivariate analysis, a younger age at diagnosis, severe transaminasemia, and a coagulation abnormality were independent prognostic factors for survival. Responses during initial treatments were also significant indicators of outcome.Our study showed the unique predominance of a UNC13D mutation and vulnerability to Epstein-Barr virus infection in Korean children with HLH and emphasizes the prognostic significance of age, liver dysfunction, and treatment responses in this disease. A multicenter prospective trial that builds on the present results is warranted to identify subgroups of patients with a poor prognosis and identify optimal treatments.
1737	Reduced-intensity conditioning hematopoietic cell transplantation is an effective treatment for patients with SLAM-associated protein deficiency/X-linked lymphoproliferative disease type 1.	24923536	X-linked lymphoproliferative disease type 1 (XLP1) is a rare immune deficiency caused by mutations in SH2D1A. Allogeneic hematopoietic cell transplantation (HCT) is often performed because of the morbidity and mortality associated with XLP1. There is limited experience using reduced-intensity conditioning (RIC) regimens for these patients. Here we report our 8-year single-center experience. Sixteen consecutive patients diagnosed with XLP1 underwent allogeneic HCT between 2006 and 2013 after a RIC regimen consisting of alemtuzumab, fludarabine, and melphalan. Patient phenotypes included hemophagocytic lymphohistiocytosis (HLH) after Epstein-Barr virus (n = 5) or human herpesvirus 6 (n = 1), macrophage activation syndrome (n = 1), interstitial pneumonitis and encephalitis (n = 1), B cell lymphoma (n = 8), and hypogammaglobulinemia (n = 2). One patient was asymptomatic. Fourteen of 16 patients received 8/8 HLA-matched unrelated or related bone marrow grafts, whereas 2 patients received mismatched unrelated grafts. Acute graft-versus-host disease (GVHD) prophylaxis consisted of methylprednisolone and cyclosporine in all but 1 patient, who additionally received methotrexate. All patients had hematopoietic recovery. There were no cases of hepatic veno-occlusive disease or pulmonary hemorrhage. One patient (6%) developed acute GVHD and later also developed chronic GVHD (6%). Five patients (31%) developed mixed chimerism. Only 1 patient with mixed chimerism (6%) experienced a decline of donor chimerism to less than 50% but returned to full donor chimerism after infusion of donor lymphocytes and a CD34(+) selected stem cell boost. Infectious complications were frequent, particularly viral reactivation. One-year survival estimated by Kaplan-Meier analysis was 80%, with long-term survival estimated at 71%. Survival was similar for patients with or without a history of HLH (86% versus 75%, respectively, P = .70). There were no occurrences of lymphoma or HLH after HCT. RIC HCT with alemtuzumab, fludarabine, and melphalan is an effective treatment for patients with XLP1, offering good survival rates regardless of prior disease manifestations, including HLH.
1738	Id transcriptional regulators in adipogenesis and adipose tissue metabolism.	24896358	Id proteins (Id1-Id4) are helix-loop-helix (HLH) transcriptional regulators that lack a basic DNA binding domain. They act as negative regulators of basic helix-loop-helix (bHLH) transcription factors by forming heterodimers and inhibit their DNA binding and transcriptional activity. Id proteins are implicated in the regulation of various cellular mechanisms such as cell proliferation, cellular differentiation, cell fate determination, angiogenesis and tumorigenesis. A handful of recent studies also disclosed that Id proteins have critical functions in adipocyte differentiation and adipose tissue metabolism. Here, we reviewed the progress made thus far in understanding the specific functions of Id proteins in adipose tissue differentiation and metabolism. In addition to reviewing the known mechanisms of action, we also discuss possible additional mechanisms in which Id proteins might participate in regulating adipogenic and metabolic pathways.
1739	Daughterless homodimer synergizes with Eyeless to induce Atonal expression and retinal neuron differentiation.	24886829	Class I Basic Helix-Loop-Helix (bHLH) transcription factors form homodimers or heterodimers with class II bHLH proteins. While bHLH heterodimers are known to have diverse roles, little is known about the role of class I homodimers. In this manuscript, we show that a linked dimer of Daughterless (Da), the only Drosophila class I bHLH protein, activates Atonal (Ato) expression and retinal neuron differentiation synergistically with the retinal determination factor Eyeless (Ey). The HLH protein Extramacrocheate (Emc), which forms heterodimer with Da, antagonizes the synergistic activation from Da but not the Da-Da linked dimer with Ey. We show that Da directly interacts with Ey and promotes Ey binding to the Ey binding site in the Ato 3׳ enhancer. Interestingly, the Ey binding site in the Ato 3׳ enhancer contains an embedded E-box that is also required for the synergistic activation by Ey and Da. Finally we show that mammalian homologs of Ey and Da can functionally replace their Drosophila counterparts to synergistically activate the Ato enhancer, suggesting that the observed function is evolutionary conserved.
1740	Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes.	248822172494836324950206	Animal host defense against infection requires the expression of defense genes at the right place and the right time. Understanding such tight control of host defense requires the elucidation of the transcription factors involved. By using an unbiased approach in the model Caenorhabditis elegans, we discovered that HLH-30 (known as TFEB in mammals) is a key transcription factor for host defense. HLH-30 was activated shortly after Staphylococcus aureus infection, and drove the expression of close to 80% of the host response, including antimicrobial and autophagy genes that were essential for host tolerance of infection. TFEB was also rapidly activated in murine macrophages upon S. aureus infection and was required for proper transcriptional induction of several proinflammatory cytokines and chemokines. Thus, our data suggest that TFEB is a previously unappreciated, evolutionarily ancient transcription factor in the host response to infection.
1741	Risk for complications in patients with hemophagocytic lymphohistiocytosis who undergo hematopoietic stem cell transplantation: myeloablative versus reduced-intensity conditioning regimens.	24871821	Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative option for patients with primary hemophagocytic lymphohistiocytosis (HLH) and for patients with secondary HLH who fail to respond to therapy. Advances in HSCT and supportive care measures have resulted in improved patient outcomes and decreased treatment-related mortality. Despite the overall improvement in outcome, HLH patients who undergo HSCT using myeloablative conditioning regimens are still at significant risk for complications. The HLH-94 study conducted by the Histiocyte Society reported a 30% TRM with increased pulmonary and hepatic complications. Recently, the use of reduced-intensity conditioning (RIC) regimens has shown favorable outcomes when compared to conventional HSCT and lower rate of acute complications. In this review we compare the potential complications of myeloablative and RIC regimens for HSCT in HLH patients.
1742	Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl.	24867218	As the major mechanism of plant growth and morphogenesis, cell elongation is controlled by many hormonal and environmental signals. How these signals are coordinated at the molecular level to ensure coherent cellular responses remains unclear. In this study, we illustrate a molecular circuit that integrates all major growth-regulating signals, including auxin, brassinosteroid, gibberellin, light, and temperature. Analyses of genome-wide targets, genetic and biochemical interactions demonstrate that the auxin-response factor ARF6, the light/temperature-regulated transcription factor PIF4, and the brassinosteroid-signaling transcription factor BZR1, interact with each other and cooperatively regulate large numbers of common target genes, but their DNA-binding activities are blocked by the gibberellin-inactivated repressor RGA. In addition, a tripartite HLH/bHLH module feedback regulates PIFs and additional bHLH factors that interact with ARF6, and thereby modulates auxin sensitivity according to developmental and environmental cues. Our results demonstrate a central growth-regulation circuit that integrates hormonal, environmental, and developmental controls of cell elongation in Arabidopsis hypocotyl.
1743	[Prognostic significance of coagulation disorders in children with hemophagocytic syndrome].	24857001	To study the prognostic significance of coagulation disorders in children with hemophagocytic syndrome (HPS).Thirty-five children with HPS were retrospectively studied to analyze the etiology, clinical characteristics, laboratory results and treatment outcomes.After treatment, 27 of the 35 HPS patients survived, and the other 8 cases died. All cases were treated according to the HLH-2004 protocol, but etoposide (VP-16) was not used in 10 of them. The response rate in patients who received VP-16 (22/25, 88%) was significantly higher than that in those not receiving VP-16 (5/10, 50%) (P<0.05). Compared with the survival group, the dead group had significantly lower platelet count, fibrinogen level, and VP-16 utilization rate (P<0.05) but significantly longer activated partial thromboplastin time and prothrombin time (P<0.05).Coagulation function can be used as an indicator of disease outcome. It is essential for improving the clinical outcome of HPS to monitor the coagulation function during treatment, detect and correct abnormalities in time, and provide treatment strictly according to the HLH-2004 protocol.
1744	Prenatal assessment of ventriculocoronary connections and ventricular endocardial fibroelastosis in hypoplastic left heart.	24854131	The outlook for newborns with hypoplastic left heart (HLH) has substantially improved over the last decade. However, differences in outcome among various anatomical subgroups have been described. We aimed to describe the incidence of ventriculocoronary communications and endocardial fibroelastosis in HLH and the possible implication on hospital survival (30 d).We retrospectively reviewed our medical records, still frames and video loops of 72 fetuses with HLH and critical aortic valve stenosis and evolving HLH from 2008 - 2013. The presence of VCAC and EFE were systematically assessed. Outcome parameters were incidence of VCAC and EFE among different anatomical subgroups of HLH and hospital survival (30 d).72 fetuses were included in this series. The incidence of VCAC was 11.1 % (8 cases) and EFE occurred in 33.3 % (24 cases). 5 fetuses with VCAC occurred in the subgroup of mitral valve stenosis/aortic valve atresia (MS/AA, 62.5 %) and 2 fetuses with VCAC occurred in the group of mitral atresia/aortic valve atresia (MA/AA, 25 %). Further classification was not possible in one case with VCAC (12.5 %). EFE predominantly occurred in the subgroup of MS/AA, MA/AA and in those cases with aortic valve stenosis and evolving HLH. The overall hospital survival on an intention-to-treat basis was 91.2 % (52/57 newborns). Hospital survival was 91 % for the subgroup of cases with MS/AA and for all other anatomical subgroups.The presence of VCAC in HLH can be diagnosed by fetal echocardiography predominantly occurring in cases with obstructed outflow and to some extent patent mitral valve. EFE is a frequent coexisting finding. Hospital survival was comparable among different anatomical subgroups and in cases with VCAC. The presence of VCAC in HLH did not limit the results of surgical palliation within the observation period of 30 days.
1745	Wolman disease associated with hemophagocytic lymphohistiocytosis: attempts for an explanation.	24844354	The lysosomal acid lipase (LAL) is the enzyme responsible of the hydrolysis of cholesteryl esters and triglycerides within endo-lysosomes. Loss of enzyme activity leads to accumulation of cholesteryl esters and triglycerides in the lysosome of most tissues. The complete deficiency of LAL is responsible of Wolman disease (WD), a severe systemic disease manifesting in the first days of life with vomiting, diarrhea, failure to thrive, hepatosplenomegaly, jaundice, anemia, and thrombocytopenia. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition which may be genetically determined or secondary to infections, malignancies, immune deficiencies, and rheumatologic disorders. So far, some inborn errors of metabolism have been associated with HLH (e.g., lysinuric protein intolerance, Gaucher's disease), and it has been anecdotally described in three WD patients, without any specific pathogenetic hypothesis. Here, we report on a WD patient, showing clear clinical, biochemical, and histological features indicative of HLH. We discuss the pathophysiological role of cholesteryl ester-induced inflammasome activation in macrophages, leading to a secondary HLH.This case indicates that WD can cause secondary HLH and suggests that a careful metabolic workup should be performed when facing to a pediatric patient with HLH.
1746	Carboxylation of cytosine (5caC) in the CG dinucleotide in the E-box motif (CGCAG|GTG) increases binding of the Tcf3|Ascl1 helix-loop-helix heterodimer 10-fold.	24835951	Three oxidative products of 5-methylcytosine (5mC) occur in mammalian genomes. We evaluated if these cytosine modifications in a CG dinucleotide altered DNA binding of four B-HLH homodimers and three heterodimers to the E-Box motif CGCAG|GTG. We examined 25 DNA probes containing all combinations of cytosine in a CG dinucleotide and none changed binding except for carboxylation of cytosine (5caC) in the strand CGCAG|GTG. 5caC enhanced binding of all examined B-HLH homodimers and heterodimers, particularly the Tcf3|Ascl1 heterodimer which increased binding ~10-fold. These results highlight a potential function of the oxidative products of 5mC, changing the DNA binding of sequence-specific transcription factors.
1747	Bartonella henselae infection inducing hemophagocytic lymphohistiocytosis in a kidney transplant recipient.	24829973	Bartonella henselae (Bh) is the cause of cat-scratch fever. When infection is symptomatic, it typically presents with singular lymphadenitis and fever. Less commonly, the infection can become disseminated and cause endocarditis, osteomyelitis, and micro-abscesses in multiple sites including liver, spleen, eyes, and brain, especially in immunocompromised patients. Hemophagocytic lymphohistiocytosis (Hlh) is a rare and severe multisystem disorder that may be triggered by infections. In one prior case, Bh, like other infections, has induced Hlh, an immune-mediated disease that can be characterized by septic-like presentation with persistent fevers, hepatosplenomegaly, and pancytopenia. In an immunocompromised transplant recipient, the onset of Hlh can be difficult to discern from a severe presentation of Bh. We report a case of criteria-proven secondary Hlh occurring after Bh infection in an 11-yr-old girl who was 13 months post-renal transplant. The patient developed multi-organ failure, and her severe clinical presentation required a thorough evaluation for infectious and non-infectious possibilities including post-transplant lymphoproliferative disorder and rejection. Early recognition of Hlh allowed for better directed therapies, leading to recovery of the patient and resolution of both Bh and Hlh.
1748	Clinical characteristics and genetic analysis of childhood acute lymphoblastic leukemia with hemophagocytic lymphohistiocytosis: a Japanese retrospective study by the Kyushu-Yamaguchi Children's Cancer Study Group.	24827398	This present study sought to analyze acute lymphoblastic leukemia (ALL) patients with hemophagocytic lymphohistiocytosis (HLH) registered in Kyushu-Yamaguchi Children's Cancer Study Group studies conducted between 1996 and 2007. Four of 357 patients, including two of 318 patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL) and two of 39 of those with T cell acute lymphoblastic leukemia (T-ALL), were identified. HLH was observed more frequently in the T-ALL patients than in the BCP-ALL patients (P = 0.061). The mean age of 13.0 years at the diagnosis of leukemia in the HLH + ALL group was significantly higher than the 6.05 years observed in the remaining ALL groups (P = 0.001). A female predisposition was noted, as all four patients were female (P = 0.043). In two of four patients, the leukemic cells exhibited deletions on the long arm of chromosome 6 (P = 0.003). Three patients suffered from HLH during maintenance therapy. Parvovirus B19 infection and cytomegalovirus reactivation were identified as causes of HLH in one and two patients, respectively. All four patients are currently in complete remission, although one developed relapse of leukemia after receiving maintenance therapy. Based on the genetic analyses, non-synonymous single nucleotide polymorphisms (SNPs) in UNC13D, syntaxin 11, and STXBP2 were identified in all patients. Clinicians should therefore be aware of the risk of HLH during maintenance therapy, especially in older T-ALL patients with SNPs in familial HLH causative genes.
1749	Nasal osteotomies: a cadaveric study of fracture lines.	24809333	The different nasal osteotomy patterns used to perform rhinoplasty are poorly described in the literature, and there is a continuous debate between surgeons on the ideal sequence and technique to obtain desired results.(1) To evaluate the necessity of a paramedian osteotomy when performing a high-low-high (HLH) osteotomy, (2) to study the fracture pattern of a high-low-low (HLL) osteotomy when combined with a paramedian osteotomy in the presence and in the absence of a transverse osteotomy, and (3) to evaluate the mobility of the central segment (located between the paramedian osteotomies) after digital pressure and the ideal osteotomy to mobilize it if needed.This was a prospective cadaveric study performed in the dissection laboratory in our tertiary referral center.Twenty cadavers were divided in 2 groups of 10. Group A had a paramedian osteotomy combined with an HLH osteotomy on 1 side and an HLH osteotomy alone on the other side. Group B had a paramedian combined with a transverse osteotomy followed by HLL osteotomy on 1 side. On the other side, we performed a paramedian combined with an HLL osteotomy. Finally, we evaluated the mobility of the central segment in group B, first with digital manipulation and then with a transverse osteotomy.The 3 authors evaluated individually the different fracture patterns. A result was considered successful when (1) the fracture followed the desired pattern, (2) a continuous line was obtained, and (3) a complete mobilization of the nasal segment was visualized.In group A, the side without a paramedian osteotomy had more unstable and greenstick fractures than the other side (P < .001). In group B, the side with a transverse osteotomy had more reliable and stable fractures than the other side (P < .05). Digital manipulation alone was not enough to mobilize the central segment in 8 of the 10 cadavers studied.Following this study we make the following suggestions: (1) to perform a paramedian osteotomy when an HLH osteotomy is needed, (2) to perform a transverse osteotomy before an HLL osteotomy when combined with paramedian osteotomy, and (3) to manipulate the central segment with a transverse osteotomy in order to mobilize it in a safe and predictable manner.NA.
1750	Chemical synthesis of the β-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.	24806200	Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique β-subunit, that form noncovalent heterodimers. Structurally, β-hCG shares a high degree of sequence similarity with β-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogeneous β-hLH and β-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of β-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (β-hCG) as well as its closely related homologue (β-hLH) as discrete glycoforms.
1751	Profile of hemophagocytic lymphohistiocytosis; efficacy of intravenous immunoglobulin therapy.	24806152	To study the profile of children with Hemophagocytic lymphohistiocytosis (HLH) and compare the outcome of treatment with intravenous immunoglobulin therapy and Dexamethasone vs. HLH-2004 protocol.The present retrospective cohort study was conducted in a tertiary care pediatric hospital in Chennai. Children with a diagnosis of HLH admitted to the hospital from June 2008 through June 2011 were included. Medical records of the subjects were reviewed and their clinical and demographic profile studied. Difference in outcome between treatment modalities was analysed.Of the 40 children studied, all had fever of 38.5 °C for more than 7 d. Splenomegaly was noted in 25 children at admission, but eventually occurred in all the patients. All children had bicytopenia. Mean laboratory values were as follows- neutrophil count 3,400/cu.mm, hemoglobin 8.75 g/dl, platelet count 84,000/cu.mm, fasting triglycerides 358 mg/dl, ferritin 8,139 mg/dl and fibrinogen 137 mg/dl. All children had evidence of hemophagocytosis in bone marrow smear. Good outcome was seen in 19/22 children treated with IVIG therapy (Group 1) vs. 10/12 children treated with HLH-2004 protocol with etoposide, cyclosporine and Dexamethasone (Group 2), P = 1.00. Good outcome was seen in 4/6 children treated with IVIG therapy followed by HLH-2004 protocol (Group 3). Serum ferritin levels of more than 3,000 mg/dl were present in 13 children. In this group, good outcome was seen in 7/8 patients treated with IVIG vs. 4/5 treated with the HLH-2004 protocol (P = 1.00).IVIG and HLH-2004 protocol may be equally effective in the management of HLH. IVIG may be a preferable initial regimen, to avoid the risk of secondary malignancy associated with etoposide.
1752	Hemophagocytic lymphohistiocytosis in imported pediatric visceral leishmaniasis in a nonendemic area.	24797953	To describe characteristics of visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (HLH) with focus on diagnostic clues and pitfalls, including the frequency of central nervous system (CNS) involvement, and to determine the efficacy of liposomal amphotericin B (L-AmB).We retrospectively analyzed clinical and laboratory features, diagnostic procedures, and treatment of 13 patients with HLH with imported visceral leishmaniasis, reported to the German HLH reference center (1999-2012).The spectrum of presentations was indistinguishable from patients with hereditary HLH or with acquired HLH because of infections with other pathogens. In 8 patients, disease onset occurred before the age of 2 years, coinciding with the typical age of manifestation of primary HLH. Two patients had mild nonspecific CNS findings. Misleading antiviral IgM (n = 6) and autoantibodies (n = 2) led to inaccurate interpretation of the etiology of HLH, sometimes with inappropriate therapeutic consequences. False negative results for Leishmania were obtained by initial bone marrow microscopy in 6/13, serology in 1/12, bone marrow culture in 2/5, and polymerase chain reaction of peripheral blood in 1/3 patients, and all bone marrow samples tested were Leishmania-positive by polymerase chain reaction (n = 7). L-AmB was administered to 12 patients, 5 of whom had no prior HLH-directed immunosuppressive therapy; sodium stibogluconate was administered to 1 patient. Persistent remission was achieved in 11 cases. Two patients required repeated or prolonged L-AmB therapy.Awareness of diagnostic pitfalls may save patients from unnecessary toxic treatment. CNS involvement is rare. L-AmB shows efficacy in visceral leishmaniasis-associated HLH.
1753	Hemophagocytic syndromes--an update.	24792320	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome and not an independent disease. HLH represents the extreme end of a severe uncontrolled hyperinflammatory reaction that can occur in many underlying conditions. Genetic forms of HLHs are due to defects in transport, processing and function of cytotoxic granules in natural killer cells and cytotoxic T lymphocytes, and are not restricted to manifestation in childhood. Acquired forms of HLH are encountered in infections, autoinflammatory and autoimmune diseases, malignancies, acquired immune deficiency. Functional tests allow for differentiation between genetic and acquired HLH. Treatment aims at suppressing hypercytokinemia and eliminating activated and infected cells. It includes immunomodulatory and immunosuppressive agents, cytostatics, T-cell and cytokine antibodies. In genetic HLH cure can only be achieved with hematopoietic stem cell transplantation. Reduced-intensity conditioning regimens have considerably improved survival.
1754	Haemophagocytic lymphohistiocytosis with lung cavity and lytic bone lesion in a 45 day infant.	24783117	Haemophagocytic lymphohistiocytosis (HLH) is a potentially fatal, hyper inflammatory condition which is caused by a highly stimulated but ineffective immune response. We are presenting here, a case of HLH which occurred in a 45 day infant. Presence of lung cavity and a lytic bone lesion in the skull, as was seen in this case, have not been reported in HLH in the literature. This raises a possibility of a simultaneous occurrence of HLH and Langerhans cell histiocytosis. In a child who presents with septicaemia but does not respond to treatment, the possibility of HLH needs to be considered.
1755	[Characterization of BZLF1 gene and its promoter Zp of EBV strains in children with EBV-associated diseases in recent 5 years in Beijing area].	24772891	This study aims to investigate the genetic characteristics of BZLF1 gene and its promoter Zp of the epidemic strains in children with primary Epstein-Barr virus (EBV)-associated diseases. Total DNA was extracted from the peripheral blood of 134 children with EBV-associated infectious mononucleosis (EBV-IM) and 32 children with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) who were admitted to Beijing Children's Hospital from 2006 to 2011. The EBNA3C, BZLF1, and Zp genes were amplified by PCR assay. Typing of EBV was performed according to the size of the amplification product of EBNA3C gene; the amplification products of BZLF1 and Zp genes were subjected to direct sequencing, and sequence analysis was performed using BioEdit 7. 0. 9. The results were as follows: (1) EBV-1 was present in 140 samples (97.2%, 140/144) and EBV-II in 4 samples (2.8%, 4/144). (2) Three BZLF1 genotypes and their 12 subtypes (including 6 newly found subtypes) were detected in this study; there were no significant differences in the frequencies of BZLF1-A and BZLF1-B between the children with EBV-IM and EBV-HLH (P = 0.083); BZLF1-A1 was the dominant genotype in children with EBV-associated diseases; t BZLF1-A mostly had three 29-bp repeats in the first intron of BZLF1 gene, and BZLF1-B mostly had 30-bp repeats (P = 0.000), with the number of repeats varying from 1 to 13. (3) Four Zp genotypes were detected in this study, including Zp-P, Zp-V3, Zp-V4, and Zp-V1; there were no significant differences in the frequencies of these Zp genotypes between children with EBV-IM and EBV-HLH (P = 0.272, 0.252, 1.0, and 1.0, respectively). (4) The linkage analysis of BZLF1 gene and its promoter Zp showed that BZLF1-A1 was highly associated with Zp-V3 (P = 0.000), while BZLF1-B4 with Zp-P (P = 0.000); EBV-I + BZLF1 A1 was highly associated with Zp-V3 (P = 0.000), while EBV-I+BZLF1-B4 with Zp-P (P = 0.000). The conclusions are as follows: (1) BZLF1-A1 is the dominant genotype in children with EBV-associated diseases; there are mostly 29-bp repeats in the first intron of BZLF1 gene for BZLF1-A genotype and 30-bp repeats for BZLF1-B genotype. (2) Zp-P and Zp-V3 are dominant Zp genotypes of EBV in children, which shared similar detection rates. (3) BZLF1-A1 is highly associated with Zp-V3, while BZLF1-B4 with Zp-P; EBV-I+BZLF1-A1 is highly associated with Zp-V3, while EBV-I+BZLF1-B4 with Zp-P.
1756	The perfect storm: hemophagocytic lymphohistiocytosis complicating disseminated yersiniosis.	24772830	Hemophagocytic lymphohistio-cytosis (HLH) is a rare but serious medical condition with variable clinical outcome. HLH presents real diagnostic and therapeutic challenges to the treating physician despite the major advances in molecular laboratory testing and the immune-chemotherapeutic interventions. Here we present a unique case ofHLH in the setting of hemoglobin H disease provoked by disseminated Yersinia enterocolitica infection. The hemoglobinopathy led to an iron overload state, which provided an optimal milieu for the siderophilic bacterium, Y. enterocolitica, to disseminate. This over whelming infection triggered an uncontrolled and dysregulated inflammatory cytokine cascade with resulting clinical sequelae. The patient was treated conservatively without immunosuppressive therapy and recovered quickly. The long-term outcome of such cases needs further definition.
1757	Genome-wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development.	24764321	The mechanisms that coordinate the regulation of autophagy with developmental signaling during multicellular organism development remain largely unknown. Here, we show that impaired function of ribosomal protein RPL-43 causes an accumulation of SQST-1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we identify 139 genes that promote autophagy activity upon inactivation. Various signaling pathways, including Sma/Mab TGF-β signaling, lin-35/Rb signaling, the XBP-1-mediated ER stress response, and the ATFS-1-mediated mitochondrial stress response, regulate the expression of autophagy genes independently of the TFEB homolog HLH-30. Our study thus provides a framework for understanding the role of signaling pathways in regulating autophagy under physiological conditions.
1758	Determination of an appropriate cut-off value for ferritin in the diagnosis of hemophagocytic lymphohistiocytosis.	24753034	Hyperferritinemia is a hallmark of hemophagocytic lymphohistiocytosis (HLH). In the HLH-2004 criteria, a ferritin value >500 µg/L is considered positive. However, this level was not determined based on evidence. We compared 123 patients with HLH and 320 patients with other hyperferritinemic conditions, calculated a receiver-operating characteristic, and determined sensitivity and specificity for different values. At 2,000 µg/L a trade-off is reached with sensitivity at 70% and specificity at 68%. If familial HLH and virus-associated acquired HLH are analyzed separately, sensitivity and specificity are similar for this level. The results may guide a potential modification of the current HLH criteria.
1759	[Allogeneic hematopoietic stem cell transplantation for hemophagocytic syndrome].	24739718	To observe the conditioning regimen, efficacy and side effects of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hemophagocytic lymphohistiocytosis (HLH).From 2010 to 2012, a total of 11 cases after allo-HSCT were evaluated including 8 cases with familial hemophagocytic lymphohistiocytosis (FHL) and 3 cases with Epstein-Barr virus (EBV) related HLH. Allo-HSCT from HLA haploidentical HSCT was performed for 3 cases and unrelated allo-HSCT for 8 cases; 7 cases underwent allo-HSCT with conditioning regimen of etoposide (VP16), busulphan (Bu), fludarabine (Flu) and antilymphocyte globulin (ATG) and 4 cases with Flu, melphalan (Mel) and ATG. Cyclosporine (CsA) or tacrolimus, mycophenolate (MMF) and methorexate (MTX) were used for prevention of graft versus host disease (GVHD). Four cases received anti-CD25 MoAbs, 7 cases received cord blood and 1 of them received haploidentical bone marrow to prevent GVHD.Three cases died after allo-HSCT. The median overall survival time of the 8 cases evaluated was 585 days (154-1 115 d). All the patients were successfully engrafted. Acute GVHD (aGVHD) occurred in 8 cases, including 3 cases of gradeI/II and 5 cases of grade III/IV. Chronic GVHD (cGVHD) occurred in 4 cases. Seven cases had cytomegalovirus (CMV) reactivation.The allo-HSCT was successful in treating primary and refractory hemophagocytic syndrome.
1760	Secondary hemophagocytic lymphohistiocytosis (HLH) from a presumed brown recluse spider bite.	24737162	Systemic loxoscelism is a rare complication after Loxosceles reclusa (brown recluse spider) envenomation. Loxosceles venom contains pro-inflammatory proteins, which have been shown to be elevated in patients with hemophagocytic lymph histiocytosis. We present a case of a 10-year-old male that developed presumed systemic loxoscelism, secondary hemophagocytic lymphohistiocytosis with hepatic dysfunction and renal failure. He was treated with dexamethasone and made a full recovery.
1761	Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures.	24732063	Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHβ or anti-oLHβ antibodies, respectively, for capture and a biotinylated anti-hFSHα (α4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90 °C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74 °C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64 °C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80 °C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93 °C and retained in vitro bioactivity. This suggests the existence of gonadotropin αβ heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined.
1762	Hemophagocytic lymphohistiocytosis syndrome in Dengue hemorrhagic fever.	247287012558919325589194	Hemophagocytic lymphohistiocytosis (HLH) is a life threatening florid activation of macrophages of the lymphoreticular system. It is reported to be associated with dengue in children in India and carries a high mortality. Patients present with high fever, worsening blood cell counts, splenomegaly, abnormal liver enzymes with features of liver failure, coagulopathy and neurological complications. The diagnosis is according to "Diagnostic Guidelines for HLH 2004", based on a triad of clinical, blood parameters and bone marrow cytology. In the present study, data of 212 children admitted with dengue were analyzed. Of 212 children, 31 children were classified as suspect HLH and advised bone marrow evaluation; of whom 23 children had marrow evidence of HLH. Worsening of blood cell counts were recorded in all children with a mean platelet count of 58,303.03 cells/cumm, low hematocrit in 95.65 %, low mean hemoglobin level of 8.37 g/dL, high erythrocyte sedimentation rate (ESR) and prolonged pro-thrombin time/international normalization ratio (PT/INR). Serum triglycerides, ferritin and transaminases were high. Of the 23 children, 19 patients received intravenous immunoglobulin (IVIG) and all of these children recovered. Dengue with multi-organ dysfunction is commonly concurrent to HLH in the marrow and hence, an early diagnosis based on clinical, laboratory and bone marrow evaluation is significant. A bone marrow evaluation confirms the diagnosis of HLH.
1763	Fulminant gastrointestinal bleeding caused by EBV-triggered hemophagocytic lymphohistiocytosis: report of a case.	24718941	Hemophagocytic lymphohistiocytosis (HLH) is a rare and often fatal hyperinflammatory syndrome characterized by fever, cytopenia, dramatically increased ferritin and hepatosplenomegaly. Here, we describe a previously healthy 39 year old pregnant woman in 30th week of her pregnancy with diarrhoea, intermittent gastrointestinal bleeding and fever of unknown focus. After cesarean section of twins in the 31st week she deteriorated with fulminant upper and lower gastrointestinal bleeding and disseminated intravascular coagulation. Gastro-, ileocolonoscopy and capsule endoscopy identified multiple bleeding punched ulcerations in the stomach, the entire small bowel and in parts of the colon. Emergency surgery with intraoperative endoscopy for uncontrolled hemorrhagic shock resulted in the resection of actively bleeding ulcers in the jejunum which temporally stabilized the critically ill patient. Jejunal histology and in situ hybridisation showed extensive ulcerations, focal lymphohistiocytic infiltration and EBV-positive immunoblasts. The diagnosis fulminant EBV-related HLH was confirmed based on the HLH-2004 diagnostic criteria and through detection of a reactivated EBV infection (up to 3 × 10(7) DNA copies/mL serum). Despite immunosuppressive therapy with steroids, cyclosporine A and etoposide in combination with Rituximab, the patient died from this sepsis-like, hyper-inflammatory syndrome in multiorgan failure with uncontrolled bleeding.
1764	Fatal missed case of hemophagocytic lymphohistiocytosis co-infected with parvovirus B19 and Epstein-Barr virus in an infant: test hyperferritinaemia early.	24713910	Hemophagocytic lymphohistiocytosis (HLH) triggered by Parvovirus B19 and Epstein-Barr virus co-infection is rare and unknown in infants. A 2-month-old male infant with fever, rash, bicytopenia and hepato-splenomegaly died owing to diagnostic dilemmas. Hence simply testing for hyperferritinaemia and hypertriglyceridemia/hypofibrinogenemia could diagnose HLH early while robust treatment be life-saving.
1765	Dopamine signaling in C. elegans is mediated in part by HLH-17-dependent regulation of extracellular dopamine levels.	24709946	In Caenorhabditis elegans, the dopamine transporter DAT-1 regulates synaptic dopamine (DA) signaling by controlling extracellular DA levels. In dat-1(ok157) animals, DA is not taken back up presynaptically but instead reaches extrasynpatic sites, where it activates the dopamine receptor DOP-3 on choligeneric motor neurons and causes animals to become paralyzed in water. This phenotype is called swimming-induced paralysis (SWIP) and is dependent on dat-1 and dop-3. Upstream regulators of dat-1 and dop-3 have yet to be described in C. elegans. In our previous studies, we defined a role for HLH-17 during dopamine response through its regulation of the dopamine receptors. Here we continue our characterization of the effects of HLH-17 on dopamine signaling. Our results suggest that HLH-17 acts downstream of dopamine synthesis to regulate the expression of dop-3 and dat-1. First, we show that hlh-17 animals display a SWIP phenotype that is consistent with its regulation of dop-3 and dat-1. Second, we show that this behavior is enhanced by treatment with the dopamine reuptake inhibitor, bupropion, in both hlh-17 and dat-1 animals, a result suggesting that SWIP behavior is regulated via a mechanism that is both dependent on and independent of DAT-1. Third, and finally, we show that although the SWIP phenotype of hlh-17 animals is unresponsive to the dopamine agonist, reserpine, and to the antidepressant, fluoxetine, hlh-17 animals are not defective in acetylcholine signaling. Taken together, our work suggests that HLH-17 is required to maintain normal levels of dopamine in the synaptic cleft through its regulation of dop-3 and dat-1.
1766	An Unusual Case of Adult-Onset Still's Disease with Hemophagocytic Syndrome, Necrotic Leukoencephalopathy and Disseminated Intravascular Coagulation.	24707428	Case. A 34-year-old African-American female with a history of adult-onset Still's disease presented to an outside hospital with oligoarthritis. She experienced a generalized tonic-clonic seizure en route via ambulance, was intubated upon arrival, and transferred to the intensive care unit for treatment of suspected pneumonia and sepsis. She subsequently developed generalized cutaneous desquamation that progressed despite the cessation of antibiotics and other potential offending drugs which required transfer to our hospital's burn unit. She was suspected to have reactive hemophagocytic syndrome based on her clinical presentation of fever, rash, polyarthritis, elevated liver enzymes, coagulopathy, splenomegaly, normocytic anemia, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, and hemophagocytosis visualized in bone marrow biopsy specimen. Magnetic resonance imaging demonstrated necrotic demyelination of the deep white matter and corona radiata. The patient developed multiorgan dysfunction and DIC without any other attributable etiology. Despite aggressive broad spectrum therapy and high dose of steroids she progressively deteriorated and eventually expired. Conclusion. Previous publications have highlighted the prevalence of necrotic leukoencephalopathy in children with familial hemophagocytic syndrome. Our patient demonstrated some uncommon features complicating her HLH including DIC and necrotic leukoencephalopathy, which are very rare entities in AOSD.
1767	A high sIL-2R/ferritin ratio is a useful marker for the diagnosis of lymphoma-associated hemophagocytic syndrome.	24705932	Lymphoma-associated hemophagocytic syndrome (LAHS), which is the major subtype of adult-onset secondary hemophagocytic lymphohistiocytosis (HLH), has a poor outcome. Although the early diagnosis and treatment of LAHS contributes to a better outcome, the lack of mass formation and the absence of distinct lymph node enlargement often delay the diagnosis of underlying lymphoma. A recent study, which statistically analyzed HLH cases in the literature, showed that the serum soluble interleukin-2 receptor (sIL-2R)/ferritin ratio could be used as a marker to diagnosis of LAHS. To verify this finding, we retrospectively analyzed the laboratory findings of 21 patients with HLH (10 benign disease-associated HLH and 11 LAHS). No significant differences were observed in the levels of LDH or CRP levels. The mean sIL-2R levels (units per milliliter) were significantly higher in the LAHS group (4,176 vs. 13,451, p = 0.0031), and ferritin levels (nanogram per milliliter) were higher in the benign disease-associated HLH group (20,462 vs. 2,561, p = 0.0031). Consequently, the mean serum sIL-2R/ferritin ratio of patients with LAHS was markedly higher than that of patients with benign disease-associated HLH (0.66 vs. 8.56, p = 0.0004). Thus, the results of this study demonstrated that the serum sIL-2R/ferritin ratio is a very useful marker for diagnosing of LAHS, which was further supported by clinical case analysis. Further studies to clarify the pathophysiology of secondary HLH caused by various triggers are needed.
1768	Human lutropin (hLH) and choriogonadotropin (CG) are assembled by different pathways: a model of hLH assembly.	24692561	The glycoprotein hormones are all structurally related heterodimers consisting of an α-subunit and a ligand-specific β-subunit that confers their unique biological activity. Crystal structures showed how the β-subunit surrounds a part of the α-subunit, and we showed the existence of the two mechanisms responsible for that assembly. In human choriogonadotropin, the β-subunit is folded before the subunits dock, and the α-subunit becomes incorporated into the dimer by a mechanism we termed "threading," passing between parts of the preassembled β-subunit. Here, we show that the human lutropin β-subunit is not folded completely prior to its interaction with the α-subunit and show that docking of the subunits enables the α-subunit to serve as a chaperone to the β-subunit. Based on data described here, we propose that the α-subunit facilitates formation of the human lutropin β-subunit by two mechanisms. First, the cystine knot of the α-subunit potentiates formation of the β-subunit cystine knot, and second, contacts between α-subunit loop 2 and a hydrophobic tail in the β-subunit facilitate formation of the seatbelt latch disulfide, which stabilizes the heterodimer. The primary influence of the α-subunit was seen when the hydrophobic tail was present or absent, but the secondary mechanism was required only when the hydrophobic tail of the β-subunit was present. During the evolution of human choriogonadotropin, neither of these α-subunit roles was necessary for folding of the β-subunit. The complex mechanism for lutropin assembly may be required to provide an additional control on its positive feedback function in vertebrate reproduction.
1769	Pathophysiology and spectrum of diseases caused by defects in lymphocyte cytotoxicity.	24680986	In experimental settings, lymphocyte cytotoxicity has been recognized as a central mechanism for immune defense against infected and neoplastic cells. More recently, molecular determinants of lymphocyte cytotoxicity have been identified through studies of rare, inherited hyperinflammatory and lymphoproliferative syndromes that include hemophagocytic lymphohistiocytosis (HLH). These studies have unraveled a set of genes pivotal for the biogenesis and directed release of perforin-containing lysosomes that mediate target cell killing, in addition to other pathways including Fas that also contribute to induction of cell death. Furthermore, studies of such human primary immunodeficiencies have highlighted non-redundant roles of perforin for maintenance of immune homeostasis. Besides providing mechanistic insights to lymphocyte cytotoxicity, studies of individuals with rare hyperinflammatory diseases are highlighting the relevance of lymphocyte cytotoxicity to more common human diseases. It is increasingly recognized that mutations abrogating lymphocyte cytotoxicity not only cause HLH, but also are associated with susceptibility to cancer and autoimmune syndromes. In addition, patients may initially be present with neurological symptoms or severe infectious disease masquerading as variable immunodeficiency syndrome. Here, we highlight new knowledge regarding the molecular mechanisms regulating lymphocyte cytotoxicity and review how mutations in genes associated with HLH cause disease. We also discuss the wider implications of impairments in lymphocyte cytotoxicity for human disease predisposition.
1770	Hemophagocytic Lymphohistiocytic Syndrome and Enteropathy-Associated T-cell Lymphoma in a Patient with Refractory Celiac Disease.	26157854	A 70-year-old woman with celiac disease presented with weight loss and diarrhea unresponsive to gluten-free diet (GFD) and prednisone. Diagnosis of type 2 refractory celiac disease (RCD) was made by small intestinal biopsies showing severe villous blunting and intraepithelial lymphocytosis. She was diagnosed with hemophagocytic lymphohistiocytic syndrome (HLH) after developing fever, pancytopenia, hypofibrinogenemia, elevated ferritin, and demonstration of hemophagocytosis on her bone marrow biopsy. An expert pathologist on lymphoma reviewed her biopsies and revised the final diagnosis to type 1 enteropathy-associated T-cell lymphoma (EATL) based on large T-cells infiltrating the lamina propria. We describe the first case of HLH associated with localized EATL and RCD.
1771	Clinical analysis and prognostic significance of haemophagocytic lymphohistiocytosis-associated anaplastic large cell lymphoma in children.	24666094	Haemophagocytic lymphohistiocytosis (HLH) has been rarely described in children treated for an anaplastic large-cell lymphoma (ALCL). We evaluated the incidence, the clinical and histological characteristics and the prognosis of HLH associated-ALCL. The medical, biological, cytological and histological data of patients treated for ALK-positive ALCL in the paediatric department of a single institution between 1975 and 2008 were analysed and assessed for HLH according to diagnosis criteria of the Histiocyte Society. Data concerning a series of 50 consecutive children with ALCL were reviewed. HLH-associated ALCL was observed in 12% of the patients. Lung involvement was significantly more frequent in HLH-associated ALCL patients than in the group without HLH (P = 0·004), as well as central nervous system (CNS) and bone marrow involvement (P = 0·001 and P = 0·007 respectively). The histological subtype in children with HLH-associated ALCL did not differ from that of the group without HLH. There was no significant difference between the two groups in 5-year EFS and OS (P = 0·91 and P > 0·99 respectively). In conclusion, HLH is not rare in paediatric ALCL. Despite a high incidence of visceral, CNS and bone marrow involvement, HLH does not seem to exert a significant impact on outcome in children treated for ALCL.
1772	[XIAP gene mutation screening in children with hemophagocytic lymphohistiocytosis].	24661516	To investigate the prevalence of mutations and sequence variations in X-linked inhibitor of apoptosis (XIAP) gene among Chinese pediatric patients with hemophagocytic lymphohistiocytosis (HLH).Sixty-five children who were diagnosed with HLH between January 2009 and December 2012 (case group), as well as 70 healthy children (control group), were enrolled in the study. The exons of XIAP gene (1-1, 1-2, 2-6) were amplified by PCR and directly sequenced. The genotypic and allelic frequencies of single nucleotide polymorphism (SNP) were analyzed.None of the HLH patients showed mutations in these exons of XIAP gene. Only one nonsynonymous SNP, rs5956583 located in exon 5, was observed, but there were no significant differences in the genotypic and allelic frequencies of this SNP between the case and control groups (P>0.05).HLH caused by XIAP mutations may be rare in children. SNP rs5956583 of XIAP gene may have little contribution to the development of childhood HLH.
1773	COX-2 overexpression increases malignant potential of human glioma cells through Id1.	24659686	Increased COX-2 expression directly correlates with glioma grade and is associated with shorter survival in glioblastoma (GBM) patients. COX-2 is also regulated by epidermal growth factor receptor signaling which is important in the pathogenesis of GBMs. However, COX-2 expression has not been previously shown to directly alter malignancy of GBMs. Id1 is a member of the helix-loop-helix (HLH) family of transcriptional repressors that act as dominant-negative inhibitors of basic-HLH factors. This factor has been shown to be regulated by COX-2 in breast carcinoma cells and recent studies suggest that Id1 may also be involved in the genesis/progression of gliomas. We now show that COX-2 increases the aggressiveness of GBM cells. GBM cells with COX-2 overexpression show increased growth of colonies in soft agar. Tumorigenesis in vivo is also increased in both subcutaneous flank and orthotopic intracranial tumor models. COX-2 overexpression induces Id1 expression in two GBM cell lines suggesting a role for Id1 in glioma transformation/tumorigenesis. Furthermore, we find direct evidence of a role for Id1 with significant suppression of in vitro transformation and in vivo tumorigenesis in COX-2-overexpressing GBM cells where Id1 has been knocked down. In fact, Id1 is even more efficient at enhancing transformation/tumorigenesis of GBM cells than COX-2. Finally, GBM cells with COX-2 or Id1 overexpression show greater migration/invasive potential and tumors that arise from these cells also display increased microvessel density, results in line with the increased malignant potential seen in these cells. Thus, COX-2 enhances the malignancy of GBM cells through induction of Id1.
1774	In vitro effect of molluscan hemocyanins on CAL-29 and T-24 bladder cancer cell lines.	24648926	The aim of this study was to investigate the anti-tumor effects of molluscan hemocyanins (Hcs) isolated from the marine snail Rapana venosa (RvH) and the garden snail Helix lucorum (HlH) on human bladder cancer cell lines. The antitumor effect of the native molecules of the above-mentioned Hcs and their subunits were examined in comparison to keyhole limpet hemocyanin (KLH), which is the most thoroughly studied Hc. The experiments were conducted using 2 human bladder cancer cell lines: CAL-29 and T-24. Doxorubicin hydrochloride (DOX) and mitomycin-C (MIT-C), which are routinely used in clinical practice to treat bladder cancer, were used for comparison. The viability of the 2 bladder cancer cell lines, used at a concentration of 20,000 cells/well, was measured by WST-1 assay at 24, 48 and 72 h after treatment with the above-mentioned Hcs and their isoforms at a concentration ranging from 0.8 to 500 μg/ml. A direct growth inhibitory effect on the tumor cells was observed mainly after treatment with the native molecule of HlH and the structural subunit, RvH1, at a concentration of 500 μg/ml. The native molecule of RvH exhibited an efficacy similar to that of KLH. However, the observed growth inhibitory effect of HlH was superior to that observed for KLH and RvH, when used at the same concentration. These findings demonstrate the antitumor effect of other Hcs, apart from KLH. Our data suggest that the native molecule of HlH and the subunit, RvH1, are alternative candidates for the treatment of human superficial bladder cancer.
1775	Hemophagocytic lymphohistiocytosis: clinical analysis of 103 adult patients.	24646466	To investigate the clinical features of adult patients with hemophagocytic lymphohistiocytosis (HLH) and to explore possible risk factors for death, we retrospectively reviewed the medical records of 103 adult HLH patients hospitalized from 1997 to 2012. We analyzed the underlying diseases, clinical characteristics, laboratory findings, outcomes, and prognostic factors. The most common cause of HLH was hematologic malignancies (n = 49), followed by infectious diseases (n = 24) and autoimmune disorders (n = 14); 24 cases were of unknown etiology. Eight patients had a combination of underlying diseases. HLH was clinically characterized by high fever (96.1%), splenomegaly (79.6%), hepatomegaly (65.0%), lymphadenopathy (53.4%), proteinuria (31.1%), skin rash (25.2%), gastrointestinal hemorrhage (14.6%), disseminated intravascular coagulation (13.6%), increased creatinine (7.8%), and central nervous system involvement (12.6%) including altered mental status (9.7%) and cranial hemorrhage (2.9%). Laboratory abnormalities included cytopenia (99.0%), serum ferritin >500 ug/L (98.4%), liver dysfunction (98.1%), hypertriglyceridemia (88.5%), hemophagocytosis in bone marrow smear (87.4%), and hypofibrinogenemia (60.9%).In addition to the treatment they received for the underlying causes, patients received therapy for HLH consisting of corticosteroids, immunosuppressive drugs, and intravenous immunoglobulin. Twenty-six patients (25.2%) recovered after treatment, and 19 of them achieved long-term remission during follow-up. Seventy-seven patients (74.8%) died because of tumor, sepsis, multiple organ failure, or HLH-related organ hemorrhage and coagulopathy. The deceased patients were more likely to be older at disease onset, male, and to present with splenomegaly and thrombocytopenia, compared to the survivors. Treatment for the underlying diseases combined with corticosteroids, immunosuppressive agents, and immunoglobulin therapy may improve the prognosis of HLH. More attention should be paid to high-risk patients to prevent the development of serious complications associated with HLH.
1776	The Notch target E(spl)mδ is a muscle-specific gene involved in methylmercury toxicity in motor neuron development.	24632433	Methylmercury (MeHg) is a ubiquitous environmental toxin that has a selective and potent impact on the nervous system, particularly during neural development yet, the mechanisms for its apparent neurodevelopmental specificity are unknown. The Notch receptor pathway has been implicated as a MeHg target in several studies. Notch signaling mediates cell-cell signals in a number of developmental contexts including neurogenesis and myogenesis, where it fundamentally acts to repress differentiation. Previous work in our lab has shown that MeHg causes preferential upregulation of a canonical Notch response gene, E(spl)mδ, in Drosophila embryos. In parallel, MeHg is seen to disrupt outgrowth of embryonic intersegmental motor nerves (ISN), which can be mimicked by expression of activated Notch in embryonic neurons. However, overexpression of E(spl)mδ in developing neurons fails to elicit motor neuron outgrowth defects, pointing to a non-autonomous role for E(spl)mδ in motor axon development. In this study we investigate a role for E(spl)mδ in conveying the toxicity of MeHg in the embryo. We find that endogenous expression of the E(spl)mδ gene localizes to developing somatic muscles in embryos. Notably, E(spl)mδ expression is seen in several muscles that are known synaptic targets for both the ISN and the segmental motor nerve (SN). We also demonstrate that the SN, similar to the ISN, exhibits disrupted axon outgrowth in response to MeHg. E(spl)mδ can induce a SN motor neuron phenotype, similar to MeHg treatment; but, only when E(spl)mδ expression is targeted to developing muscles. E(spl)mδ overexpression in developing muscles also results in aberrant muscle morphology, which is not apparent with expression of the closely related E(spl)mγ in developing muscles. Our data point to a role for the Notch target E(spl)mδ in mediating MeHg toxicity in embryonic development by disrupting the coordinated targeting of motor neurons to their muscle targets.
1777	Updates on histiocytic disorders.	24610771	Histiocytic disorders are rare entities that are becoming more recognized as our understanding of the molecular pathogenesis lead to novel diagnostic tests and targeted drug development. A symposium held at the American Society of Pediatric Hematology/Oncology (ASPHO) 2013 Annual Meeting discussed new insights into histiocytic disorders. This review highlights the symposium presentations, divided into three sections encompassing Langerhans cell histiocytosis (LCH), hemophagocytic lymphohistiocytosis (HLH) and Rosai Dorfman disease (RDD) including subsections on pathogenesis, clinical diagnostic criteria and novel insights into treatment. Details of other histiocytic disorders as well as the standard treatment guidelines have been published elsewhere and are beyond the scope of this discussion [Haupt et al. (2013). Pediatr Blood Cancer 60:175-184; Henter et al. (2007). Pediatr Blood Cancer 48:124-131].
1778	Identification of clinical features of lymphoma-associated hemophagocytic syndrome (LAHS): an analysis of 69 patients with hemophagocytic syndrome from a single-center in central region of China.	24610542	To identify the clinical features of lymphoma-associated hemophagocytic syndrome (LAHS), we retrospectively analyzed the clinical characteristics, laboratory findings and survival data of 16 LAHS patients from 69 adult hemophagocytic syndrome (HPS) patients. The results showed that the most common clinical manifestations and laboratory parameters were fever (100%), ferritin ≥ 500 g/L (100%), peripheral cytopenia in two or more lineages (100%), fibrinogen (Fbg) < 1.5 g/L (93.8%) and splenomegaly (81.3%) in LAHS patients. The percentages of patients with Fbg < 1.5 g/L, PLT < 40 × 10(9)/L and LDH ≥ 1,000 U/L in the LAHS group were significantly higher than those in non-LAHS patients (P = 0.010, 0.000, and 0.001, respectively). Survival analysis showed that HLH patients with rheumatological reasons had better prognosis (OS; median not reached), followed by patients in the infection group (350 days) and those with unexplained causes (140 days). LAHS had the worst prognosis (only 37 days). The symptoms of LAHS patients are usually confused with other HPS. Patients with LAHS had higher probabilities to have Fbg < 1.5 g/L, PLT < 40 × 10(9)/L, LDH ≥ 1,000 U/L and poor prognosis, so early diagnosis and systemic treatments are required.
1779	A functionally significant polymorphism in ID3 is associated with human coronary pathology.	24603695	We previously identified association between the ID3 SNP rs11574 and carotid intima-media thickness in the Diabetes Heart Study, a predominantly White diabetic population. The nonsynonymous SNP rs11574 results in an amino acid substitution in the C-terminal region of ID3, attenuating the dominant negative function of ID3 as an inhibitor of basic HLH factor E12-mediated transcription. In the current investigation, we characterize the association between the functionally significant polymorphism in ID3, rs11574, with human coronary pathology.The Multi-Ethnic Study of Atherosclerosis (MESA) is a longitudinal study of subclinical cardiovascular disease, including non-Hispanic White (n = 2,588), African American (n = 2,560) and Hispanic (n = 2,130) participants with data on coronary artery calcium (CAC). The Coronary Assessment in Virginia cohort (CAVA) included 71 patients aged 30-80 years, undergoing a medically necessary cardiac catheterization and intravascular ultrasound (IVUS) at the University of Virginia. ID3 SNP rs11574 risk allele was associated with the presence of CAC in MESA Whites (P = 0.017). In addition, the risk allele was associated with greater atheroma burden and stenosis in the CAVA cohort (P = 0.003, P = 0.04 respectively). The risk allele remained predictive of atheroma burden in multivariate analysis (Model 1: covariates age, gender, and LDL, regression coefficient = 9.578, SE = 3.657, p = 0.0110; Model 2: covariates Model 1, presence of hypertension, presence of diabetes, regression coefficient = 8.389, SE = 4.788, p = 0.0163).We present additional cohorts that demonstrate association of ID3 SNP rs11574 directly with human coronary artery pathology as measured by CAC and IVUS: one a multiethnic, relatively healthy population with low levels of diabetes and the second a predominantly White population with a higher incidence of T2DM referred for cardiac catheterization.
1780	A study of haematological and bone marrow changes in symptomatic patients with human immune deficiency virus infection with special mention of functional iron deficiency, anaemia of critically ill and haemophagocytic lymphohistiocytosis.	24600136	Haematological abnormalities are among the most common complications of HIV. These involve all lineages of blood cells. Bone marrow studies form integral part of complete workup of the HIV positive patients specially when they present as case of pyrexia of unknown origin (PUO), refractory anaemia and pancytopenia.55 HIV infected symptomatic patient requiring bone marrow examination were included in the study. Relevant clinical history, baseline haematological investigations including full blood count, CD4 cell counts using flow cytometry were recorded.Median ANC values in males were found to be significantly lower than females (p = 0.046). CD4 cell count statistically significantly correlated with age, TLC, ANC & platelet count. Anaemia was present in 45 patients and out of which 66.66% patients had normocytic normochromic anaemia. Iron deficiency anaemia was present in (37.77%) patients and anaemia of chronic disease in (62.22%) patients. 2 patients had anaemia of the critically ill. Two patients had non-Hodgkin's lymphoma (NHL) and showed lymphoma deposit in the bone marrow. Gelatinous degeneration was seen in 3 patients. Ill formed epithelioid cell granulomas were seen in 7 cases, and 2 cases were positive for acid fast bacilli (AFB). Haemophagocytosis was seen in 8 cases; two cases later diagnosed as a case of infection induced HLH. Leishmania donovani (LD) bodies seen in 2 cases.Bone marrow study is an important investigation in HIV infected symptomatic patients with peripheral haematological abnormalities.
1781	The transcriptome of utricle hair cell regeneration in the avian inner ear.	24599453	Sensory hair cell loss is the major cause of hearing and balance disorders. Mammals are incapable of sustained hair cell regeneration, but lower vertebrates can regenerate these mechano-electrical transducers. We present the first comprehensive transcriptome (by mRNA-Seq) of hair cell regeneration in the chick utricle. We provide pathway and pattern annotations and correlate these with the phenotypic events that occur during regeneration. These patterns are surprisingly synchronous and highly punctuated. We show how these patterns are a new resource for identifying components of the hair cell transcriptome and identify 494 new putative hair-cell-specific genes and validate three of these (of three tested) by immunohistochemical staining. We describe many surprising new components and dynamic expression patterns, particularly within NOTCH signaling. For example, we show that HES7 is specifically expressed during utricle hair cell regeneration and closely parallels the expression of HES5. Likewise, the expression of ATOH1 is closely correlated with HEYL and the HLH inhibitory transcription factors ID1, ID2, and ID4. We investigate the correlation between fibroblast growth factor signaling and supporting cell proliferation and show that FGF20 inhibits supporting cell proliferation. We also present an analysis of 212 differentially expressed transcription factor genes in the regenerative time course that fall into nine distinct gene expression patterns, many of which correlate with phenotypic events during regeneration and represent attractive candidates for future analysis and manipulation of the regenerative program in sensory epithelia and other vertebrate neuroepithelia.
1782	Prognostic factors and outcomes of adults with hemophagocytic lymphohistiocytosis.	24581757	To describe the prognostic factors and outcomes of adults with hemophagocytic lymphohistiocytosis (HLH), a rare disorder caused by pathologic activation of the immune system.The study population consisted of a consecutive cohort of adult (age ≥18 years) patients treated at Mayo Clinic in Rochester, Minnesota, from January 1, 1996, through December 31, 2011, in whom a diagnosis of HLH was suspected and subsequently confirmed by retrospective review using the HLH-04 diagnostic criteria.Of 250 adult patients suspected of having HLH, 62 met the HLH-04 diagnostic criteria and were included in the final analysis. The median age was 49 years (range, 18-87 years), and 42 (68%) were male. The underlying cause of HLH was malignant tumor in 32 patients (52%), infection in 21 patients (34%), autoimmune disorder in 5 patients (8%), and idiopathic disease in 4 patients (6%). After a median follow-up of 42 months, 41 patients (66%) had died. The median overall survival of the entire cohort was 2.1 months. The median overall survival of patients with tumor-associated HLH was 1.4 months compared with 22.8 months for patients with non-tumor-associated HLH (P=.01). The presence of a malignant tumor and hypoalbuminemia were significant predictors of inferior survival on multivariate analysis.In this large series of adults with secondary HLH treated at a single tertiary care center, patients with low serum albumin levels and tumor-associated HLH had a markedly worse survival. Hemophagocytic lymphohistiocytosis remains elusive and challenging to clinicians who must maintain a high index of suspicion. The recent discovery of several novel diagnostic and therapeutic modalities may improve outcomes of adult patients with HLH.
1783	A novel PRF1 gene mutation in a fatal neonate case with type 2 familial hemophagocytic lymphohistiocytosis.	24578718	Hemophagocytic lymphohistiocytosis (HLH) occurs in the primary form (genetic or familial) or secondary form (acquired). The familial form of HLH (FHL) is a potentially fatal autosomal recessive disorder that occurs because of constitutional defects in cell-mediated cytotoxicity. Here, we report a fatal neonatal case of type 2 FHL (FHL2) that involved a novel frameshift mutation. Clinically, the newborn presented with severe sepsis-like features and required mechanical ventilation and continuous venovenous hemodiafiltration. Flow cytometry analysis showed marked HLH and complete absence of intracytoplasmic perforin expression in cytotoxic cells; therefore, we performed molecular genetic analyses for PRF1 mutations, which showed that the patient had a compound heterozygous mutation in PRF1, that is, c.65delC (p.Pro22Argfs*2) and c.1090_1091delCT (p.Leu364Glufs*93). Clinical and genetic assessments for FHL are required for neonates with refractory fever and progressive multiple organ failure, particularly when there is no evidence of microbiological or metabolic cause.
1784	A novel mutation of the transcobalamin II gene in an infant presenting with hemophagocytic lymphohistiocytosis.	24563082	Transcobalamin II (TC II) deficiency is a rare disorder of cobalamin (CBL, vitamin B12) metabolism that occurs due to mutations in transcobalamin gene (TCN2). Hemophagocytic lymphohistiocytosis (HLH) in contrast is a syndrome characterized by uncontrolled immune response with hyperinflammation. A 2-month-old male baby was admitted with complaints of fever, cough, diarrhea, and respiratory distress. The parents were first cousins. The baby exhibited five of the eight diagnostic criteria for HLH-2004 and was diagnosed as HLH. A second bone marrow aspiration demonstrated megaloblastic changes in the erythroid series. The patient's vitamin B12 level was normal; however, hyperhomocysteinemia was present. A genetic deficiency of TC II was suspected. The patient and his parents were tested for TCN2 mutation. He had a homozygote mutation that was not included in Human 'Gene Mutation Database Cardiff'. The patient was treated with intramuscular vitamin B12, which was followed by improvement in both clinical and laboratory findings. He was 12 months old at the time of this report, with normal physical and neuromotor development. In this case presenting with the clinical and laboratory findings of HLH, TC II deficiency was diagnosed. A new mutation was found that was not reported before. Potential causative mechanisms of HLH induced by defects of cobalamin synthesis merit further investigation.
1785	Recombinant human follicle-stimulating hormone (r-hFSH) plus recombinant luteinizing hormone versus r-hFSH alone for ovarian stimulation during assisted reproductive technology: systematic review and meta-analysis.	24555766	The potential benefit of adding recombinant human luteinizing hormone (r-hLH) to recombinant human follicle-stimulating hormone (r-hFSH) during ovarian stimulation is a subject of debate, although there is evidence that it may benefit certain subpopulations, e.g. poor responders.A systematic review and a meta-analysis were performed. Three databases (MEDLINE, Embase and CENTRAL) were searched (from 1990 to 2011). Prospective, parallel-, comparative-group randomized controlled trials (RCTs) in women aged 18-45 years undergoing in vitro fertilization, intracytoplasmic sperm injection or both, treated with gonadotrophin-releasing hormone analogues and r-hFSH plus r-hLH or r-hFSH alone were included. The co-primary endpoints were number of oocytes retrieved and clinical pregnancy rate. Analyses were conducted for the overall population and for prospectively identified patient subgroups, including patients with poor ovarian response (POR).In total, 40 RCTs (6443 patients) were included in the analysis. Data on the number of oocytes retrieved were reported in 41 studies and imputed in two studies. Therefore, data were available from 43 studies (r-hFSH plus r-hLH, n=3113; r-hFSH, n=3228) in the intention-to-treat (ITT) population (all randomly allocated patients, including imputed data). Overall, no significant difference in the number of oocytes retrieved was found between the r-hFSH plus r-hLH and r-hFSH groups (weighted mean difference -0.03; 95% confidence interval [CI] -0.41 to 0.34). However, in poor responders, significantly more oocytes were retrieved with r-hFSH plus r-hLH versus r-hFSH alone (n=1077; weighted mean difference +0.75 oocytes; 95% CI 0.14-1.36). Significantly higher clinical pregnancy rates were observed with r-hFSH plus r-hLH versus r-hFSH alone in the overall population analysed in this review (risk ratio [RR] 1.09; 95% CI 1.01-1.18) and in poor responders (n=1179; RR 1.30; 95% CI 1.01-1.67; ITT population); the observed difference was more pronounced in poor responders.These data suggest that there is a relative increase in the clinical pregnancy rates of 9% in the overall population and 30% in poor responders. In conclusion, this meta-analysis suggests that the addition of r-hLH to r-hFSH may be beneficial for women with POR.
1786	Prognostic factors of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children: report of the Japan Histiocytosis Study Group.	24535916	Despite several advances in the treatment of Epstein-Barr virus (EBV) in recent years, patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) do not always show satisfactory outcomes. We here conducted a nationwide survey in Japan to identify prognostic factors of EBV-HLH in children with this disease in an effort to improve the management and the outcomes of these patients.Between January 2003 and June 2008, we enrolled 98 children younger than 18 years of age who were diagnosed with EBV-HLH. We then studied the clinical characteristics and laboratory findings at the time of diagnosis with the aim to identify prognostic factors for EBV-HLH.The mean age of onset of EBV-HLH was 3.9 ± 2.8 years. Most of our patients presented with fever, hepatosplenomegaly, lymphadenopathy, and hemophagocytosis of bone marrow. Sixty-two percent of patients showed T cell clonality, and 97% had EBV infection in either T or natural killer cells. Most patients (60%) were treated with a multi-agent chemotherapeutic regimen, including corticosteroid, etoposide, and cyclosporine. After initial treatment, 90.3% of patients were in remission, and 7 patients (8.2%) experienced recurrence of EBV infection. Among several prognostic factors, patients with both hyperbilirubinemia (>1.8 mg/dl) and hyperferritinemia (>20,300 ng/ml) at the time of diagnosis had significantly poorer outcomes than those with low serum bilirubin and ferritin levels.These findings suggest that the therapeutic strategy for children with EBV-HLH could be tailored according to the laboratory findings at diagnosis.
1787	Two-faced haemophagocytic lymphohistiocytosis: comparative review of two cases of adult haemophagocytic lymphohistiocytosis.	2452881725081055	Haemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal disease of children and adults. Cytokine dysfunction, uncontrolled accumulation of activated T-cells and histiocytes, and the inability to terminate the immune response lead to the clinical manifestations of extreme inflammation and end-organ damage. HLH is notoriously underreported because of its ability to mimic many other common diseases. Here, we outline two cases of HLH, one primary and the other secondary, to highlight some of the differences and to discuss therapeutic principles and emerging concepts.
1788	Cytophagic histiocytic panniculitis, hemophagocytic lymphohistiocytosis and undetermined autoimmune disorder: reconciling the puzzle.	24524345	Cytophagic histiocytic panniculitis is a rare disease, associated with either nonmalignant conditions or subcutaneous panniculitis-like T-cell lymphoma, and often also associated with hemophagocytic lymphohistiocytosis (HLH). We report the case of a 11-year-old boy with a history of secondary HLH who, after a local trauma, developed a painful, indurated plaque over the right thigh associated with relapsing HLH. Histopathologic findings from skin biopsy specimens revealed significant lobular panniculitis with benign histiocytes showing hemophagocytosis. High-dose intravenous methylprednisolone and cyclosporine A treatment was highly effective. A genetic study after a new, relapsing episode of HLH revealed an heterozygous missense mutation on STX 11 gene inherited from the mother.
1789	Hemophagocytic lymphohistiocytosis caused by primary Epstein-Barr virus in patient with Crohn's disease.	24520429	We present a case of a 19-year-old man with a 6-year history of Crohn's disease (CD), previously treated with 6-mercaptopurine, who was admitted to our department for Epstein-Barr virus (EBV) infection and subsequently developed a hemophagocytic lymphohistiocytosis (HLH). HLH is a rare disease which causes phagocytosis of all bone marrow derived cells. It can be a primary form as a autosomic recessive disease, or a secondary form associated with a variety of infections; EBV is the most common, the one with poorer prognosis. The incidence of lymphoproliferative disorders was increased in patients with inflammatory bowel disease (IBD) treated with thiopurines. Specific EBV-related clinical and virological management should be considered when treating a patient with IBD with immunosuppressive therapy. Moreover EBV infection in immunosuppressed patient can occur with more aggressive forms such as encephalitis and diffuse large B cell lymphoma. Our case confirms what is described in the literature; patients with IBD, particularly patients with CD receiving thiopurine therapy, who present 5 d of fever and cervical lymphadenopathy or previous evidence of lymphopenia should be screened for HLH.
1790	Successful rituximab treatment of refractory hemophagocytic lymphohistiocytosis and autoimmune hemolytic anemia associated with systemic lupus erythematosus.	24517558	High-dose steroids, immunosuppressants such as cyclophosphamide and cyclosporine, and high-dose intravenous immunoglobulin have all been used to control hemophagocytic lymphohistiocytosis (HLH) or autoimmune hemolytic anemia (AIHA) associated with systemic lupus erythematosus (SLE); however, some patients are refractory to treatment. Rituximab has successfully resolved many of the refractory manifestations of SLE. Here, we report a case of HLH and AIHA associated with SLE that was refractory or intolerable to conventional therapy, but was successfully treated with rituximab.
1791	Pancytopenia as an early indicator for Stevens-Johnson syndrome complicated with hemophagocytic lymphohistiocytosis: a case report.	24512039	Stevens-Johnson syndrome (SJS) is a severe skin and mucosal bullous disease. When complicated with Hemophagocytic lymphohistiocytosis (HLH), the condition is especially life-threatening.Here we report the case of a 4-year-old boy suffering from SJS with extensive erythema multiforme and bulla. Despite active intervention and supportive care, the boy experienced increased skin lesions and a higher fever. Meanwhile, decreases in white blood cell count and hemoglobin were observed. Hyperferritinemia, increased soluble CD25 level, decreased NK cell activity and hemophagocytosis in the boy's bone marrow confirmed the diagnosis of HLH. After high-dose intravenous immunoglobulin and methylprednisone pulse therapy, the boy was discharged in good condition.Simultaneous occurrence of HLH and SJS is very uncommon and the condition is life-threatening. Pancytopenia can be a precocious indicator and enables to start a prompt diagnosis and treatment.
1792	Hemophagocytic syndrome in children and adults.	24509696	Hemophagocytic syndrome, also known as hemophagocytic lymphohistiocytosis (HLH), is a heterogenic syndrome, which leads to an acute, life-threatening inflammatory reaction. HLH occurs both in children and adults, and can be triggered by various inherited as well as acquired factors. Depending on the etiology, HLH can be divided into genetic (i.e., primary) and acquired (i.e., secondary) forms. Among genetic HLH forms, one can distinguish between familial HLH and other genetically conditioned forms of HLH. Acquired HLH can be typically triggered by infections, autoimmune diseases, and malignancies. The most common symptoms of HLH are unremitting fever, splenomegaly, and peripheral blood cytopenia. Some severely ill patients present with central nervous system involvement. Laboratory tests reveal hyperferritinemia (often >10,000 μg/L), increased serum concentration of soluble receptor α for interleukin-2 (>2,400 U/L), hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hyponatremia, hypoproteinemia, and elevated liver transaminases and bilirubin. Prognosis in HLH is very serious. Genetic HLH is always lethal if adequate therapy is not administered. Similarly, severe acquired cases often lead to death without appropriate treatment. Since HLH can be encountered by various specialists in the medical field, basic knowledge of this entity such as diagnostic criteria and treatment should be familiar to all physicians.
1793	Epstein-barr virus-induced hemophagocytic lymphohistiocytosis.	24505517	Hemophagocytic lymphohistiocytosis (HLH) is one of the complications of Epstein-Barr virus (EBV) infection. Although the patients who have developed HLH following EBV have normal immune system, there are a few patients with EBV-induced immune deficiency who develop HLH as well. Here, we describe the case of a 10-year-old girl with neurological complications caused by EBV-induced HLH. The patient received rituximab, leading to weakening inflammation associated with EBV infection and suppression of disease through quick treatment.
1794	Helix-loop-helix/basic helix-loop-helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop.	24505057	Cell elongation is promoted by different environmental and hormonal signals, involving light, temperature, brassinosteroid (BR), and gibberellin, that inhibit the atypical basic helix-loop-helix (bHLH) transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1). Ectopic accumulation of IBH1 causes a severe dwarf phenotype, but the cell elongation suppression mechanism is still not well understood. Here, we identified a close homolog of IBH1, IBH1-LIKE1 (IBL1), that also antagonized BR responses and cell elongation. Genome-wide expression analyses showed that IBH1 and IBL1 act interdependently downstream of the BRASSINAZOLE-RESISTANT1 (BZR1)-PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-DELLA module. Although characterized as non-DNA binding, IBH1 repressed direct IBL1 transcription, and they both acted in tandem to suppress the expression of a common downstream helix-loop-helix (HLH)/bHLH network, thus forming an incoherent feed-forward loop. IBH1 and IBL1 together repressed the expression of PIF4, known to stimulate skotomorphogenesis synergistically with BZR1. Strikingly, PIF4 bound all direct and down-regulated HLH/bHLH targets of IBH1 and IBL1. Additional genome-wide comparisons suggested a model in which IBH1 antagonized PIF4 but not the PIF4-BZR1 dimer.
1795	[Report of a case with secondary acute promyelocytic leukemia after therapy for hemophagocytic lymphohistiocytosis and review of literature].	24495767	To explore the characteristics and risk of etoposide-related leukemia in the treatment of hemophagocytic lymphohistiocytosis (HLH).Clinical characteristics of a case with secondary acute promyelocytic leukemia (APL) were summarized and 10 cases of secondary leukemia after treatment for HLH from literature were analyzed.The child was diagnosed with Epstein-Barr virus associated HLH and received HLH-2004 protocol. The cumulative dose of etoposide (VP16) was 3520 mg/m(2). The patient was diagnosed with APL after 28 months of HLH.He achieved complete remission after induction chemotherapy of all-trans-retinoic acid and darubicin. Consolidated chemotherapy was continued. There were 10 reports of etoposide-related leukemia after treatment for HLH in the literature.Review of 11 cases treated with VP16, of which cumulative doses were 900-20 500 mg/m(2). The interval period between HLH and secondary leukemia was 24 months. The types of secondary leukemia included 1 case with acute lymphoblastic leukemia, 1 case with myelodysplastic syndrome and 9 cases of acute myeloid leukemia. The abnormalities of chromosome included 3 patients with 11q23, 3 APL patients with t (15, 17).Seven patients survived and 4 died.The latency period of etoposide-related leukemia is short. Acute myeloid leukemia and balanced chromosomal abnormality are common in etoposide-related leukemia. The risk factors for development of secondary leukemia are related to cumulative drug doses of etoposide, treatment schedules and co-administration of other antineoplastic agents.It is appropriate to keep suitable range of the cumulative dose of etoposide in HLH therapy in order to reduce the risk of therapy related leukemia.
1796	The 31-kDa caspase-generated cleavage product of p130Cas antagonizes the action of MyoD during myogenesis.	24472550	Myogenesis is regulated by the basic helix-loop-helix (bHLH) myogenic regulatory factor MyoD, which induces muscle-specific gene expression by binding to the E-box sequence as a heterodimer with ubiquitous bHLH E2A (E12/E47) proteins. Here, we report that a 31-kDa caspase-generated cleavage product of Crk-associated substrate (p130Cas), herein called 31-kDa, is downregulated during muscle cell differentiation. 31-kDa contains a helix-loop-helix (HLH) domain that shows greater sequence homology with Id (inhibitor of DNA binding) proteins than with bHLH proteins. This HLH domain, lacking the basic region required for DNA binding, mediated the direct interaction of 31-kDa with MyoD. Overexpression of 31-kDa in C3H10T1/2 cells inhibited not only the transcriptional activation of p21(Waf1/Cip1) and E-box-dependent muscle-specific genes by MyoD and/or E2A but also MyoD-induced myosin heavy chain expression and myogenic conversion. In sum, our results suggest a role for 31-kDa as a negative regulator of MyoD in the muscle differentiation program.
1797	The 253-kb inversion and deep intronic mutations in UNC13D are present in North American patients with familial hemophagocytic lymphohistiocytosis 3.	2447039924616126	The mutations in UNC13D are responsible for familial hemophagocytic lymphohistiocytosis (FHL) type 3. A 253-kb inversion and two deep intronic mutations, c.118-308C > T and c.118-307G > A, in UNC13D were recently reported in European and Asian FHL3 patients. We sought to determine the prevalence of these three non-coding mutations in North American FHL patients and evaluate the significance of examining these new mutations in genetic testing.We performed DNA sequencing of UNC13D and targeted analysis of these three mutations in 1,709 North American patients with a suspected clinical diagnosis of hemophagocytic lymphohistiocytosis (HLH).The 253-kb inversion, intronic mutations c.118-308C > T and c.118-307G > A were found in 11, 15, and 4 patients, respectively, in which the genetic basis (bi-allelic mutations) explained 25 additional patients. Taken together with previously diagnosed FHL3 patients in our HLH patient registry, these three non-coding mutations were found in 31.6% (25/79) of the FHL3 patients. The 253-kb inversion, c.118-308C > T and c.118-307G > A accounted for 7.0%, 8.9%, and 1.3% of mutant alleles, respectively. Significantly, eight novel mutations in UNC13D are being reported in this study. To further evaluate the expression level of the newly reported intronic mutation c.118-307G > A, reverse transcription PCR and Western blot analysis revealed a significant reduction of both RNA and protein levels suggesting that the c.118-307G > A mutation affects transcription.These specified non-coding mutations were found in a significant number of North American patients and inclusion of them in mutation analysis will improve the molecular diagnosis of FHL3.
1798	[Hemophagocytic syndrome associated with Epstein-Barr virus. Presentation of a case and treatment in the Resuscitation Unit].	24468010	We report the case of a 17 year old male patient, who was admitted to the Resuscitation Unit with the diagnosis of hemophagocytic syndrome, associated with infection by Epstein-Barr virus with unfavorable outcome. Hemophagocytic syndrome is a pathological immune activation syndrome due to the production/uncontrolled modulation of some cytokines. Its clinical signs and symptoms, defined by consensus criteria HLH-2004, are not pathognomonic, and often appear sequentially, thus suspicion should be followed by aggressive supportive therapy combined with early specific treatment of the triggering factor, as it is the only way to improve survival in patients with multiple organic failure as a result.
1799	Posterior reversible encephalopathy syndrome in pediatric patients undergoing treatment for hemophagocytic lymphohistiocytosis: clinical outcomes and putative risk factors.	24466550	Hemophagocytic lymphohistiocytosis (HLH) is a rare multiorgan disease of toxic immune activation caused by the interaction of cytotoxic T cells and innate immune cells and frequently involves the central nervous system (CNS). Posterior reversible encephalopathy syndrome (PRES) might develop during treatment with the HLH-2004 protocol from the Histiocyte Society. The aims of this study were to evaluate clinical outcomes and putative risk factors for prediction of PRES related to HLH.We reviewed the medical records of 28 patients with HLH who were treated between April 2005 and April 2012. We compared various clinical and laboratory parameters in patients without or with PRES to evaluate putative risk factors related to development of PRES.Six (21.4%) of the patients experienced PRES during treatment with the HLH-2004 protocol. Clinical and laboratory manifestations were not different compared with other conditions causing PRES. The main mechanism of PRES may be related to the HLH-2004 protocol and a high pro-inflammatory state. Most patients recovered quickly from neurologic manifestations without significant long-term sequelae. Preceding hypertension, an increase in ferritin level >50% compared with 1 week before development of PRES and hyponatremia were statistically significant factors.PRES is clinically reversible and has a favorable outcome in patients with HLH. Awareness of PRES and a differential diagnosis of other causes of neurologic complications, including CNS involvement of HLH, can help avoid unnecessary treatment or delayed management. Patients with preceding hypertension, hyponatremia, and rising ferritin levels during HLH treatment should be closely monitored for PRES.
1800	Clinical characteristics of hemophagocytic lymphohistiocytosis following Kawasaki disease: differentiation from recurrent Kawasaki disease.	24466549	Our aim was to investigate the clinical pattern of hemophagocytic lymphohistiocytosis following Kawasaki disease (HLH-KD), to enable differentiation of HLH from recurrent or refractory KD and facilitate early diagnosis.We performed a nationwide retrospective survey and reviewed the clinical characteristics of patients with HLH-KD, including the interval between KD and HLH, clinical and laboratory findings, treatment responses, and outcomes, and compared them with historical data for both diseases.Twelve patients with HLH-KD, including 5 previously reported cases, were recruited. The median age was 6.5 years (range, 9 months-14.7 years). Eight patients were male and 4 were female. The median interval between the first episode of KD and the second visit with recurrent fever was 12 days (3-22 days). Of the 12 children, 2 were initially treated with intravenous IgG (IVIG) for recurrent KD when they presented at the hospital with recurrent fever. Eventually, 10 children received chemotherapy under an HLH protocol and 2 received supportive treatment. Two patients died of combined infections during chemotherapy, 1 was lost to follow up, and 9 remain alive. The overall survival rate at 4 years was 81.1% with a median follow up of 45.1 months.A diagnosis of HLH-KD should be considered when symptoms similar to recurrent KD develop within 1 month of the first episode of KD. Our findings will help physicians differentiate between HLH and the recurrent form of KD.
1801	Hyperferritinemia in neonatal and infantile human parechovirus-3 infection in comparison with other infectious diseases.	24462418	Human parechovirus-3 (HPeV-3) has been associated with severe clinical manifestations in neonates and infants in the form of sepsis or hemophagocytic lymphohistiocytosis (HLH)-like illness. To clarify the clinical features of HPeV-3 infection, we compared clinical signs and laboratory findings among enteroviruses (EVs), HPeV-3, and other infections. Participants were 26 febrile infants in whom EVs (n = 20) or HPeV-3 (n = 6) were isolated from throat swab or fecal specimens. Clinical and laboratory data were compared among EVs, HPeV-3, respiratory syncytial virus (RSV) infection (n = 15), and bacterial meningitis (n = 8) groups. Apnea was frequently seen in the HPeV-3 group although there were no significant differences in other clinical symptoms. Leukocyte count was significantly lower in the HPeV-3 group than in the EV and RSV group. Platelet count was significantly lower in the HPeV-3 group than in the RSV group. Serum ferritin levels in the HPeV-3 group (mean, 2437 ng/ml) and EV group (mean, 552 ng/ml) were significantly higher than in the RSV group (mean 237 ng/ml; P = 0.008 and P = 0.002, respectively). The frequency of patients with clearly high ferritin levels ≥1000 ng/ml was comparatively higher in the HPeV-3 group (4/6) than the EV group (3/20) (P = 0.03). In the HPeV-3 group, ferritin levels were high on Days 4-5. Elevated ferritin levels, decreased leukocyte and platelet counts could offer diagnostic clues to HPeV-3 infection in infant. These laboratory findings might be associated with aberrant immune response to HPeV-3, which could contribute to the development of sepsis or HLH-like illness in neonates.
1802	Fatal hemophagocytic lymphohistiocytosis associated with locally acquired dengue virus infection - New Mexico and Texas, 2012.	24452132	Dengue is caused by infection with any of four mosquito-transmitted dengue viruses (DENV-1-4) and is characterized by fever, headache, myalgia, and leukopenia. Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyperinflammatory syndrome that can be familial or acquired, and is characterized by persistent fever, pancytopenia, hepatosplenomegaly, and increased serum ferritin. Acquired HLH is most frequently associated with Epstein Barr virus infection but also has been associated with dengue. This report describes a fatal case of acquired HLH that was apparently triggered by infection with DENV-3. The patient developed an acute febrile illness in August 2012 during a 1-month vacation in New Mexico. After returning to her home in Texas, she was initially diagnosed with West Nile virus (WNV) infection, developed pancytopenia, liver failure, and disseminated intravascular coagulopathy, and died. DENV-3 was detected in a premortem bone marrow biopsy in which erythrophagocytosis was evident. This case underscores the need for clinicians in the United States to be vigilant for dengue and request diagnostic testing for suspected cases, which should be reported to public health authorities.
1803	Differences between lutropin-mediated and choriogonadotropin-mediated receptor activation.	24438591	The human lutropin/choriogonadotropin receptor (hLHR) for the gonadotropic hormones human luteinizing hormone (hLH; lutropin) and human choriogonadotropin (hCG) is crucial for normal sexual development and fertility. We aimed to unravel differences between the two hLHR hormones in molecular activation mechanisms at hLHR. We utilized a specific hLHR variant that lacks exon 10 (hLHR-delExon10), which maintains full cAMP signaling by hCG, but decreases hLH-induced receptor signaling, resulting in a pathogenic phenotype. Exon 10 encodes 27 amino acids within the hinge region, which is an extracellular segment that is important for signaling and hormone interaction. Initially, we assumed that the lack of exon 10 might disturb intermolecular trans-activation of hLH, a mechanism that has been reported for hCG at hLHR. Coexpression of signaling-deficient hLHR and binding-deficient hLHR can be used to examine the mechanisms of receptor signaling, in particular intermolecular cooperation and intramolecular cis-activation. Therefore, hLHR-delExon10 was combined with the hLHR Lys605→Glu mutant, in which signaling is abolished, and the hLHR mutant Cys131→Arg, in which binding is deficient. We found that hCG signaling was partially rescued, indicating trans-activation. However, the hLH signal could not be restored via forced trans-activation with any construct. Fluorescence cross-correlation spectroscopy detected oligomerization in all combinations, indicating that these functional differences cannot be explained by monomerization of hLHR-delExon10. Thus, our data demonstrate not only that the different behavior of hLH at hLHR-delExon10 is unlikely to be related to modified intermolecular receptor activation, but also that hLH may exclusively stimulate the targeted hLHR by cis-activation, whereas hCG is also capable of inducing trans-activation.
1804	Extremely elevated cerebrospinal fluid protein levels in a child with neurologic symptoms: beware of haemophagocytic lymphohistiocytosis.	24433830	Neurologic symptoms can be the initial manifestation of haemophagocytic lymphohistiocytosis (HLH). In this case study, we present a 3-year old boy with a clinical picture of encephalitis, a cerebrospinal fluid (CSF) protein level up to 1165 mg/dl and diffuse cerebral MRI abnormalities. The diagnosis of HLH was established only 6 weeks after initial presentation. The boy recovered after HLH therapy with persisting mild cognitive defects. Genetic investigation demonstrated X-linked lymphoproliferative disease (XLP) as the underlying cause of HLH. The extremely elevated protein level in CSF in this case has not yet been reported in patients with HLH.
1805	Hemophagocytic lymphohistiocytosis complicating erythroleukemia in a child with monosomy 7: a case report and review of the literature.	24416604	Herein, the first case of childhood erythrophagocytosis following chemotherapy for erythroleukemia in a child with monosomy 7 is reported. A 5-year-old boy presented with anemia, thrombocytopenia, and hepatosplenomegaly in whom erythroleukemia was diagnosed. Prolonged pancytopenia accompanied by persistent fever and huge splenomegaly and hepatomegaly became evident after 2 courses of chemotherapy. On bone marrow aspiration, macrophages phagocytosing erythroid precursors were observed and the diagnosis of HLH was established; additionally, monosomy 7 was detected on bone marrow cytogenetic examination. In conclusion, monosomy 7 can lead to erythrophagocytosis associated with erythroid leukemia and should be considered among the chromosomal abnormalities contributing to the association.
1806	Hemophagocytic Syndrome and Critical Illness: New Insights into Diagnosis and Management.	24407034	Hemophagocytic lymphohistiocytosis (HLH) comprises a heterogeneous group of diseases that are characterized by a hyperinflammatory state due to uncontrolled T cell, macrophage, and histiocyte activation, accompanied by excessive cytokine production. This rare condition is almost uniformly fatal unless promptly recognized and treated. Much progress has been made in the last two decades in our understanding of the mechanisms underlying familial, and to a lesser extent, acquired cases of HLH. Recurrent mutations in more than 10 different genes have now been identified, involving biological pathways converging on intracellular vesicle trafficking and cytolytic granule exocytosis. Mechanisms underlying the majority of patients with acquired HLH, however, remain elusive, hampering both diagnostic evaluation and therapeutic management of these patients. Given that the majority of intensive care unit (ICU) patients with sepsis or multiorgan failure share many features of HLH, it is especially critical for pediatric and adult intensivists to be able to recognize patients with bona fide HLH and initiate treatment without delay. In this article, we review our current understanding of the pathophysiology, clinical testing, diagnosis, and treatment of patients with HLH, especially as it pertains to the care of critically ill patients in pediatric and medical ICUs.
1807	The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA.	24403087	Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.
1808	An unusual presentation of galactosemia: hemophagocytic lymphohistiocytosis.	24385729	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening condition. Uncontrolled proliferation of activated lymphocytes secreting high amounts of inflammatory cytokines seems to be the main pathogenesis. The diagnosis of HLH can often be difficult. It may presents in many forms such as fever of unknown origin, hepatitis, acute liver failure, and sepsis-like illness. Here we present a newborn galactosemia case presented with HLH. Close monitoring of the diagnostic criteria of HLH during the course of galactosemia-associated hemophagocytosis, both before and after dietary treatment, should be performed in order to fully determine if the triggering mechanism is infection or accumulation of metabolites.None declared.Hemofagositik lenfohistiyositozis (HLH), nadir görülen, yaşamı tehdit eden bir durumdur. Patogenezinde; yüksek miktarda sitokin üreten aktive lenfositlerin kontrolsüz proliferasyonunun olduğu düşünülmektedir. HLH tanısını koymak genellikle güçtür. Hastalar nedeni bilinmeyen ateş, hepatit, akut karaciğer yetmezliği, sepsis gibi çok farklı klinik bulgularla başvurabilir. Burada HLH ile başvuran galaktozemili bir yenidoğan vakasını sunduk. Galaktozemi ile ilişkili HLH vakalarında tanı kriterlerinin diyet tedavisi öncesi ve sonrasında yakın takip edilmesi hastalığı tetikleyen mekanizmanın enfeksiyon mu yoksa metabolit birikimi mi olduğunu daha iyi açıklayabilmek için gereklidir.
1809	Hemophagocytic lymphohistiocytosis in infants: a single center experience from India.	24383954	There is paucity of outcome data for hemophagocytic lymphohistiocytosis (HLH) in infants from India, especially post stem cell transplant (SCT). We report outcome data of eight infants diagnosed with HLH. Mean age was 7.1 months (range 2-11). Mutation analysis was possible in seven patients. One patient had Griscelli syndrome. In three patients, no known mutation could be identified, while in remaining three homozygous mutations in Perforin, Munc and STX11 gene were identified. All were treated as per HLH 2004 protocol. Four died during induction phase. One patient abandoned therapy. Two underwent SCT, while one is awaiting SCT. First patient is alive and disease-free at 22 months postmatched sibling donor SCT. Second underwent unrelated double cord blood transplant, but died 5 months posttransplant due to renal failure. It is feasible to offer SCT for infants with familial HLH in the developing world although barriers like sepsis and disease refractoriness remain.
1810	Trichosporon asahii, sepsis, and secondary hemophagocytic lymphohistiocytosis in children with hematologic malignancy.	24383917	Trichosporon asahii (T. asahii) is an uncommon fungal pathogen rarely seen in patients with hematologic malignancies. Although appropriate therapy is started, infection with T. asahii usually leads to mortality. Here, we describe two patients developed severe T. asahii infection and secondary HLH. Despite rapid identification of T. asahii and negative blood cultures achieved by prompt initiation of treatment with voriconazole, fever and pancytopenia, persisted and both developed hepatosplenomegaly, and their clinical state worsened. Bone marrow aspiraton revealed hemophagocytosis. Elevated ferritin, triglyceride levels were seen. The first patient did not receive HLH directed therapy and died with multiple organ dysfunctions. Prompt diagnosis and treatment of secondary HLH led to rapid improvement in clinical and laboratory abnormalities in the second patient and kept her alive. We suggest that HLH may present as a secondary condition, accompanying a severe infection with T. asahii may, at least in part, contribute to high mortality rates in these cases.
1811	Graded defects in cytotoxicity determine severity of hemophagocytic lymphohistiocytosis in humans and mice.	24379813	Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease of hyperinflammation resulting from immune dysregulation due to inherited defects in the cytolytic machinery of natural killer and T cells. In humans, mutations in seven genes encoding proteins involved in cytolytic effector functions have so far been identified that predispose to HLH. However, although most affected patients develop HLH eventually, disease onset and severity are highly variable. Due to the genetic heterogeneity and variable time and nature of disease triggers, the immunological basis of these variations in HLH progression is incompletely understood. Several murine models of primary HLH have been established allowing to study HLH pathogenesis under more defined conditions. Here we directly compare the clinical HLH phenotype in six HLH-prone mouse strains with defects in the granule-dependent cytotoxic pathway. A severity gradient of HLH manifestations could be identified that is defined by the genetically determined residual lytic activity of cytotoxic T lymphocytes (CTL) and their ability to control lymphocytic choriomeningitis virus, which was used as a trigger for disease induction. Importantly, analysis of cohorts of HLH patients with severe bi-allelic mutations in the corresponding genes yielded a similar severity gradient in human HLH as reflected by the age at disease onset. Our findings define HLH as a threshold disease determined by subtle differences in the residual lytic activity of CTL.
1812	Hemophagocytic lymphohistiocytosis (HLH) in a 25-year-old presenting with multisystem organ failure.	24371860	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of extreme inflammation caused by pathologic activation of the immune system. Diagnosis of HLH is challenging as the clinical presentation is similar to common medical entities such as sepsis. When a source of the extreme inflammation is not found, HLH should be considered in the differential diagnosis. In HLH, inflammatory markers such as soluble CD25 and ferritin levels are elevated. Ferritin assay is widely available at most institutions; a level greater than 10,000 is highly suggestive of HLH.2 Delayed diagnosis and failure to initiate cytotoxic chemotherapy will result in a fatal outcome.
1813	Perforin: an important player in immune response.	26155110	Perforin is a glycoprotein responsible for pore formation in cell membranes of target cells. Perforin is able to polymerize and form a channel in target cell membrane. Many research groups focus on the role of perforin in various diseases, immune response to bacterial and viral infections, immune surveillance and immunopathology. In addition, perforin is involved in the pathogenesis of autoimmune diseases and allogeneic transplant rejection. Natural killer (NK) cells and CD8-positive T-cells are the main source of perforin. However, CD4-positive T-cells are also able to express a low amount of perforin, when classic cytotoxicity is ineffective or disturbed. Polymerized perforin molecules form channels enabling free, non-selective, passive transport of ions, water, small-molecule substances and enzymes. In consequence, the channels disrupt protective barrier of cell membrane and destroy integrity of the target cell. This review will focus on mechanisms of action and structure of perforin. Also, in this review we discuss the problem of abnormal perforin production in diseases such as: hemophagocytic lymphohistiocytosis (HLH), leukemias and lymphomas, infectious diseases and autoimmune diseases. Better understanding of the role of these molecules in health and disease will open a new field of research with possible therapeutic implications.
1814	Hemophagocytic lymphohistiocytosis masquerading as progressive chronic lymphocytic leukemia.	24371766	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal syndrome characterized by a non-malignant expansion of the macrophage population in the setting of a heightened cytokine response with subsequent widespread hemophagocytosis. It can occur as either genetic or acquired forms; the latter of which frequently occurs in the setting of infection, autoimmune disease, or malignancy. We present the second known case of HLH associated Chronic Lymphocytic Leukemia (CLL) in the absence of infectious etiology and review the current literature.
1815	Evaluation of the plasma micro RNA expression levels in secondary hemophagocytic lymphohistiocytosis.	24363881	Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hyper inflammatory disease. Micro RNAs (miRNA) are about 22 nucleotide-long, small RNAs encoded with genes, and they have regulatory functions in immune response.To determine the miRNA expression levels of 11 secondary HLH patients, we evaluated the associations of miRNA levels with pathogenesis, clinical presentation, and prognosis of the disease.Patients who were diagnosed with secondary HLH from January 2011 to December 2012 were included in this study. We profiled the expressions of 379 miRNAs in plasma of both HLH patients and healthy controls. Patients were evaluated regarding with age, clinical findings, miRNA expresions, laboratory data, treatment, and prognosis, by using descriptive statistics.A total of 11 secondary HLH patients and 11 healthy children were included in this study. miR-205-5p was expressed in all case and controls and expression level of miR-205-5p was found 6.21 fold higher than control group (p=0.01). We detected the second highest expression percent in miR-194-5p with 81% of cases and controls. Expression level of miR-194-5p was found to have 163 fold higher than controls (p= 0.009). miR-30c-5p showed 77% expression percent in cases and controls together. The expression level of this miRNA was detected 9 fold decreased in HLH patients compared to healthy children (p= 0.031).We showed that miR-205-5p, miR-194-5p and miR-30c-5p could be useful plasma biomarkers for HLH. Further research is needed in larger and homogenous study groups, especially for these miRNAs as biomarkers for HLH.
1816	Marrow assessment for hemophagocytic lymphohistiocytosis demonstrates poor correlation with disease probability.	243437382559625925738202	To evaluate the amount of hemophagocytosis in 64 marrow core biopsy specimens and aspirates from 58 patients with clinical suspicion for secondary hemophagocytic lymphohistiocytosis (HLH) or reported findings of hemophagocytosis.A review of medical records assigned patients to a low-risk (45 patients) or high-risk (13 patients) HLH group, and association with histologic findings was examined using the Fisher exact test.The amount of hemophagocytosis in aspirate or the core biopsy specimen did not correlate with disease probability (P = .17 and P = .63, respectively). Of the clinical/laboratory criteria assessed, the most significant correlations with HLH were highly elevated ferritin (P = .01), cytopenias (P = .02), and fever (P = .009).Our findings indicated that marrow histologic findings alone do not reliably predict the probability of HLH, and an isolated finding of hemophagocytosis, even when present in a high amount, lacks specificity for HLH.
1817	Not another case of mono: Epstein-Barr virus (EBV) associate hemophagocytic lymphohistiocytosis (HLH).	24340129	Male, 30 FINAL DIAGNOSIS: Hemophagocytic lymphohistiocytosis (LHL) Symptoms: Abdominal pain • fever • hypotension • pancytopenia- Clinical Procedure:- Specialty: Infectious Diseases.Rare disease.Hemophagocytic lymphohistiocytosis (HLH) is a result of dysregulated cellular response system. Primary HLH is an autosomal recessive disorder of childhood, with defects in cellular cytotoxicity. Secondary HLH is an acquired syndrome that presents in young adulthood secondary to a variety of inflammatory conditions: viral infections, rheumatologic conditions, or malignant processes. The inflammatory nature of certain conditions triggers a cytokine release in individuals who have abnormal T cell activation.A 30-year-old Hispanic male presented with worsening abdominal pain for 5 months and was found to have fever, pancytopenia, and hypotension. Serial CT scans of the abdomen/pelvis showed splenomegaly but no abscesses, areas of infection, or masses. Infectious causes were considered but results of all cultures and tests were negative except for a high Epstein-Barr viral load. The patient deteriorated and required intubation on hospital day 28. Repeat bone marrow biopsy on day 32 suggested a diagnosis of hemophagocytic lymphohistiocytosis, although there was no evidence of hemophagocytosis within the bone marrow. The patient continued to deteriorate and was too unstable to receive treatment with chemotherapy. He died on hospital day 34.This case highlights the importance of early consideration and treatment of secondary HLH in an individual presenting with progressive fever, hepatomegaly, and cytopenias.
1818	Id4 dependent acetylation restores mutant-p53 transcriptional activity.	24330748	The mechanisms that can restore biological activity of mutant p53 are an area of high interest given that mutant p53 expression is observed in one third of prostate cancer. Here we demonstrate that Id4, an HLH transcriptional regulator and a tumor suppressor, can restore the mutant p53 transcriptional activity in prostate cancer cells.Id4 was over-expressed in prostate cancer cell line DU145 harboring mutant p53 (P223L and V274F) and silenced in LNCaP cells with wild type p53. The cells were used to quantitate apoptosis, p53 localization, p53 DNA binding and transcriptional activity. Immuno-precipitation/-blot studies were performed to demonstrate interactions between Id4, p53 and CBP/p300 and acetylation of specific lysine residues within p53.Ectopic expression of Id4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, the transcriptional targets of p53. Mutant p53 gained DNA binding and transcriptional activity in the presence of Id4 in DU145 cells. Conversely, loss of Id4 in LNCaP cells abrogated wild type p53 DNA binding and transactivation potential. Gain of Id4 resulted in increased acetylation of mutant p53 whereas loss of Id4 lead to decreased acetylation in DU145 and LNCaP cells respectively. Id4 dependent acetylation of p53 was in part due to a physical interaction between Id4, p53 and acetyl-transferase CBP/p300.Taken together, our results suggest that Id4 regulates the activity of wild type and mutant p53. Id4 promoted the assembly of a macromolecular complex involving CBP/P300 that resulted in acetylation of p53 at K373, a critical post-translational modification required for its biological activity.
1819	Fludarabine-based reduced-intensity conditioning regimen for hematopoietic stem cell transplantation in primary hemophagocytic lymphohistiocytosis.	24330187	Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition that clinically characterized by fever, hepatosplenomegaly, and cytopenia. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment option for patients diagnosed with primary HLH.In this prospective study, we analyzed the outcome of 10 pediatric patients with primary HLH who had received HSCT, using reduced-intensity conditioning (RIC) regimen from 2007 to 2012. The median age at transplantation was 22.6 months (range: 6-60). All of the patients received the same RIC regimen based on the use of fludarabine in combination with melphalan and horse antithymocyte globulin (ATG). Cyclosporine and methylprednisolone were used as graft-vs.-host disease (GvHD) prophylaxis.Hematopoietic engraftment occurred in all patients. At the present time, 8 patients with a median follow-up of 39 months are still alive and all of them are disease free. Acute and chronic GvHD developed in 6 and 2 patients, retrospectively. Two patients died of sepsis and chronic GvHD during the study.Because of pretransplant infections caused by underlying immunodeficiency in patients with primary HLH, the use of less toxic regimen with RIC seems to be highly effective in this regard. Recipients of RIC transplant, with either full or mixed chimerism, had a long-term survival rate with no manifestation of primary HLH symptoms.
1820	Hemophagocytic lymphohistiocytosis: pathogenesis and treatment.	24319239	Hemophagocytic lymphohistiocytosis (HLH) is not an independent disease but rather a life-threatening clinical syndrome that occurs in many underlying conditions and in all age groups. HLH is the consequence of a severe, uncontrolled hyperinflammatory reaction that in most cases is triggered by an infectious agent. Persistent stimulation of lymphocytes and histiocytes results in hypercytokinemia, leading to the characteristic symptoms of HLH. Genetic defects in familial HLH and in immunodeficiency syndromes associated with albinism affect the transport, processing, and function of cytotoxic granules in natural killer cells and cytotoxic T lymphocytes. This leads to defective killing of target cells and a failure to contract the immune response. The defects are increasingly found also in adolescents and adults. Acquired HLH occurs in autoinflammatory and autoimmune diseases (macrophage activation syndrome) and in patients with iatrogenic immunosuppression or with malignancies, but also in otherwise healthy persons with infections. Treatment of HLH aims at suppressing hypercytokinemia and eliminating the activated and infected cells. In genetic HLH, hematopoietic stem cell transplantation (HSCT) is needed for the correction of the immune defect. Treatment modalities include immunosuppressive, immunomodulatory, and cytostatic drugs; T-cell antibodies; and anticytokine agents. Using immunochemotherapy, familial HLH, which had been invariably fatal, has become a curable disease with more than 50% survivors. Reduced intensity conditioning for HSCT, which is associated with less transplantation-related mortality, will further improve cure rates.
1821	Pediatric acute liver failure and immune dysregulation.	2431550724837861	NaN
1822	A comparison of outcomes from in vitro fertilization cycles stimulated with either recombinant luteinizing hormone (LH) or human chorionic gonadotropin acting as an LH analogue delivered as human menopausal gonadotropins, in subjects with good or poor ovarian reserve: a retrospective analysis.	24314801	To compare rates of pregnancy and IVF parameters in subjects who were stimulated with FSH plus recombinant human luteinizing hormone or menopausal gonadotropins. To determine whether responses to type of LH differ in poor or good responders.Retrospective analysis at a university-based fertility center. Subjects were women with good and poor ovarian reserve, who underwent in vitro fertilization during a 2 year period, as part of a long agonist (N=122), or microdose flair (N=79) protocol. Measurements included FSH and LH dose, number of oocytes collected, number of embryos obtained, and pregnancy and clinical pregnancy rates.Patients treated with r-hLH (n=105) had higher numbers of eggs retrieved and of embryos while using less FSH than their hMG-treated (n=96) counterparts. Pregnancy and clinical pregnancy rates were significantly higher with r-hLH than with hMG protocols (p=0.008 and 0.009, respectively). If patients had a baseline serum FSH level ≥10IU/L, clinical pregnancy rates were higher when r-hLH was used. When the antral follicle count was below 6 no significant differences in stimulation parameters or outcomes were detected between the groups.r-hLH may be beneficial when compared to hMG and used for in-vitro fertilization, except in subjects with baseline follicle counts less than 6. Further data should be obtained.
1823	A Caenorhabditis elegans locomotion phenotype caused by transgenic repeats of the hlh-17 promoter sequence.	24312354	Transgene technology is one of the most heavily relied upon tools in modern biological research. Expression of an exogenous gene within cells, for research and therapeutic applications, nearly always includes promoters and other regulatory sequences. We found that repeats of a non-protein coding transgenic sequence produced profound changes to the behavior of the nematode Caenorhabditis elegans. These changes were produced by a glial promoter sequence but, unexpectedly, major deficits were observed specifically in backward locomotion, a neuron-driven behavior. We also present evidence that this behavioral phenotype is transpromoter copy number-dependent and manifests early in development and is maintained into adulthood of the worm.
1824	Monoallelic mutations of the perforin gene may represent a predisposing factor to childhood anaplastic large cell lymphoma.	24309606	Anaplastic large cell lymphoma (ALCL) accounts for approximately 15% of all pediatric non-Hodgkin lymphomas. It has distinct clinical features, including frequent involvement of extranodal sites and rare localization to the central nervous system. As some presenting features of ALCL are in common with the hemophagocytic syndrome, we previously analyzed a small series of patients with ALCL for PRF1 mutations and found that 27% of them carried mutations. We now expanded our preliminary study by increasing the cohort of ALCL patients to a total of 84 consecutive cases, in whom we extended mutation analysis to the genes SH2D1A, PRF1 e UNC13D, all related to familial HLH. Furthermore, perforin expression in tumor cells was investigated on paraffin-embedded tissues by immunohistochemical analysis. Mutations were observed in 23/84 patients (27.4%). Twenty-one patients (25%) carried a total of 10 different mutations of PRF1; they were monoallelic in 20 patients, biallelic in 1. No mutations were found in the gene SH2D1A. Two additional patients had missense mutations of the UNC13D gene. These data show that monoallelic germline mutations of PRF1 are frequent in patients with childhood ALCL, suggesting that partially impaired cytotoxic machinery may represent a predisposing factor for ALCL. Involvement is less frequent for UNC13D and absent for SH2D1A.
1825	Risk factors for early fatal outcomes among children with hemophagocytic lymphohistiocytosis (HLH): a single-institution case-series in Vietnam.	24308730	Hemophagocytic lymphohistiocytosis (HLH) is a rare and fatal hematological syndrome that causes a disturbance of the immune system. Overall mortality of HLH is greater than 50% and the majority of patients who die do so within the first 8 weeks of chemotherapy treatment. To find clinical parameters relating to high-risk HLH patients, this study examined associations between an early fatal outcome and potential prognostic clinical factors and laboratory findings on admission. Eighty-nine pediatric HLH patients were prospectively recruited in Children's Hospital No. 1, Ho-Chi-Minh City, Vietnam, during the period from January 2010 to August 2012. Associations between early fatal outcome and clinical and laboratory findings, including a cerebrospinal fluid examination and virological test on admission, were examined. During the 8-week therapy, 25 (28%) HLH patients died. Persistent fever (>2 weeks), severe thrombocytopenia (<75 × 10(9)/L), hyperbilirubinemia, and prolonged activated partial thromboplastin time (APTT) (>33 sec) were significant risk factors of early fatal outcome. Multivariate logistic regression analysis revealed that thrombocytopenia and prolonged APTT (P for trend was 0.054 and 0.013, respectively) were independently associated with the early fatal outcome. Persistent fever, severe thrombocytopenia, hyperbilirubinemia, and prolonged APTT on admission will be useful and practical predictors to determine high-risk HLH patients.
1826	A novel reduced-intensity conditioning regimen for unrelated umbilical cord blood transplantation in children with nonmalignant diseases.	24296492	Reduced-intensity conditioning (RIC) regimens have the potential to decrease transplantation-related morbidity and mortality. However, engraftment failure has been prohibitively high after RIC unrelated umbilical cord blood transplantation (UCBT) in chemotherapy-naïve children with nonmalignant diseases (NMD). Twenty-two children with a median age of 2.8 years, many with severe comorbidities and prior viral infections, were enrolled in a novel RIC protocol consisting of hydroxyurea, alemtuzumab, fludarabine, melphalan, and thiotepa followed by single UCBT. Patients underwent transplantation for inherited metabolic disorders (n = 8), primary immunodeficiencies (n = 9), hemoglobinopathies (n = 4) and Diamond Blackfan anemia (n = 1). Most umbilical cord blood (UCB) units were HLA-mismatched with median infused total nucleated cell dose of 7.9 × 10(7)/kg. No serious organ toxicities were attributable to the regimen. The cumulative incidence of neutrophil engraftment was 86.4% (95% confidence interval [CI], 65% to 100%) in a median of 20 days, with the majority sustaining > 95% donor chimerism at 1 year. Cumulative incidence of acute graft-versus-host disease (GVHD) grades II to IV and III to IV by day 180 was 27.3% (95% CI, 8.7% to 45.9%) and 13.6% (95 CI, 0% to 27.6%), respectively. Cumulative incidence of extensive chronic GVHD was 9.1% (95% CI, 0% to 20.8%). The primary causes of death were viral infections (n = 3), acute GVHD (n = 1) and transfusion reaction (n = 1). One-year overall and event-free survivals were 77.3% (95% CI, 53.7% to 89.8%) and 68.2% (95% CI, 44.6% to 83.4%) with 31 months median follow-up. This is the first RIC protocol demonstrating durable UCB engraftment in children with NMD. Future risk-based modifications of this regimen could decrease the incidence of viral infections. (www.clinicaltrials.gov/NCT00744692).
1827	Hypo-glycosylated human follicle-stimulating hormone (hFSH(21/18)) is much more active in vitro than fully-glycosylated hFSH (hFSH(24)).	24291635	Hypo-glycosylated hFSH(21/18) (possesses FSHβ(21) and FSHβ(18)bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH(24) was prepared by combining the fully-glycosylated FSHβ(24) variant with hCGα and isolating the heterodimer. The hFSH(21/18) glycoform preparation was significantly smaller than the hFSH(24) preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH(21/18) was 9- to 26-fold more active than fully-glycosylated hFSH(24) in FSH radioligand assays. Significantly greater binding of (125)I-hFSH(21/18) tracer than hFSH(24) tracer was observed in all competitive binding assays. In addition, higher binding of hFSH(21/18) was noted in association and saturation binding assays, in which twice as much hFSH(21/18) was bound as hFSH(24). This suggests that more ligand binding sites are available to hFSH(21/18) in FSHR than to hFSH(24). Hypo-glycosylated hFSH(21/18) also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH(24) specific binding proceeded very slowly for almost the first hour of incubation.
1828	Cytomegalovirus associated haemophagocytic lymphohistiocytosis in the immunocompetent adult managed according to HLH-2004 diagnostic using clinical and serological means only.	24265923	Haemophagocytic lymphohistiocytosis (HLH) describes a rare, poorly recognised and under-diagnosed immunopathological syndrome whereby there is a highly stimulated yet ineffective multisystem inflammatory response [1]. I present the first case in English literature of Cytomegalovirus (CMV) associated HLH diagnosed by clinical and serological means, and the fourth case of CMV associated haemophagocytic lymphohistiocytosis in an immunocompetent adult, according to HLH-2004 diagnostic guidelines. I include a literature review of CMV associated HLH in adults and raise awareness of checking serum ferritin in patients who present with a sepsis like syndrome. Additionally, this article discusses the merits of HLH-2004 diagnostic work-up without bone marrow biopsy, using clinical and serological means only. I support the reclassification of HLH alongside the other hyperinflammatory syndromes of SIRS, sepsis, septic shock, and MODS to improve understanding and recognition.
1829	bHLH-PAS proteins in cancer.	24263188	Mammalian basic HLH (helix-loop-helix)-PER-ARNT-SIM (bHLH-PAS) proteins are heterodimeric transcription factors that sense and respond to environmental signals (such as pollutants) or to physiological signals (for example, hypoxia and circadian rhythms) through their two PAS domains. PAS domains form a generic three-dimensional fold, which commonly contains an internal cavity capable of small-molecule binding and outer surfaces adept at protein-protein interactions. These proteins are important in several pro-tumour and antitumour pathways and their activities can be modulated by both natural metabolites and oncometabolites. Recently determined structures and successful small-molecule screening programmes are now providing new opportunities to discover selective agonists and antagonists directed against this multitasking family of transcription factors.
1830	Etoposide selectively ablates activated T cells to control the immunoregulatory disorder hemophagocytic lymphohistiocytosis.	24259502	Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects in this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug that inhibits topoisomerase II, is the mainstay of treatment for HLH, although its therapeutic mechanism remains unknown. We used a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin-deficient mice, to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells, and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide's immunomodulatory effects were similar in wild-type and perforin-deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have usefulness as a novel immune modulator in a broad array of immunopathologic disorders.
1831	[Research advances in molecular genetics and treatment of familial hemophagocytic lymphohistiocytosis].	24229589	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by pancytopenia and multiple organ infiltrations of lymphocytes and histiocytes with proliferation and hemohpagocytic activity. HLH is classified as primary (or familial) and secondary. Familial HLH is common in infants and young children, and is related to genetic defects. This article aims to review research advances on PRF1, UNC13D, STX11 and STXBP2, as well as the other 5 genes associated with familial HLH based on molecular genetics, and to summarize diagnosis and treatment methods for this disease.
1832	Hemophagocytosis in a case with Crimean-Congo hemorrhagic fever and an overview of possible pathogenesis with current evidence.	24217086	Hemophagocytic lymphohistiocytosis (HLH) is a clinicopathologic condition characterized by high fever, hepatosplenomegaly, cytopenia, hyperferritinemia, and increased hemophagocytic macrophage proliferation and activation in the reticuloendothelial system. Primary HLH is familial and is a fatal disease that begins during early childhood. Secondary HLH may be acquired after intense activation of the immune system due to infection. Clinical and biologic symptoms result from cytokines secreted by T-lymphocytes and macrophages. Subtypes of primary HLH are caused by genetic defects in several cell types, including perforin-dependent cytotoxic T-lymphocytes and natural killer (NK) cells. Secondary HLH is often associated with intracellular pathogen infections. Crimean-Congo hemorrhagic fever (CCHF) is caused by a tick-borne virus, Nairovirus, from the Bunyaviridae family. It is characterized by a poor prognosis and has a high mortality. We report the case of a 14-year-old boy living in a CCHF-endemic area with no history of tick exposure. He presented with fever, and laboratory tests showed bicytopenia and hemophagocytosis in the bone marrow aspiration. Blood samples were polymerase chain reaction (PCR)-negative for CCFH but immunoglobulin (Ig)M-positive. In conclusion, patients with hemophagocytosis should be assessed for CCHF during the evaluation of cytopenia.
1833	Immunologic difference between hypersensitivity to mosquito bite and hemophagocytic lymphohistiocytosis associated with Epstein-Barr virus infection.	24204658	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, virus-triggered immune disease. Hypersensitivity to mosquito bite (HMB), a presentation of Chronic Active Epstein-Barr Virus infection (CAEBV), may progress to HLH. This study aimed to investigate the immunologic difference between the HMB episodes and the HLH episodes associated with EBV infection. Immunologic changes of immunoglobulins, lymphocyte subsets, cytotoxicity, intracellular perforin and granzyme expressions, EBV virus load and known candidate genes for hereditary HLH were evaluated and compared. In 12 HLH episodes (12 patients) and 14 HMB episodes (4 patients), there were both decreased percentages of CD4+ and CD8+ and increased memory CD4+ and activated (CD2+HLADR+) lymphocytes. In contrast to HMB episodes that had higher IgE levels and EBV virus load predominantly in NK cells, those HLH episodes with virus load predominantly in CD3+ lymphocyte had decreased perforin expression and cytotoxicity that were recovered in the convalescence period. However, there was neither significant difference of total virus load in these episodes nor candidate genetic mutations responsible for hereditary HLH. In conclusion, decreased perforin expression in the HLH episodes with predominant-CD3+ EBV virus load is distinct from those HMB episodes with predominant-NK EBV virus load. Whether the presence of non-elevated memory CD4+ cells or activated lymphocytes (CD2+HLADR+) increases the mortality rate in the HLH episodes remains to be further warranted through larger-scale studies.
1834	Combined small RNA and degradome sequencing reveals microRNA regulation during immature maize embryo dedifferentiation.	24183719	Genetic transformation of maize is highly dependent on the development of embryonic calli from the dedifferentiated immature embryo. To better understand the regulatory mechanism of immature embryo dedifferentiation, we generated four small RNA and degradome libraries from samples representing the major stages of dedifferentiation. More than 186 million raw reads of small RNA and degradome sequence data were generated. We detected 102 known miRNAs belonging to 23 miRNA families. In total, we identified 51, 70 and 63 differentially expressed miRNAs (DEMs) in the stage I, II, III samples, respectively, compared to the control. However, only 6 miRNAs were continually up-regulated by more than fivefold throughout the process of dedifferentiation. A total of 87 genes were identified as the targets of 21 DEM families. This group of targets was enriched in members of four significant pathways including plant hormone signal transduction, antigen processing and presentation, ECM-receptor interaction, and alpha-linolenic acid metabolism. The hormone signal transduction pathway appeared to be particularly significant, involving 21 of the targets. While the targets of the most significant DEMs have been proved to play essential roles in cell dedifferentiation. Our results provide important information regarding the regulatory networks that control immature embryo dedifferentiation in maize.
1835	The emergence of CD20-/CD19- tumor cells after rituximab therapy for Epstein-Barr virus-associated post-transplant lymphoproliferative disorder complicated with hemophagocytic lymphohistiocytosis.	24169729	Post-transplant lymphoproliferative disorder (PTLD) is a well-recognized aggressive disease commonly associated with Epstein-Barr virus (EBV) infection after hematopoietic stem cell transplantation (HSCT). Although rituximab (RTX) is incorporated into the first-line therapy for EBV-PTLD patients, the outcome of the clinically overt disease is still not optimal mainly due to the regrowth of tumor cells. The proliferation of CD20-/CD19+ tumor cells is increasingly reported in RTX-treated EBV-PTLD patients, whereas the emergence of CD20-/CD19- tumor cells is barely recognized. Here, we report a fatal case of an 18-year-old patient who developed EBV-PTLD after allogeneic HSCT for anaplastic large-cell lymphoma. On day 60 after HSCT, the patient developed abdominal pain, watery diarrhea, and low-grade fever. Colon biopsy revealed the proliferation of CD20+/CD19+/EBV-encoded RNA (EBER)+ tumor cells, and an increase of EBV DNA was detected in peripheral blood (PB). He was treated with RTX for EBV-PTLD and was cleared of EBV DNA in PB. However, he manifested high-grade fever, pancytopenia, and elevated soluble interleukin-2 receptor with a prominent hemophagocytosis in bone marrow aspirates and was treated with etoposide for hemophagocytic lymphohistiocytosis (HLH) complication. He then developed EBV DNA positivity in PB and finally died of Bacteroides fragilis sepsis subsequent to bloody stool and ileus on day 163. Autopsy revealed erosion and bleeding in the whole colon with the proliferation of CD20-/CD19-/EBER+ tumor cells. Immunohistochemical analysis uncovered the CD3-/CD56-/CD79a+/CD79b+ B-cell origin of tumor cells. This case clinically demonstrates the removal of both CD20 and CD19 antigens from EBER+ B cells in an RTX-treated EBV-PTLD patient with HLH complication.
1836	Acute pain transfusion reaction.	24161631	A 34-year-old woman with a diagnosis of hemophagocytic lymphohistocytosis (HLH) received a double umbilical cord blood transplantation following a myeloablative chemotherapy preparative regimen with busulfan and cyclophosphamide. HLH is a rare, potentially fatal hematologic disorder characterized by the overactivation of histocytes and T lymphocytes, leading to organ infiltration and acute illness. On day 25 post-transplantation, the patient required a platelet transfusion for a platelet count of 6,000 per ml (normal range = 150,000-450,000 per ml). The patient's blood type prior to the cord blood transplantation was B positive and, although both umbilical cord blood donors were O positive, the patient was still B positive per blood bank testing on that day. Although the recipient of an allogenic stem cell transplantation will eventually become the blood type of the donor, the time for this process to occur varies for each person. That process must be monitored by the blood bank for the purpose of cross-matching blood products to decrease hemolysis as much as possible. The patient was premedicated with the facility's standard for platelet transfusions: acetaminophen 650 mg and diphenhydramine 25 mg about 30 minutes prior to the platelet transfusion.
1837	The roles of HLH transcription factors in epithelial mesenchymal transition and multiple molecular mechanisms.	24158354	Epithelial-to-mesenchymal transition (EMT) is presently recognized as an important event and the initiating stage for tumor invasion and metastasis. Several EMT inducers have been identified, among which the big family of helix-loop-helix (HLH) transcription factors are rising as a novel and promising family of proteins in EMT mediation, such as Twist1, Twist2, E47, and HIFs, etc. Due to the variety and complexities of HLH members, the pathways and mechanisms they employ to promote EMT are also complex and characteristic. In this review, we will discuss the roles of various HLH proteins in the regulation and sustenance of the EMT and multiple cellular mechanisms, attempting to provide a novel and broadened view towards the link between HLH proteins and EMT.
1838	Hemophagocytic lymphohistiocytosis associated with dermatomyositis: a case report.	241561772460078924600790	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon life-threatening disorder of pathologic immune activation. HLH associated with autoimmune disease is recognized although the mechanism remains uncertain. HLH associated with dermatomyositis is very rare and just as rarely reported in medical literature. A high index of suspicion is warranted in the setting of fever, cytopenias, abnormal liver function, and markedly elevated ferritin.A 61-year-old male presented with proximal weakness, rash, periorbital edema, and elevated creatine kinase (CK). He also had pancytopenia, abnormal liver functions, and elevated ferritin. His bone marrow biopsy confirmed HLH. He was treated with high-dose dexamethasone and etoposide and seemed to have been recovering. Soon after his second infusion, he died of a complication from an unrelated endoscopic procedure.Early recognition of HLH is critical to the establishment of appropriate management and the prevention of fatal outcomes. We should be aware of the cardinal signs and symptoms of HLH and it should be considered in patients presenting with inflammatory myopathies associated with cytopenias, abnormal liver functions, and markedly elevated ferritin level.
1839	Targeted next-generation sequencing: a novel diagnostic tool for primary immunodeficiencies.	24139496	Primary immunodeficiency (PID) disorders are a heterogeneous group of inherited disorders caused by a variety of monogenetic immune defects. Thus far, mutations in more than 170 different genes causing PIDs have been described. A clear genotype-phenotype correlation is often not available, which makes a genetic diagnosis in patients with PIDs complex and laborious.We sought to develop a robust, time-effective, and cost-effective diagnostic method to facilitate a genetic diagnosis in any of 170 known PID-related genes by using next-generation sequencing (NGS).We used both targeted array-based and in-solution enrichment combined with a SOLiD sequencing platform and a bioinformatic pipeline developed in house to analyze genetic changes in the DNA of 41 patients with PIDs with known mutations and 26 patients with undiagnosed PIDs.This novel NGS-based method accurately detected point mutations (sensitivity and specificity >99% in covered regions) and exonic deletions (100% sensitivity and specificity). For the 170 genes of interest, the DNA coverage was greater than 20× in 90% to 95%. Nine PID-related genes proved not eligible for evaluation by using this NGS-based method because of inadequate coverage. The NGS method allowed us to make a genetic diagnosis in 4 of 26 patients who lacked a genetic diagnosis despite routine functional and genetic testing. Three of these patients proved to have an atypical presentation of previously described PIDs.This novel NGS tool facilitates accurate simultaneous detection of mutations in 161 of 170 known PID-related genes. In addition, these analyses will generate more insight into genotype-phenotype correlations for the different PID disorders.
1840	Clinical, laboratory and molecular signs of immunodeficiency in patients with partial oculo-cutaneous albinism.	24134793	Hypopigmentation disorders that are associated with immunodeficiency feature both partial albinism of hair, skin and eyes together with leukocyte defects. These disorders include Chediak Higashi (CHS), Griscelli (GS), Hermansky-Pudlak (HPS) and MAPBP-interacting protein deficiency syndromes. These are heterogeneous autosomal recessive conditions in which the causal genes encode proteins with specific roles in the biogenesis, function and trafficking of secretory lysosomes. In certain specialized cells, these organelles serve as a storage compartment. Impaired secretion of specific effector proteins from that intracellular compartment affects biological activities. In particular, these intracellular granules are essential constituents of melanocytes, platelets, granulocytes, cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Thus, abnormalities affect pigmentation, primary hemostasis, blood cell counts and lymphocyte cytotoxic activity against microbial pathogens. Among eight genetically distinct types of HPS, only type 2 is characterized by immunodeficiency. Recently, a new subtype, HPS9, was defined in patients presenting with immunodeficiency and oculocutaneous albinism, associated with mutations in the pallidin-encoding gene, PLDN.Hypopigmentation together with recurrent childhood bacterial or viral infections suggests syndromic albinism. T and NK cell cytotoxicity are generally impaired in patients with these disorders. Specific clinical and biochemical phenotypes can allow differential diagnoses among these disorders before molecular testing. Ocular symptoms, including nystagmus, that are usually evident at birth, are common in patients with HPS2 or CHS. Albinism with short stature is unique to MAPBP-interacting protein (MAPBPIP) deficiency, while hemophagocytic lymphohistiocytosis (HLH) mainly suggests a diagnosis of CHS or GS type 2 (GS2). Neurological disease is a long-term complication of CHS, but is uncommon in other syndromic albinism. Chronic neutropenia is a feature of HPS2 and MAPBPIP-deficiency syndrome, whereas it is usually transient in CHS and GS2. In every patient, an accurate diagnosis is required for prompt and appropriate treatment, particularly in patients who develop HLH or in whom bone marrow transplant is required. This review describes the molecular and pathogenetic mechanisms of these diseases, focusing on clinical and biochemical aspects that allow early differential diagnosis.
1841	Hemophagocytic lymphohistiocytosis: critical reappraisal of a potentially under-recognized condition.	24127015	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon, potentially life threatening, hyper inflammatory syndrome of diverse etiologies. Cardinal signs include prolonged fever, organomegaly, and persistent unexplained cytopenias. In spite of the well known diagnostic criteria put forth by HLH society, this continues to pose great diagnostic challenge in both pediatric and adult intensive care settings. We describe 4 adult (2 males, 2 females, aged 19, 29, 40, and 17 years) and 3 pediatric (2 males, 1 female, aged 1 month, 6 months, and 12 years) patients with secondary HLH who satisfied the HLH-2004 diagnostic criteria. Definite evidence of hemophagocytosis was noted in 4 patients on initial bone marrow examination. The underlying etiologies were as follows: Rickettsia tsutsugamushi (case 1), autoimmune disorder (case 2), systemic onset juvenile idiopathic arthritis (sJIA) (case 3), unknown bite (possibly a venomous snake) (case 4), Plasmodium vivax (case 5), Cytomegalo virus (case 6), and Mycobacterium tuberculosis (case 7). In one patient, hemophagocytosis was presumed to have been exacerbated by administration of granulocyte monocyte colony stimulating factor (GMCSF) for severe neutropenia. Two patients died with disseminated intravascular coagulation (DIC) and multi organ failure within few days of HLH diagnosis. Immunosuppressive therapy was started in 3 patients, and etoposide was started in one patient only. Due to lack of specificity of diagnostic criteria, diagnosing and differentiating HLH from its closest mimickers like sepsis/septic shock may be quite challenging in critically ill patients. Therefore, increasing awareness among physicians is essential for early diagnosis and effective therapy to reduce the mortality.
1842	Evolutionary aspects of variability in bHLH orthologous families: insights from the pearl oyster, Pinctada fucata.	24125650	Basic helix-loop-helix (bHLH) transcription factors play significant roles in multiple biological processes in metazoan cells. In recent work, we showed that three orthologous HLH families, pearl, amber, and peridot, have apparently been lost in the Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens lineages. To further address the gain and loss of bHLH proteins during bilaterian evolution, we examined the genome of the pearl oyster, Pinctada fucata, which has recently been sequenced. We characterized the putative full set 65 bHLH genes and showed that genes previously categorized into the orthologous family PTFb, actually fall into two distinct orthologous families, 48-related-1 and 48-related-2. We also identified a novel orthologous family, clockwork orange. Based on these newly identified orthologous family members and on orphan bHLH factors, we propose that genes encoding bHLH factors in bilaterians are not as evolutionarily stable as previously thought.
1843	Structure activity relationships of peptidic analogs of MyoD for the development of Id1 inhibitors as antiproliferative agents.	24123584	Id proteins, inhibitors of DNA binding proteins, have highly conserved dimerization motif known as the helix-loop-helix (HLH) domain that acts as a negative regulator of basic HLH (bHLH) transcription factors. In signaling pathways, Id proteins play an important role in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of cellular differentiation-associated genes in cancer. Recently, we reported an inhibitor of Id1, peptide 3C, which showed good affinity to Id1 protein and exhibited inhibitory effects in cancer cells. In this study, Ala (A)-substituted analogs of peptide 3C were synthesized by SPPS, purified by RP-HPLC, and characterized by MALDI-TOF MS. Binding of each peptide to Id1 or Id1-HLH (the HLH domain of Id1) was monitored by surface plasmon resonance (SPR)-based biosensor. Biological effect of each peptide in MCF-7 breast cancer cells was analyzed by MTT cell viability assay. The secondary structure of substituted analogs of peptide 3C was investigated by circular dichroism (CD) spectroscopy. SPR results revealed that A-substituted analogs of peptide 3C showed weaker binding to Id1 than that of peptide 3C, indicating that the six amino acid residues in the N-terminal of peptide 3C were all essential for binding to Id1 and the importance of amino acid residue was I(2)  > Q(6)  > Y(1)  > G(4)  > L(5)  > E(3). In addition, substitution of E(3) in peptide 3C with D, Q, and R did not improve the binding potency of peptide 3C. MTT assay demonstrated that neither A-substituted nor position 3-substituted analogs of peptide 3C showed increased antiproliferative effect in MCF-7 cancer cells. CD results indicated that peptide 3C exhibited the highest content of α-helical structure (39.37%), suggesting that the α-helical structure may contribute to its binding potency for Id1 and Id1-HLH. SAR results provided important information for the development of peptidic inhibitors of Id1 as anticancer agents and demonstrated peptide 3C as a promising lead for further modifications.
1844	Spectrum of imaging appearances in Australian children with central nervous system hemophagocytic lymphohistiocytosis.	24119957	Hemophagocytic lymphohistiocytosis (HLH) is a rare multisystem disorder characterised by the proliferation and infiltration of lymphocytes and histiocytes. Central nervous system (CNS) infiltration is particularly devastating. Neuroradiological findings have been reported predominantly as individual case reports due to the rarity of the condition. To our knowledge there have been no published studies of imaging in Australian patients. This study aimed to retrospectively describe and illustrate the MRI appearances of CNS involvement by HLH in a cohort of seven Australian children from two paediatric centres between 2000 and 2011. MRI appearances demonstrate intersubject and intrasubject variability over time, likely reflecting the severity of CNS infiltration and associated demyelination. Familiarity with MRI patterns is important for assessing and monitoring disease activity.
1845	Hemophagocytic lymphohistiocytosis (HLH): a review of literature.	24105023	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease in children and an exceptionally rare occurrence in adults. It is categorized broadly into primary (familial) or the secondary types; the latter being associated most commonly with an underlying malignancy. HLH carries a high rate of mortality, and the treatment itself is associated with significant morbidity and risk of mortality. A high degree of suspicion for the diagnosis, early treatment and aggressive supportive care is critical for management. We present a comprehensive review of literature describing the clinical features, diagnosis, management and outcome of HLH.
1846	Autoimmune lymphoproliferative syndrome misdiagnosed as hemophagocytic lymphohistiocytosis.	24101757	Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder of apoptosis, most commonly due to mutations in the FAS (TNFRSF6) gene. It presents with chronic lymphadenopathy, splenomegaly, and symptomatic multilineage cytopenias in an otherwise healthy child. Unfortunately, these clinical findings are also noted in other childhood lymphoproliferative conditions, such as leukemia, lymphoma, and hemophagocytic lymphohistiocytosis, which can confound the diagnosis. This report describes a 6-year-old girl with symptoms misdiagnosed as hemophagocytic lymphohistiocytosis and treated with chemotherapy before the recognition that her symptoms and laboratory values were consistent with a somatic FAS mutation leading to ALPS. This case should alert pediatricians to include ALPS in the differential diagnosis of a child with lymphadenopathy, splenomegaly, and cytopenias; obtain discriminating screening laboratory biomarkers, such as serum vitamin B-12 and ferritin levels; and, in the setting of a highly suspicious clinical scenario for ALPS, pursue testing for somatic FAS mutations when germ-line mutation testing is negative.
1847	Recombinant human soluble thrombomodulin is active against hemophagocytic lymphohistiocytosis associated with acquired immunodeficiency syndrome.	24101336	A 39-year-old man was admitted to our hospital to initiate highly active anti-retroviral therapy (HAART) for documented acquired immune deficiency syndrome. The HIV load was 2.5 million copies/mL and the CD4-positive lymphocyte count was only 52 cells/µL at presentation. The HAART regimen consisted of lamivudine and abacavir as the backbone, plus raltegravir and lopinavir/ritonavir as the base. The day after initiating HAART, his body temperature rose to 102.4 °F (39.1 °C), accompanied by elevated levels of liver enzymes, neutropenia, coagulopathies, and an extremely high serum ferritin level, prompting us to suspect hemophagocytic lymphohistiocytosis (HLH) and disseminated intravascular coagulation (DIC). To correct the coagulation abnormalities, recombinant thrombomodulin (rTM) was initiated at 375 U/kg. Surprisingly, fever resolved almost immediately, in parallel with dramatic decreases in serum levels of ferritin and liver enzymes and prompt normalization of coagulopathy with only two doses of rTM. The patient subsequently developed amebiasis, which was successfully treated using metronidazole. In summary, the use of rTM dramatically improved not only DIC, but also HLH, suggesting potent anti-inflammatory effects of the agent. Although further clinical reports and trials are needed, rTM appears to provide an additional therapeutic option in the management of HLH.
1848	Deadpan contributes to the robustness of the notch response.	24086596	Notch signaling regulates many fundamental events including lateral inhibition and boundary formation to generate very reproducible patterns in developing tissues. Its targets include genes of the bHLH hairy and Enhancer of split [E(spl)] family, which contribute to many of these developmental decisions. One member of this family in Drosophila, deadpan (dpn), was originally found to have functions independent of Notch in promoting neural development. Employing genome-wide chromatin-immunoprecipitation we have identified several Notch responsive enhancers in dpn, demonstrating its direct regulation by Notch in a range of contexts including the Drosophila wing and eye. dpn expression largely overlaps that of several E(spl) genes and the combined knock-down leads to more severe phenotypes than either alone. In addition, Dpn contributes to the establishment of Cut expression at the wing dorsal-ventral (D/V) boundary; in its absence Cut expression is delayed. Furthermore, over-expression of Dpn inhibits expression from E(spl) gene enhancers, but not vice versa, suggesting that dpn contributes to a feed-back mechanism that limits E(spl) gene expression following Notch activation. Thus the combined actions of dpn and E(spl) appear to provide a mechanism that confers an initial rapid output from Notch activity which becomes self-limited via feedback between the targets.
1849	Sustained elevation of serum interleukin-18 and its association with hemophagocytic lymphohistiocytosis in XIAP deficiency.	24084330	X-linked lymphoproliferative syndrome (XLP) is a rare primary immunodeficiency characterized by increased vulnerability to Epstein-Barr virus infection. XLP type 1 is caused by mutations in SH2D1A, whereas X-linked inhibitor of apoptosis (XIAP) encoded by XIAP/BIRC4 is mutated in XLP type 2. In XIAP deficiency, hemophagocytic lymphohistiocytosis (HLH) occurs more frequently and recurrence is common. However, the underlying mechanisms remain mostly unknown. We describe the characteristics of the cytokine profiles of serum samples from 10 XIAP-deficient patients. The concentration of interleukin (IL)-18 was strikingly elevated in the patients presented with HLH, and remained high after the recovery from HLH although levels of other pro-inflammatory cytokines approached the normal range. Longitudinal examination of two patients demonstrated marked exacerbation of IL-18 levels during every occasion of HLH. These findings may suggest the association between HLH susceptibility and high serum IL-18 levels in XIAP deficiency.
1850	[Infantile visceral leishmaniasis, an etiology of easily curable hemophagocytic lymphohistiocytosis syndrome].	24076317	Infantile visceral leishmaniasis associated hemophagocytic lymphohistiocytosis (HLH) is a rare clinicopathological entity, difficult to diagnose and fatal if untreated. The diagnosis should be considered in young infants with fever and splenomegaly. We report two cases of HLH caused by visceral leishmaniasis. In the first case, a 3-month-old boy was admitted with fever and pancytopenia, leading to the diagnosis of HLH based on complete clinical and biological features including hemophagocytosis on bone marrow smears. Investigations for an underlying genetic, metabolic disease and an infectious trigger were negative. Primary or genetic hemophagocytic syndrome was suspected and immunosuppressive treatment (steroids and cyclosporin) was instituted. A second bone marrow examination performed 1 month later revealed leishmania. The boy was treated with liposomal amphotericin and recovered rapidly. In the second case, a 10-year-old child was hospitalized with fever, pancytopenia, and a tumoral syndrome. He had a history of recurrent infections. The bone marrow biopsy showed leishmania and treatment with liposomal amphotericin was delivered. After 3 days of treatment, the improvement was judged inadequate and the boy presented biological signs of HLH. He was treated with steroids. An underlying primary immunodeficiency (interleukin-12/interferon-γ axis disorder) was secondarily diagnosed.
1851	Clinical and laboratory characteristics of severe fever with thrombocytopenia syndrome in Chinese patients.	24076112	Severe fever with thrombocytopenia syndrome (SFTS) associated with severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging infectious disease. 12 patients with severe fever with thrombocytopenia syndrome in our study were presented mainly with fever and severe malaise. The clinical manifestations typically became worse on the 6th or 7th day. The average fever time is 9.11 ± 1.54 days. Most of them had multiorgan dysfunction, and part of them had hemophagocytic lymphohistiocytosis histiocytosis (HLH). The characteristic laboratory findings in the early stage were the drop of white blood cells (WBC), platelets (PLT) and serum Ca++, while increase of aspartate amino transferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH). CD3+CD4+ were significantly decreased, while CD3-CD56+ were significantly increased, whereas CD3+CD8+ were constantly elevated throughout the disease course. Ten to 14 days after illness onset, symptoms were improved, accompanied by resolution of laboratory abnormalities. These results indicate that severe fever with thrombocytopenia syndrome has an acute onset and self-limited course. It is a systemic infection. The host immune response caused tissues and organs injury. The improvement of symptoms and laboratory tests is consistent with the elimination of the virus and recover of immune response. Further investigation should be done in order to better understand this disease and guide the clinical treatment.
1852	Hemophagocytic Lymphohistiocytosis Complicating T-Cell Lymphoma in a Patient with HIV Infection.	24073345	Hemophagocytic lymphohistiocytosis (HLH), while uncommon, may be a devastating complication of lymphoma and/or human immunodeficiency virus (HIV) infection. While several of the diagnostic criteria for HLH are relatively nonspecific, particularly in the setting of a systemic inflammatory response, more diagnostic specificity may be achieved with marked elevations in serum ferritin (e.g., >100,000 ng/mL). Increased suspicion of HLH, particularly in the setting of persistent, unexplained fevers, pancytopenia, and transaminitis, should prompt consideration of HLH. Earlier diagnosis and initiation of therapy have the potential to alter the natural history and poor prognosis of this disorder. We present a patient with HIV infection who developed relapsed T-cell lymphoma complicated by hemophagocytic lymphohistiocytosis.
1853	Hemophagocytic lymphohistiocytosis associated with parechovirus 3 infection.	24072243	Hemophagocytic lymphohistiocytosis (HLH) denotes the common final pathway of a potentially fatal hyperinflammatory condition of diverse etiologies. We describe the first case of documented HLH associated with human parechovirus 3. A monoallelic Ala91Val mutation was found in the PRF1 gene, but the contribution of this mutation to HLH remains controversial. The diagnosis, based on accepted criteria, was established early in the course of the disease and led to successful treatment and complete recovery. The awareness of this new association is clinically important in facilitating early treatment, preventing organ damage, and increasing the likelihood of complete recovery.
1854	Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop.	24068570	Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGβ, hCGβn, hCGβcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGβ-, 8 hCGα-, and 13 αβ-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1%; epitope β1), 12 with low (<1.0%; epitopes β2/4), and 13 with high (>>1%; epitopes β3/5) hLH cross-reactivity. The majority of hCGβ epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of β-sheet loops 1 and 3 (epitopes β2-6; 27 mAbs) and the second around the cystine knot (e.g., epitopes β1, β7, and β10; 9 mAbs). Four mAbs recognized epitopes on hCGβcf-only (e.g., epitopes β11 and β13) and six mAbs epitopes on the remote hCGβ-carboxyl-terminal peptide (epitopes β8 and β9 corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGβ-only, or hCG together with hCGβ or hCG together with hCGβ and hCGβcf. Sandwich assays that measure hCG plus hCGβ and eventually hCGβcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGβcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope β1 (Asp10, Asp60, and Gln89) in combination with epitopes β2 or β4 located at the top of β-sheet loops 1 + 3 of hCGβ involving aa hCGβ20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.
1855	[The clinical condition and diagnosis of EBV-T/NK-LPD (CAEBV, EBV-HLH etc.)].	24064853	NaN
1856	Clinical implication of F-18 FDG PET/CT in patients with secondary hemophagocytic lymphohistiocytosis.	24061788	The contribution that F-18 fluoro-2-deoxyglucose positron emission tomography/computed tomography (F-18 FDG) PET/CT makes to the diagnosis of malignancy in patients with hemophagocytic lymphohistiocytosis (HLH) is still uncertain. The aim of this study was to evaluate the diagnostic performance of F-18 FDG PET/CT for the detection of underlying malignancy, to investigate the correlation between PET and laboratory parameters, and to identify prognosis-related factors in patients with secondary HLH. We enrolled 14 patients who were diagnosed with HLH and referred for F-18 FDG PET/CT to exclude malignancy. The diagnostic performance of F-18 FDG PET/CT for malignancy detection was assessed. The correlations between PET and laboratory parameters were determined. The prognostic significance of the following factors was evaluated: PET and laboratory parameters, age in years, presence of underlying malignancy, and fever and splenomegaly. Six of the 14 patients had malignancies (four with lymphoma, one with multiple myeloma, and one with colonic malignancy). Sensitivity, specificity, and diagnostic accuracy of F-18 FDG PET/CT for malignancy detection were 83, 62.5, and 71.4 %, respectively. F-18 FDG uptake in the bone marrow and spleen was positively correlated with neutrophil count and C-reactive protein. All of the PET parameters, but none of the clinical or laboratory parameters, were significantly associated with patient outcome, as determined by univariate analysis. Given the small sample size, F-18 FDG PET/CT was useful for detecting underlying malignancy, and PET parameters correlated with laboratory parameters that reflected inflammatory status. F-18 FDG PET/CT might provide prognostic information for the management of patients with secondary HLH.
1857	Flightless I homolog negatively regulates ChREBP activity in cancer cells.	24055811	The glucose-responsive transcription factor carbohydrate responsive element binding protein (ChREBP) plays an important role in regulating glucose metabolism in support of anabolic synthesis in both hepatocytes and cancer cells. In order to further investigate the molecular mechanism by which ChREBP regulates transcription, we used a proteomic approach to identify proteins interacting with ChREBP. We found several potential ChREBP-interacting partners, one of which, flightless I homolog (FLII) was verified to interact and co-localize with ChREBP in HCT116 colorectal cancer and HepG2 hepatocellular carcinoma cells. FLII is a member of the gelsolin superfamily of actin-remodeling proteins and can function as a transcriptional co-regulator. The C-terminal 227 amino acid region of ChREBP containing the DNA-binding domain interacted with FLII. Both the N-terminal leucine-rich repeat (LRR) domain and C-terminal gelsolin homolog domain (GLD) of FLII interacted and co-localized with ChREBP. ChREBP and FLII localized in both the cytoplasm and nucleus of cancer cells. Glucose increased expression and nuclear localization of ChREBP, and had minimal effect on the level and distribution of FLII. FLII knockdown using siRNAs increased mRNA and protein levels of ChREBP-activated genes and decreased transcription of ChREBP-repressed genes in cancer cells. Conversely, FLII overexpression negatively regulated ChREBP-mediated transcription in cancer cells. Our findings suggest that FLII is a component of the ChREBP transcriptional complex and negatively regulates ChREBP function in cancer cells.
1858	Down-regulation of CD5 expression on activated CD8+ T cells in familial hemophagocytic lymphohistiocytosis with perforin gene mutations.	24051121	Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled activation of T cells and macrophages with overproduction of cytokines. Familial HLH type 2 (FHL2) is the most common form of primary HLH and is caused by mutations in PRF1. We have recently described a significant increase in the subpopulation of CD8(+) T cells with clonal expansion and CD5 down-regulation in Epstein-Barr virus associated-HLH, which represented a valuable tool for its diagnosis. However, this unusual phenotype of CD8(+) T cells has not been investigated fully in patients with FHL2. We performed immunophenotypic analysis of peripheral blood and measured serum pro-inflammatory cytokines in five patients with FHL2. All patients showed significantly increased subpopulations of activated CD8(+) T cells with down-regulation of CD5, which were negligible among normal controls. Analysis of T-cell receptor Vβ repertoire suggested the reactive and oligoclonal expansion of these cells. The proportion of the subset declined after successful treatment concomitant with reduction in the serum levels of cytokines in all patients except one who continued to have a high proportion of the subset and died. These findings suggest that down-regulation of CD5 on activated CD8(+) T cells may serve as a useful marker of dysregulated T cell activation and proliferation in FHL2.
1859	Vascular endothelial growth factor and angiopoietin production by primate follicles during culture is a function of growth rate, gonadotrophin exposure and oxygen milieu.	24045779	What is the time course of production of vascular endothelial growth factor-A (VEGF-A), angiopoietin (ANGPT)-1 and ANGPT-2 by primate follicles during encapsulated three-dimensional culture, and what conditions affect their production?Primate follicles produce VEGF-A and ANGPT-2 in vitro, particularly after developing to the antral stage, with VEGF production influenced by FSH concentration and O(2) tension.Folliculogenesis, i.e. the development of primordial follicles into mature, antral follicles, requires the creation of a vascular network in the follicle wall via a process called angiogenesis. Angiogenic factors including VEGFs and ANGPTs have documented roles in angiogenesis. However, direct studies on the production and regulation of angiogenic factors by individual, growing follicles are limited.Ovaries (n = 9 pairs) were obtained from rhesus macaques during the early follicular phase of the menstrual cycle (cycle days 1-4). Secondary (125-225 µm) follicles were isolated mechanically, encapsulated into alginate (0.25% w/v) and cultured for 40 days.Individual follicles were cultured in a 5 or 20% O(2) environment in alpha minimum essential medium supplemented with recombinant human (h) FSH. Half of the follicles had recombinant hLH added to the media from Days 30 to 40. Follicle diameters were measured weekly. Follicles were categorized at Week 5 as no-grow (NG; <250 μm in diameter), slow-grow (SG; 251-499 μm) and fast-grow (FG; >500 μm). VEGF-A, ANGPT-1 and -2 concentrations in media were measured by ELISA.VEGF concentrations were low throughout the culture for NG follicles. SG and FG follicles had detectable VEGF concentrations at Week 2, which continued to rise throughout culture. VEGF concentrations were distinct (P < 0.05) among all three follicle categories during Weeks 4 and 5. VEGF concentrations were higher (P < 0.05) in SG follicles in the presence of high/mid-dose FSH at 5% O(2). In contrast, there were no dose-dependent differences in VEGF production for FG follicles based on FSH concentrations or O(2) tension. At Week 5, follicles that produced metaphase II oocytes, following exposure to an ovulatory hCG dose, secreted higher concentrations of VEGF than those containing germinal vesicle-intact oocytes. Media concentrations of ANGPT-1 were low throughout culture for all three follicle categories. ANGPT-2 concentrations were low throughout culture for NG follicles. In contrast, ANGPT-2 concentrations of SG and FG follicles continued to rise from Weeks 1 to 4. During Weeks 2-4, ANGPT-2 concentrations in FG follicles were significantly higher than those of SG and NG follicles (P < 0.05).This study reports VEGF-A, ANGPT-1 and -2 production by in vitro-developed individual primate (macaque) follicles, that is limited to the interval from the secondary to small antral stage. After VEGF and ANGPT-1 assays, the limited remaining samples did not allow assessment of the independent effects of gonadotrophin and O(2) on the ANGPT-2 production by cultured follicles. Findings await translation to human follicles.The above findings provide novel information on the process of primate follicle maturation. We hypothesize that a symbiotic relationship between elevated concentrations of ANGPT-2 and VEGF allows FG antral follicles to excel in follicle maturation, e.g. by promoting its vascularization. Elevated ANGPT-2 may also offer possible insight into future oocyte quality as early as Week 2, compared with Week 4 for VEGF and follicle size.The study was funded by the following grants: NIH U54 RR024347/HD058294/PL1-EB008542 (Oncofertility Consortium), NIH U54-HD018185 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest to declare.
1860	[Visceral leishmaniasis in two children after vacation in Southern Europe].	24044542	Pancytopenia, fever and splenomegaly are frequent causes for referrals to paediatric haematology departments, on the suspicion of acute leukaemia. We report two cases of Danish children with the tropical disease visceral leishmaniasis (VL) contracted on short vacations in Southern Europe. One of the patients developed secondary haemophagocytic lymphohistocytosis (HLH). Both children were successfully treated with liposomal amphotericin B. In Denmark, VL is a rare but important differential diagnosis to acute leukaemia and HLH, and should be ruled out after journeys to endemic areas, including Southern Europe.
1861	Etoposide-containing conditioning regimen reduces the occurrence of hemophagocytic lymphohistiocytosis after SCT.	24037021	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease of severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages that secrete high amounts of inflammatory cytokines. HLH occurring after SCT is difficult to diagnose. It is characterized by severe clinical manifestations and high mortality. Despite current therapeutic approaches, outcomes remain poor. We analyzed the incidence and risk factors of HLH after SCT and the response to treatment and prognosis of 554 patients with HLH after SCT. The cumulative incidence of HLH after SCT was 4.3% (24/554). Use of etoposide in the conditioning regimen was only factor that reduced HLH after SCT (P=0.027). All patients who received autologous transplantation were successfully treated. Patients with liver dysfunction (for example, high total bilirubin level, prolonged prothrombin time and high level of fibrinogen degradation products) had a poor response to treatment for HLH. Physicians should be cautious of HLH, while not using etoposide for conditioning regimen.
1862	An intermediate alemtuzumab schedule reduces the incidence of mixed chimerism following reduced-intensity conditioning hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis.	24035782	Reduced-intensity conditioning (RIC) improves the outcomes of hematopoietic cell transplantation (HCT) in patients with hemophagocytic lymphohistiocytosis (HLH). Proximal (ie, close to graft infusion) dosing of alemtuzumab is associated with a high incidence of mixed chimerism, whereas distal (ie, distant from graft infusion) dosing is associated with less mixed chimerism but more acute graft-versus-host disease (GVHD). The alemtuzumab dose per kilogram of body weight also influences these outcomes. We hypothesized that an intermediate alemtuzumab dosing schedule would reduce mixed chimerism and maintain a low incidence of acute GVHD. In this study, 24 consecutive HCTs were performed in patients with HLH or a related disorder using a novel intermediate alemtuzumab schedule of 1 mg/kg starting on day -14. The cumulative incidences (CIs) of mixed chimerism, upfront acute GVHD grades II-IV, and receipt of additional hematopoietic cell products after HCT were compared in patients treated with a distal alemtuzumab schedule (n = 15) and those treated with a proximal alemtuzumab schedule (n = 33). All patients received fludarabine and melphalan. The CI of mixed chimerism was 31% in the intermediate group, 72% in the proximal group (P < .01), and 75% in the distal group patients who received ≥2 mg/kg alemtuzumab (P = .03). The CI of acute GVHD grades II-IV before the development of mixed chimerism was 4% in the intermediate group, 0% in the proximal group, and 13% in the distal group (P = .04, proximal versus distal). The 1-year CI of administration of additional hematopoietic cell products for mixed chimerism (donor lymphocyte infusion ± hematopoietic stem cell boost ± repeat HCT) was 14% in the intermediate group, 53% in the proximal group (P = .01), and 38% in the distal ≥2 mg/kg alemtuzumab group (P = .02). Our findings indicate that intermediate RIC reduces the incidence of mixed chimerism, is associated with a low incidence of upfront acute GVHD, and decreases the need for additional hematopoietic cell products after HCT.
1863	Hemophagocytic syndrome with atypical presentation in an adolescent.	24027258	A 14-year-old adolescent presented with a prolonged fever, abnormal liver function, anaemia, thrombocytopaenia, but a good general status. Diagnosis of hemophagocytic lymphohistiocytosis (HLH) was suspected, in spite of the initial indolent course. Secondary causes were excluded, but no specific mutation indicative of primary HLH was found. The patient started with specific therapy, but progressed with reactivations and later with persistently active disease. Haematopoietic stem cell transplantation was not successful and the adolescent died 7 months after diagnosis.
1864	Patterns of evolutionary selection pressure in the immune signaling protein TRAF3IP2 in mammals.	24021976	TRAF3 interacting protein 2 (TRAF3IP2) is important for immune responses to pathogens, inflammatory signals and autoimmunity in mammals. In the present study, we collected 19 mammalian TRAF3IP2 sequences and investigated the various types of selection pressure acting on them. Maximum likelihood estimations of nonsynonymous (dN) to synonymous (dS) substitution (dN/dS) ratios for the aligned coding sequences indicated that, as a whole, TRAF3IP2 has been subject to purifying selection. However, the N-terminus of the protein has been subject to higher selection pressure than the C-terminal domain. While eight amino acid residues within the N-terminus appear to have evolved under positive selection, no evidence for such selection was found in the C-terminus. The positively selected residues, which fall outside the currently known functional sites within TRAF3IP2, may have novel functions. The different selection pressures acting on the N- and C-terminal regions are consistent with their protein structures: the C-terminal structure is an ordered structure, whereas the N-terminus is disordered. Taken together with the results of previous studies, it is plausible that positive selection on the N-terminus of TRAF3IP2 may have occurred by competitive coevolution between mammalian hosts and viruses.
1865	Reversible cerebral vasoconstriction syndrome identification of prognostic factors.	24021453	Reversible cerebral vasoconstriction syndrome (RCVS) is described as a clinical and radiological entity characterized by thunderclap headaches, a reversible segmental or multifocal vasoconstriction of cerebral arteries with or without focal neurological deficits or seizures. The purpose of this study is to determine risk factors of poor outcome in patients presented a RCVS.A retrospective multi-center review of invasive and non-invasive neurovascular imaging between January 2006 and January 2011 has identified 10 patients with criterion of reversible segmental vasoconstriction syndrome. Demographics data, vascular risks and evolution of each of these patients were analyzed.Seven of the ten patients were females with a mean age of 46 years. In four patients, we did not found any causative factors. Two cases presented RCVS in post-partum period between their first and their third week after delivery. The other three cases were drug-induced RCVS, mainly vaso-active drugs. Cannabis was found as the causative factor in two patient, Sumatriptan identified in one patient while cyclosporine was the causative agent in also one patient. The mean duration of clinical follow-up was 10.2 months (range: 0-28 months). Two patients had neurological sequelae: one patient kept a dysphasia and the other had a homonymous lateral hemianopia. We could not find any significant difference of the evolution between secondary RCVS and idiopathic RCVS. The only two factors, which could be correlated to the clinical outcome were the neurological status at admission and the presence of intraparenchymal abnormalities (ischemic stroke, hematoma) in brain imaging.Fulminant vasoconstriction resulting in progressive symptoms or death has been reported in exceptional frequency. Physicians had to remember that such evolution could happen and predict them by identifying all factors of poor prognosis (neurological status at admission, the presence of intraparenchymal abnormalities).
1866	The usefulness of flow cytometric analysis of cytokines in peripheral blood and bone marrow plasma.	24018453	Recently attention has been paid to the role of cytokines in clinical pathology, since they can mediate a wide variety of biological effects. For these reasons multiplex methods have been developed to simultaneously measure numerous cytokines in individual small volume specimens. The aim of the study was to assess the usefulness of flow cytometric analysis of cytokines in peripheral blood and bone marrow plasma with Cytometric Bead Array (CBA) kits.The study involved 59 children. Tests were performed in peripheral blood and bone marrow plasma. Human Inflammatory Cytokine Kit (IL-8, -1β, -6, -10, TNF, -12p70) and Human Th₁/Th₂/Th₁₇ Cytokine Kit (IL-2, -4, -6, -10, TNF, INF-γ, -17A) (BD Bioscience) were used. Samples were analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter).In patients diagnosed for hemophagocytic lymphohistiocytosis (HLH) (n=10) and for acute lymphoblastic leukemia (ALL) (n=12) Human Inflammatory Cytokine Kit was used. In almost all samples individual cytokines were detected in a wide range of concentrations (0.47 - 653.74 pg/ml). In samples from patients suffering from allergy (n=12) and in healthy children (n=25) Human Th₁/Th₂/Th₁₇ Cytokine Kit was used. Detection of individual cytokines was much lower: concentration range 0.09-30.17 pg/ml.Based on our analysis the CBA test is suitable for analysis of several cytokines in small volumes of samples. A simple flow cytometer can be used for this test. The CBA test is more suitable for samples with expected increased levels of cytokines. When the levels of cytokines are low, the sensitivity of the CBA test can be too low.
1867	Methods for the expression, purification, preparation, and biophysical characterization of constructs of the c-Myc and Max b-HLH-LZs.	24006055	Specific heterodimerization and DNA binding by the b-HLH-LZ transcription factors c-Myc and Max is central to the activation and repression activities of c-Myc that lead to cell growth, proliferation, and tumorigenesis (Adhikary and Eilers, Nat Rev Mol Cell Biol 6:635-645, 2005; Eilers and Eisenman, Genes Dev 22:2755-2766, 2008; Grandori et al., Annu Rev Cell Dev Biol 16:653-699, 2000; Whitfield and Soucek, Cell Mol Life Sci 69:931-934, 2011). Although many c-Myc-interacting partner proteins are known to interact through their HLH domain (Adhikary and Eilers, Nat Rev Mol Cell Biol 6:635-645, 2005), current knowledge regarding the structure and the determinants of molecular recognition of these complexes is still very limited. Moreover, recent advances in the development and use of b-HLH-LZ dominant negatives (Soucek et al., Nature 455:679-683, 2008) and inhibitors of c-Myc interaction with its protein partners (Bidwell et al., J Control Release 135:2-10, 2009; Mustata et al., J Med Chem 52:1247-1250, 2009; Prochownik and Vogt, Genes Cancer 1:650-659, 2010) or DNA highlight the importance of efficient protocols to prepare such constructs and variants. Here, we provide methods to produce and purify high quantities of pure and untagged b-HLH-LZ constructs of c-Myc and Max as well as specific c-Myc/Max heterodimers for their biophysical and structural characterization by CD, NMR, or crystallography. Moreover, biochemical methods to analyze the homodimers and heterodimers as well as DNA binding of these constructs by native electrophoresis are presented. In addition to enable the investigation of the c-Myc/Max b-HLH-LZ complexes, the protocols described herein can be applied to the biochemical characterization of various mutants of either partner, as well as to ternary complexes with other partner proteins.
1868	Hypoxia/ischemia up-regulates Id2 expression in neuronal cells in vivo and in vitro.	23994058	Inhibitor of DNA binding/differentiation 2 (Id2) belongs to a family of transcriptional modulators characterized by a helix-loop-helix (HLH) motif that lacks the basic amino acid domain necessary to bind DNA. The aim of this study was to obtain a better understanding of the role of Id2 in hypoxia/ischemia (H/I)-induced neuronal apoptosis. Following H/I induction in a rat model of middle cerebral artery occlusion (MCAO)/reperfusion, the number of TUNEL-positive cells in cerebral cortices of the penumbra area increased gradually, while the Id2 mRNA and protein expression were also significantly up-regulated. The hypoxia-mimetic, cobalt chloride (CoCl2)-treated rat neuroblastoma B35 cell line also demonstrated enhanced Id2 mRNA and protein expression as well as increased number of cells in the sub-G1 populations after H/I exposure. Consistently, the expression of Bax, a proapoptotic protein, was also up-regulated in vivo and in vitro. Moreover, triple immunofluorescence demonstrated the obvious co-localization of Id2, TUNEL and NeuN in neurons of the penumbra area. These data suggest that H/I up-regulates Id2 expression in neuronal cells, and Id2 might play an important role in H/I-induced neuronal apoptosis.
1869	STAT3 or USF2 contributes to HIF target gene specificity.	23991099	The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein.
1870	Cognitive and psychosocial function post hematopoietic stem cell transplantation in children with hemophagocytic lymphohistiocytosis.	23987797	This study investigated the cognitive and psychosocial outcomes in childhood survivors of hemophagocytic lymphohistiocytosis after hematopoietic stem cell transplantation.Twenty-one children were assessed on standardized measures of cognitive and psychosocial functioning and compared with an unaffected sibling control group (n = 14). Parent and teacher reports were obtained to provide additional information.The average full-scale intelligence quotient for the patient cohort was 81 (95% CI, 72-90), which was significantly lower than both the population average of 100 (P = .001) and the average for the unaffected sibling control group (99.2, P = .002). Fifty-six percent of school-aged children were receiving additional support at school, with the majority needing high levels of support. These children also experienced significant psychosocial difficulties. Lower socioeconomic status was associated with poorer cognitive outcomes, but age at transplantation, time to transplantation, type of conditioning, and presence of mixed chimerism were not. Ten (48%) of 21 children had evidence of neurologic involvement at diagnosis, but surprisingly, this was not significantly associated with adverse neurologic outcomes, and some children who did not have any apparent neurologic involvement at diagnosis had severe learning difficulties at follow-up.In summary, childhood survivors of hemophagocytic lymphohistiocytosis are at risk of long-term cognitive and psychosocial difficulties. Prospective and systematic long-term follow-up of these patients is essential for early identification and effective management of these problems.
1871	The Drosophila enhancer of split gene complex: architecture and coordinate regulation by notch, cohesin, and polycomb group proteins.	23979932	The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains 12 genes that inhibit neural development. In a cell line derived from the central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E (spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. Here we show that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture we find that the E(spl)-C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. We posit that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain.
1872	Hemophagocytic syndrome in children with acute monoblastic leukemia-another cause of fever of unknown origin.	23975227	Intensification of antileukemic treatment and progress in supportive management have improved the survival rates of children with acute myeloid leukemia (AML). However, morbidity and early mortality in these patients are still very high, especially in children with acute monoblastic leukemia (AML FAB M5). Inflammatory syndromes complicating the management of these children after application of cytosine arabinoside and due to hyperleukocytosis at initial presentation have been reported. Hemophagocytic lymphohistiocytosis (HLH) has been described as a serious and life-threatening acute complication during treatment of different oncologic entities; however, data on HLH in children with AML FAB M5 are extremely rare.A retrospective study of all children with AML FAB M5 treated at our institution between 1993 and 2013 was performed to describe the clinical characteristics of patients who developed an inflammatory syndrome with HLH during oncologic treatment.Three of 10 children developed an inflammatory syndrome with fever, elevation of C-reactive protein, hyperferritinemia, elevation of soluble interleukin-2, and hemophagocytosis during prolonged aplasia following the first cycle of chemotherapy not responding to broad-spectrum antibiotics. No infectious agents could be identified; the initial symptoms occurred 17, 18, and 28 days after diagnosis of AML, respectively. The children immediately responded to dexamethasone; however, the same syndrome was observed again after the second cycle of chemotherapy and, in one patient, also after the third cycle.Treating physicians should be aware of an inflammatory syndrome resembling HLH in children with monoblastic leukemia since this problem might extremely complicate management and supportive care of these children. The co-incidence of monoblastic leukemia with HLH might be explained by cytokines released from the monoblastic leukemic cells themselves.
1873	Fatal hemophagocytic syndrome as a manifestation of immune reconstitution syndrome in a patient with acquired immunodeficiency syndrome.	23974460	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and potentially life-threatening condition characterized by uncontrolled hyper inflammation caused by various inherited or acquired immune deficiencies. We report a case of a 42-year-old man, newly diagnosed with HIV on the basis of a low CD4 T lymphocyte count (17/mm³) and HIV viral load >100,000 copies/mL by polymerase chain reaction tests who was undergoing an anti-retroviral regimen (emitricitabine, tenofovir disoproxil fumarate, ritonavir, and darunavir) and opportunistic infection prophylaxis (clarithromycin and atovaquone). He was concomitantly diagnosed with hemophagocytic syndrome, also known as HLH. He developed increasingly severe pancytopenia while on treatment with anti-retroviral drugs.
1874	Mixed hematopoietic or T-cell chimerism above a minimal threshold restores perforin-dependent immune regulation in perforin-deficient mice.	23974195	Defects in perforin and related genes lead to a loss of normal immune regulation and underlie hemophagocytic lymphohistiocytosis (HLH), which requires hematopoietic cell transplantation for long-term cure. However, transplantation may be complicated by the development of mixed chimerism and uncertainty regarding the risk of HLH recurrence. To help clarify this risk and investigate how perforin influences immune activation, we studied perforin-mediated immune regulation in the context of mixed chimerism using a murine model of HLH. We found that there is a distinct threshold of ∼10% to 20% perforin expression with either mixed hematopoietic or CD8(+) T cell chimerism, above which immune regulation was reestablished. These findings demonstrate that perforin-mediated immunoregulation functions in trans and are consistent with a feedback model in which cytotoxic T cells control immune activation by killing dendritic cells. These findings also suggest rational targets for maintenance of minimal posttransplant chimerism and for therapeutic strategies involving gene correction.
1875	X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.	23973892	X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.
1876	A novel method to predict regulatory regions based on histone mark landscapes in macrophages.	23973299	Macrophages as phagocytes and professional antigen presenting cells play critical roles in both innate and adaptive immunity. Main transcription factors acting during their differentiation and function are known, but the behavior and co-operation of these factors still remained unexplored. We introduce a new approach to map nucleosome-free regions using exclusively active enhancer and core promoter marking histone modification ChIP-seq data. We could detect approximately 56,000 potential active enhancers/promoters showing different lengths and histone modification shapes. Beside the highly enriched PU.1 and C/EBP sites, we could also detect binding sites for RUNX and AP-1, as well as for the MiT (MITF-TFE) family and MEF2 proteins. The PU.1 and C/EBP transcription factors are known for transforming cells into macrophages. The other transcription factors found in this study can play a role in macrophages as well, since it is known that the MiT family proteins are responsible for phagocytic activity and the MEF2 proteins specify monocytic differentiation over the granulocyte direction. Our results imply that this method can provide novel information about transcription factor organization at enhancers and core promoters as well as about the histone modifications surrounding regulatory regions in any immune or other cell types.
1877	Hemophagocytic Syndrome Associated with Mycoplasma pneumoniae Pneumonia.	23970989	Mycoplasma pneumoniae (Mp) sometimes causes immunological complications in children. We present a rare case of hemophagocytic syndrome (HPS) caused by Mp in a previously healthy 7-year-old Japanese girl. A chest radiograph obtained to evaluate the source of her fever showed infiltration in the lower right lung with mild splenomegaly. We could diagnose the patient with HPS on the basis of the hemophagocytic-lymphohistiocytosis- (HLH) 2004 criteria. She met the criteria for fever, splenomegaly, neutrophil count (<1,000/ μ L), platelet count (<10.0 × 10(4)/ μ L), fasting triglyceride level (>265 mg/dL), and ferritin level (>500 ng/mL). Furthermore, a peripheral blood smear showed an increased number of monocytes/macrophages with erythrophagocytosis. Treatment with clarithromycin and prednisolone, which was initiated soon after the diagnosis, was successful. Mp infection might partly progress to HPS in certain conditions. Clinicians should be aware of HPS caused by Mp and start appropriate treatment as soon as possible if the disease is suspected.
1878	[HLH-2004 protocol: diagnostic and therapeutic guidelines for childhood hemophagocytic lymphohistiocytosis].	23965886	NaN
1879	Traumatic brain injury in a rural and an urban Tanzanian hospital--a comparative, retrospective analysis based on computed tomography.	23954733	In a resource-poor environment such as rural East Africa, expensive medical devices such as computed tomographic (CT) scanners are rare. The CT scanner at the rural Haydom Lutheran Hospital (HLH) in Tanzania therefore offers a unique chance to observe possible differences with urban medical centers in the disease pattern of trauma-related cranial pathologies. The purpose of this study was to compare traumatic brain injuries (TBIs) between a rural and an urban area of Tanzania.HLH has 350 beds and one CT scanner. The urban Aga Khan Hospital is a private hospital with 80 beds and one CT scanner. This was a retrospective study. Data of 248 patients at HLH and of 432 patients at Aga Khan Hospital with TBI could be collected.The prevalence of TBI was significantly higher in the rural area compared to the urban area (34.2% vs. 21.9%, P < 0.0001). TBI due to violence was noted to occur more frequently at HLH, whereas road traffic accidents were more frequent at the Aga Khan Hospital. The number of patients showing a normal CT result was significantly higher in the urban area (53.0% vs. 35.9%, P < 0.0001). Bone fractures (35.9% vs. 15.7%, P < 0.0001) and pneumocephalus (6.9% vs. 0.9%, P < 0.0001) were diagnosed significantly more frequently in the rural survey. Soft tissue swelling (11.6% vs. 1.2%, P < 0.0001) and frontal sinus injuries (7.4% vs. 0.4%, P < 0.0001) were observed significantly more often in the urban setting.This study documents the burden of TBI and the differences in TBI-related CT diagnoses and their incidence between urban and rural areas in Eastern Africa. These results are important as they demonstrate that patients with severe TBI are not a primarily urban concern. Management of TBI should be included in the training curricula for health personnel alike irrespective of whether their workplace is primarily urban or rural.
1880	Hemophagocytic lymphohistiocytosis: advances in pathophysiology, diagnosis, and treatment.	23953723	NaN
1881	Association between childhood nephrotic syndrome and hemophagocytic lymphohistiocytosis.	23949593	Hemophagocytic lymphohistiocytosis (HLH) is caused by an excessive activation of nonmalignant macrophages. Renal lesions have been described in association with, but always after, HLH diagnosis.We describe a previously healthy 26-month-old girl who presented originally with steroid-responsive nephrotic syndrome (NS), but after 4 months, on the first NS relapse, experienced numerous complications (many of them reported to accompany NS as single events). Clinical and laboratory signs of HLH evolved with time and led to deterioration of her condition and death, within 5 months of her original presentation.To our knowledge, this is the first report of NS antedating the presentation of HLH.
1882	Clinical and genetic features of 5 Chinese patients with X-linked lymphoproliferative syndrome.	23944711	In this study, we report the clinical and genetic features of Chinese patients with X-linked lymphoproliferative syndrome (XLP). Male patients with fulminant infectious mononucleosis (FIM), Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) or persistent EBV viremia were enrolled in this study. Direct sequencing was used to detect SH2D1A/XIAP gene mutations. The patients' clinical features were assessed by retrieval of data from medical records. Twenty-one male patients with FIM, EBV-associated HLH or persistent EBV viremia were evaluated. Four patients had SH2D1A mutations, and one patient had an XIAP mutation. All five of these patients had symptoms of HLH and EBV infection. Among the five patients, the youngest one was only 1 month old at onset. One patient exhibited hypogammaglobulinemia. Of four patients evaluated for immunological function, all exhibited reduced CD4/CD8 ratios. Three patients had rapid disease progression and died. One patient received haematopoietic stem cell transplantation and is well. The overall clinical phenotypes of Chinese patients with XLP matched previous reports. For patients with severe EBV-associated HLH, our results indicate the need to examine the possibility of XLP.
1883	Haemophagocytic lymphohistiocytosis presenting as liver failure following Epstein-Barr and prior hepatitis A infections.	23943807	Haemophagocytic lymphohistiocytosis (HLH) is associated with high mortality even after prompt diagnosis. We present a young man with HLH triggered by two common viral diseases, infectious mononucleosis and hepatitis A. This patient presented with fever, rapidly progressive liver failure, anasarca and cholestasis, followed by anaemia and neutropenia. His carbohydrate antigen 19-9 reached over 9000 U/mL. Initial bone marrow and liver biopsies did not show histological features of malignancy or HLH. The patient was finally diagnosed and treated almost 1 year after the initial symptoms started, and had an excellent response with etoposide and dexamethasone. This case is unusual because it was triggered following mononucleosis in a patient with positive total antibodies against hepatitis A, with rapidly developing liver failure, and also because the patient's response was excellent despite the delay in treatment. It underscores the importance of suspecting HLH when severe systemic illness develops after a viral infection, even in the absence of clear histological features.
1884	The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans.	23925298	Autophagy is a cellular recycling process that has an important anti-aging role, but the underlying molecular mechanism is not well understood. The mammalian transcription factor EB (TFEB) was recently shown to regulate multiple genes in the autophagy process. Here we show that the predicted TFEB orthologue HLH-30 regulates autophagy in Caenorhabditis elegans and, in addition, has a key role in lifespan determination. We demonstrate that hlh-30 is essential for the extended lifespan of Caenorhabditis elegans in six mechanistically distinct longevity models, and overexpression of HLH-30 extends lifespan. Nuclear localization of HLH-30 is increased in all six Caenorhabditis elegans models and, notably, nuclear TFEB levels are augmented in the livers of mice subjected to dietary restriction, a known longevity-extending regimen. Collectively, our results demonstrate a conserved role for HLH-30 and TFEB in autophagy, and possibly longevity, and identify HLH-30 as a uniquely important transcription factor for lifespan modulation in Caenorhabditis elegans.
1885	Hemophagocytic lymphohistiocytosis after allogeneic bone marrow transplantation during chronic norovirus infection.	23922241	Hemophagocytic lymphohistiocytosis (HLH) is a macrophage activating syndrome that is known to develop in patients with autoimmune disease, malignancies or infection, for example with Epstein-Barr virus, cytomegalovirus or varicella zoster virus. We describe a 24-month old boy with acute myelogenous leukaemia relapse and allogeneic bone marrow transplantation, who developed HLH on day +40 during chronic infection with norovirus. Here, we report for the first time the development of HLH in combination with chronic norovirus infection after allogeneic bone marrow transplantation in a hematopoietic malignancy.
1886	Use of recombinant thrombomodulin in disseminated intravascular coagulation complicated hemophagocytic lymphohistiocytosis.	23912823	Hemophagocytic lymphohistiocytosis (HLH) is frequently lethal in its early phase due to complicating disseminated intravascular coagulation (DIC). The authors report a 14-mo-old girl with severe DIC complicating Epstein-Barr virus associated HLH. She was successfully treated with immunochemotherapy consisting mainly of etoposide and additional recombinant thrombomodulin (r-TM), a newly developed anticoagulant. Although the efficacy of r-TM cannot be proven in a single case report, additional anticoagulation therapy with r-TM is safe and may reduce early deaths in patients with DIC-complicated severe HLH. More clinical experience is required, although r-TM is currently licensed only in Japan.
1887	Hemophagocytic lymphohistiocytosis mimicking surgical symptoms and complications: lessons learned from four cases.	23895965	Hemophagocytic lymphohistiocytosis (HLH) is a severe immunological disorder that leads to a massive inflammatory reaction that may prove rapidly fatal. We show that HLH may present by masquerading as surgical disease or as a postoperative complication leading to delays in diagnosis and treatment.A case series of four children with acute surgical presentation and prolonged unexplained postoperative sepsis, who were diagnosed with HLH.Four children with different clinical presentations (1. neonatal abdominal distension, 2. ileostomy closure and Hirschsprung's disease, 3. iatrogenic sigmoid perforation and Crohn's disease, and 4. streptococcal toxic shock syndrome with primary peritonitis) were diagnosed with HLH at our regional pediatric surgical centre in the last two years. All developed signs of prolonged postoperative sepsis with hepatosplenomegaly and pancytopenia, requiring intensive care support. In the absence of explanation for their symptoms and deteriorating clinical condition, a total of six 'negative' exploratory laparotomies were performed. Eventually, HLH was diagnosed with bone marrow aspiration after an average of 23 days (range 17-40), following the finding of significantly elevated ferritin (up to 293150 ng/ml) and triglyceride levels. All children improved with initiation of high-dose steroid treatment followed by etoposide and cyclosporin.HLH may rarely present with symptoms and signs of surgical disease or complicate post-operative recovery. This diagnosis should be considered in children with unexplained prolonged fever, hepatosplenomegaly and pancytopenia, especially if associated with high ferritin levels. HLH can prove rapidly fatal without appropriate treatment.
1888	Differential clinical characteristics of acute liver failure caused by hemophagocytic lymphohistiocytosis in children.	23848458	Children with acute liver failure (ALF) caused by hemophagocytic lymphohistiocytosis (HLH) may be at risk of undergoing unnecessary liver transplantation (LT). The aim of this study was to compare the characteristics of ALF caused by HLH with those of ALF of unknown etiology in children.The clinical features and laboratory findings for eight children with ALF caused by HLH (ALF-HLH group) and 27 children with ALF of unknown etiology (ALF-UK group) were retrospectively compared by reviewing medical records.The ALF-HLH group had a higher incidence of pleural effusion, C-reactive protein elevation (especially >5 mg/dL), thrombocytopenia, anemia, fever, splenomegaly, and hypoalbuminemia (<2.5 mg/dL), and a higher in-hospital mortality rate. No significant differences were found in the white blood cell count, liver enzymes, coagulation profile, or incidence of hepatomegaly.LT should be performed only after it is proven that ALF is not caused by HLH, if a child with ALF shows the differential clinical features of ALF caused by HLH. Further research with larger sample sizes, however, is needed.
1889	Feasibility of reduced-intensity conditioning followed by unrelated cord blood transplantation for primary hemophagocytic lymphohistiocytosis: a nationwide retrospective analysis in Japan.	23843148	A nationwide retrospective analysis was performed on patients who received allogeneic hematopoietic stem cell transplantation for primary or familial hemophagocytic lymphohistiocytosis (HLH) in Japan. The present analysis investigated whether reduced-intensity conditioning (RIC) followed by cord blood transplantation (CBT) (RIC-CBT) is feasible, compared to the outcomes of myeloablative conditioning and bone marrow transplantation. Based on the JSHCT data, 53 patients were analyzed. The overall survival rate (OS) was 65.4 ± 6.6 %. RIC-CBT (n = 13) was not inferior to other methods. Patients with a performance status of PS 4 (ECOG scale) with HLH-associated severe organ dysfunction during the initiation of conditioning had extremely poor outcomes. The OS rate in the RIC-CBT patients, excluding those with a performance status 4, was 80.0 ± 12.6 %. RIC may reduce treatment-related mortality; in addition, patients with engraftment failure, which is the main adverse event following RIC-CBT, were successfully rescued with secondary CBT. Unrelated cord blood may represent an alternative source if a patient has no related donor. As a RIC regimen for CBT, 140 mg/m(2) melphalan with fludarabine and anti-lymphocyte globulin or anti-thymocyte globulin may be feasible, but further dosage optimization should be performed in controlled clinical trials.
1890	Evaluation of the role of secretory sphingomyelinase and bioactive sphingolipids as biomarkers in hemophagocytic lymphohistiocytosis.	23828274	Hemophagocytic lymphohistiocytosis (HLH) is a rare systemic inflammatory syndrome that results from unrestrained immune cell activation. Despite significant advances in the understanding of the pathophysiology of HLH, interventions remain limited for this often-fatal condition. Secretory sphingomyelinase (S-SMase) is a pro-inflammatory lipid hydrolase that is upregulated in several inflammatory conditions, including HLH. S-SMase promotes the formation of ceramide, a bioactive lipid implicated in several human disease states. However, the role of the S-SMase/ceramide pathway in HLH remains unexplored. To further evaluate the role of S-SMase upregulation in HLH, we tested the serum of patients with HLH (n = 16; primary = 3, secondary = 13) and healthy control patients (n = 25) for serum S-SMase activity with tandem sphingolipid metabolomic profiling. Patients with HLH exhibited elevated levels of serum S-SMase activity, with concomitant elevations in several ceramide species and sphingosine, while levels of sphingosine-1-phosphate were significantly decreased. Importantly, the ratio of C16 -ceramide:sphingosine was uniquely elevated in HLH patients that died despite appropriate treatment, but remained low in HLH patients that survived, suggesting that this ratio may be of prognostic significance. Together, these results demonstrate upregulation of the S-SMase/ceramide pathway in HLH, and suggest that the balance of ceramide and sphingosine determine clinical outcomes in HLH. .
1891	Hemophagocytic lymphohistiocytosis in total parenteral nutrition dependent children: description of 5 cases and practical tips for management.	23823121	Although total parenteral nutrition (TPN) is mandatory in children with intestinal failure, this treatment is not risk free. The main complications of TPN include catheter-related sepsis, thrombosis, hepatic cholestasis and cirrhosis, metabolic bone disease, and, rarely, reactive hemophagocytic lymphohistiocytosis (HLH). The pathogenesis of HLH in patients with TPN is not known, although some authors hypothesized that it can result from the activation of macrophages because of "fat overload." We reported 5 cases of HLH that occurred in patients with 4 different underlying disorders, all requiring TPN for a long term. In our series, an underlying immunological defect or a serious infection (sepsis) can have triggered HLH. Therefore, it could be reasonable to hypothesize that besides TPN in itself, minor immune defects and infections may act together by overcoming a threshold of immune stimulation, which ultimately leads to HLH.
1892	Haemophagocytic lymphohistiocytosis mimicking septic shock after the initiation of chemotherapy for squamous cell carcinoma of the neck.	23814201	Haemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal disorder resulting from a highly stimulated immune response with uncontrolled accumulation of lymphocytes and macrophages in multiple organs. Both the inherited and acquired forms of this disease exist; the latter can sometimes occur secondary to different malignancies. In this report, we present a middle-aged Hispanic man who presented with features of septic shock during the course of chemotherapy for squamous cell carcinoma of the neck. Despite aggressive treatment for septic shock, he rapidly deteriorated and died after 30 h of admission. Autopsy findings confirmed a diagnosis of HLH. HLH should be recognised as a serious adverse event during chemotherapy for different malignancies including squamous cell carcinoma of the neck.
1893	Clinical characteristics and outcomes of chédiak-Higashi syndrome: a nationwide survey of Japan.	23804531	Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by immunodeficiency, neurological dysfunction, and oculocutaneous albinism. Recently, several clinical CHS phenotypes have been reported. Here, we report results of a nationwide survey performed to clarify clinical characteristics and outcomes of CHS patients in Japan.Questionnaires were sent to 287 institutions to collect data regarding CHS patients diagnosed between 2000 and 2010, including results of lysosomal trafficking regulator (LYST) gene analysis. Cytotoxicity and degranulation activity of cytotoxic T lymphocytes were analyzed in available patient samples.A total of 15 patients diagnosed with CHS were eligible for enrollment in this study. Of these, 10 (67%) had recurrent bacterial infections, five (33%) developed life-threatening hemophagocytic lymphohistiocytosis (HLH), and one patient had complicated malignant lymphoma. Hematopoietic stem cell transplantation (HSCT) was performed for six patients including three with HLH, and 10 of the enrolled patients have survived at the time of this writing. LYST analysis was performed for 10 patients; seven different mutations were detected in seven patients, whereas no mutation was identified in three patients. Cytotoxicity and degranulation activity were impaired in patients with and without LYST mutation.Results of this survey indicate that one or two patients with CHS were newly diagnosed each year in Japan. The incidence of HLH was not as high as expected. Mutations of genes other than LYST were suspected in some cases. We conclude that determining indication for HSCT for CHS patients should be based on genetic and cytotoxic analysis.
1894	Hemophagocytic lymphohistiocytosis in a newborn infant born to a mother with Sjögren syndrome antibodies.	23803677	We encountered a neonatal patient with hemophagocytic lymphohistiocytosis (HLH) whose mother was positive for anti-Ro/SSA and anti-La/SSB antibodies. Complete atrioventricular block was found in a male patient at 29 weeks of gestation. The patient was born at 40 weeks of gestation. He showed severe circulatory disturbance at 22 h after the birth, and he also had elevated serum levels of aspartate aminotransferase (1027 IU l(-1)), alanine aminotransferase (121 IU l(-1)), lactic dehydrogenase (3490 IU l(-1)), ferritin (9769.7 ng ml(-1)) and soluble interleukin-2 (IL-2) receptor (3230 U ml(-1)). We could not find any known HLH genetic abnormality in the patient, but he fulfilled seven of the eight criteria for HLH. Serum levels of IL-6 and IL-8 had been already elevated in his cord blood, and serum levels of granulocyte-macrophage colony-stimulating factor and IL-8 were significantly increased on the second day of life. His symptoms regressed with the administration of hydrocortisone. We presumed that transplacental transfer of maternal antibodies could be related to the occurrence of HLH.
1895	Recombinant human thrombopoietin is an effective treatment for thrombocytopenia in hemophagocytic lymphohistiocytosis.	23778995	The aim of this study was to investigate the effectiveness and safety in the treatment of thrombocytopenia in hemophagocytic lymphohistiocytosis (HLH) by recombinant human thrombopoietin (rhTPO). A prospective randomized study was conducted between March 2010 and December 2011 in 40 patients with adult HLH whose blood platelet counts (BPC) were lower than 40 × 10(9)/L. The 40 patients were randomly assigned into the rhTPO group or control group based on sex, age, primary disease, and BPC (20 in each group). All patients were given conventional systemic therapy for HLH. The rhTPO group was administered by subcutaneous injection of rhTPO at a dose of 300 IU/kg Qd. The BPC, platelet transfusion, bleeding, and survival rate in the two groups were monitored and compared. There was no significant difference in BPC between the two groups before the treatment. Two weeks after the treatment, the BPC of the rhTPO group was significantly higher than that of the control group at every time point (P < 0.05). Although there was no significant difference in skin and mucous membrane bleeding between the rhTPO group and control group, however, the number of patients presented with gastrointestinal bleeding, urinary tract bleeding, and pulmonary bleeding in the control group were higher than that in the rhTPO group (P = 0.013). The frequency of platelet transfusion in the control group (7.25 per patient, 145 in 19 patients) was significantly higher than that in the rhTPO group (2.25 per patient, 45 in 14 patients) (P < 0.01). There was no significant difference in the survival rate between the two groups. The average recovery time of platelets to normal levels in the rhTPO groups was shorter than that in the control group (the rhTPO group vs the control group: 13.43 ± 4.62 D vs 18.00 ± 3.98 D, P = 0.013). In the early stage of HLH treatment, rhTPO combined with conventional systemic therapy can restore the BPC to normal level within a shorter period of time, reduce the frequency of platelet transfusion and severe bleeding.
1896	Defective UNC13D gene-associated familial hemophagocytic lymphohistiocytosis triggered by visceral leishmaniasis: a diagnostic challenge.	23774160	Visceral leishmaniasis (VL) triggered genetic hemophagocytic lymphohistiocytosis (HLH) is clinically challenging.One-year-old VL-HLH patient improved after liposomal-amphotericin-B therapy, but subsequently deteriorated, although bone marrow amastigotes disappeared. Symptoms resolved after 8 weeks of HLH-2004 therapy but recurred upon cessation. Homozygous UNC13D gene 627delT mutation was identified however stem cell donor was unavailable. The patient died at age 4 years after central nervous system attacks and HLH recurrences.VL in HLH patients does not exclude a genetic etiology and requires structured clinical management. VL should be excluded in all HLH patients in endemic regions before immunochemotherapy, which is recommended for VL-HLH patients unresponsive to VL treatment and/or reactivated.
1897	Mycobacterium avium Complex-Associated Hemophagocytic Lymphohistiocytosis in a Sickle Cell Patient: An Unusual Fatal Association.	23762672	Hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome, characterized clinically by fever, splenomegaly, cytopenia, and high ferritin. Infectious causes have been associated with secondary HLH, with viruses being the most common. We report a case of Mycobacterium avium complex-associated HLH in a sickle cell anemia patient. To the best of our knowledge, this association has never been reported in sickle cell anemia.
1898	Hodgkin's lymphoma preceded by haemophagocytic lymphohistiocytosis.	23761512	A 71-year-old woman was admitted because of persistent fever for 2 weeks. A diagnosis of haemophagocytic lymphohistiocytosis (HLH) was made on the basis of persistent high fever, pancytopoenia, hyperferritinaemia, increased soluble interleukin-2 receptor (sIL-2R) levels and histiocytosis and hemophagocytosis in the bone marrow. CT showed neither infection nor lymphadenopathy. After administration of prednisolone, haematological findings improved and the fever resolved; however, the patient developed persistent fever after 6 months. In addition, levels of lactate dehydrogenase and sIL-2R increased again. CT scans revealed diffuse lymphadenopathy, and Hodgkin's lymphoma was diagnosed by lymph node biopsy. Progression of pancytopenia was observed, and bone marrow examination showed a relapse of HLH. After six courses of chemotherapy were given for Hodgkin's lymphoma, complete remission was achieved with no evidence of pancytopenia. Hodgkin's lymphoma may be the underlying cause in HLH cases of unknown aetiology. Hence, the clinical course should be carefully monitored even in the absence of lymphadenopathy.
1899	Primary immunodeficiencies predisposed to Epstein-Barr virus-driven haematological diseases.	23758097	Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, maintains lifelong subclinical persistent infections in humans. In the circulation, EBV primarily infects the B cells, and protective immunity is mediated by EBV-specific cytotoxic T cells (CTLs) and natural killer (NK) cells. However, EBV has been linked to several devastating diseases, such as haemophagocytic lymphohistiocytosis (HLH) and lymphoproliferative diseases in the immunocompromised host. Some types of primary immunodeficiencies (PIDs) are characterized by the development of EBV-associated complications as their predominant clinical feature. The study of such genetic diseases presents an ideal opportunity for a better understanding of the biology of the immune responses against EBV. Here, we summarize the range of PIDs that are predisposed to EBV-associated haematological diseases, describing their clinical picture and pathogenetic mechanisms.
1900	Neonatal hemophagocytic lymphohistiocytosis associated with a vertical transmission of coxsackievirus B1.	23757031	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by fever, cytopenias, hepatosplenomegaly, and coagulopathy with the background of hypercytokinemia. Early diagnosis and etoposide therapy are not established for affected newborns. An afebrile infant suffered from apnea 4 days after birth, showing leukocytosis, thrombocytopenia, coagulopathy, and cerebrospinal fluid pleocytosis. Serum levels of ferritin and sIL-2R were high. Bone marrow studies revealed activated/hemophagocytosing macrophages. Coxsackievirus B1 (CB1) was isolated from the throat and stool. Serum anti-CB1 antibody titers were elevated in the patient (4 → 16; 6 → 43 days after birth) and mother (128; 10 days after delivery). Normal expressions of perforin and CD107a precluded inherited HLH. The vertically transmitted CB1-HLH was successfully treated without administration of corticosteroid, cyclosporine, or etoposide. Serum cytokine levels showed dominant expression of monokines (IL-1β/6/8, and TNF-α) but not IFN-γ, which is the central player of inherited HLH. The cytokine profile might represent a unique pathophysiology of enterovirus-driven neonatal HLH.
1901	Impacts of chronic low-level nicotine exposure on Caenorhabditis elegans reproduction: identification of novel gene targets.	23735997	Effects and mechanisms of chronic exposure to low levels of nicotine is an area fundamentally important however less investigated. We employed the model organism Caenorhabditis elegans to investigate potential impacts of chronic (24h) and low nicotine exposure (6.17-194.5 μM) on stimulus-response, reproduction, and gene expressions. Nicotine significantly affects the organism's response to touch stimulus (p=0.031), which follows a dose-dependent pattern. Chronic nicotine exposure promotes early egg-laying events and slightly increased egg productions during the first 72 h of adulthood. The expressions of 10 (egl-10, egl-44, hlh-14, ric-3, unc-103, unc-50, unc-68, sod-1, oxi-1, and old-1) out of 18 selected genes were affected significantly. Other tested genes were cat-4, egl-19, egl-47, egl-5, lin-39, unc-43, pink-1, and age-1. Changes in gene expression were more evident at low dosages than at relatively high levels. Genes implicated in reproduction, cholinergic signaling, and stress response were regulated by nicotine, suggesting widespread physiological impacts of nicotine.
1902	Hemophagocytic lymphohistiocytosis in a 19 year old critically ill patient.	23730021	Haemophagocytic lymphohistiocytosis (HLH) describes a clinical syndrome of hyperinflammation resulting in an uncontrolled and ineffective immune response. It presents with a clinical picture of likely sepsis, i.e., fever, laboratory evidence of inflammatory response, coagulopathy and thrombocytopaenia should be appropriately investigated and managed for sepsis, but the possible diagnosis of HLH should be borne in mind. Awareness of the clinical symptoms and of diagnostic criteria for HLH is crucial to starting immunosuppressive/immunomodulatory and cytotoxic drugs in time. We present a case of HLH in a 19 year old male who presented with fever, neurological symptoms, cytopaenias, laboratory markers of inflammation and bone marrow aspirate showed hemophagocytosis.
1903	Preventing lysosomal fat indigestion.	237284622360431623604321	Autophagy contributes to lipid catabolism through direct mobilization and breakdown of cellular lipid stores. Two recent studies reveal the regulatory mechanisms activated by cells during starvation to ensure that the cellular compartments involved in autophagic lipid catabolism are ready to receive, process and use these lipids. The regulators represent attractive therapeutic targets to help fight lipid-excess-associated diseases.
1904	Extra-intestinal malignancies in inflammatory bowel disease: results of the 3rd ECCO Pathogenesis Scientific Workshop (III).	23721759	The incidence of lymphoproliferative disorders (LD) is increasing in developed countries. Patients with inflammatory bowel disease (IBD) exposed to thiopurines are at additional risk of three specific forms of LD: Epstein-Barr-Virus-related post-transplant like LD, hepato-splenic T-cell lymphoma and post-mononucleosis lymphoproliferation. The risk of the two latter forms of LD can be reduced when considering specific immunosuppressive strategies in young males. It is still unclear whether the risk of uterine cervix abnormalities is increased in IBD women, irrespective of the use of immunosuppressants. Given the excess risk demonstrated in various other contexts of immunosuppression, it is currently recommended that all women with IBD, particularly those receiving immunosuppressants, strictly adhere to a screening program of cervical surveillance and undergo vaccination against HPV, when appropriate. Patients with IBD receiving immunosuppressants are at increased risk of skin cancers. The risk of non-melanoma skin cancer is notably increased in patients receiving thiopurines. Recent data suggest that the risk of melanoma is mildly increased in patients exposed to anti-TNF therapy. All IBD patients should adhere to a program of sun protection and dermatological surveillance, whose details should take into account the other non-IBD-related risk factors.
1905	Hematopoietic stem cell transplantation with a reduced-intensity conditioning regimen in pediatric patients with Griscelli syndrome type 2.	23714271	Partial albinism with variable immunodeficiency are the two major characteristics of Griscelli syndrome type 2 (GS-2). This syndrome is usually associated with a high mortality rate and commonly results in early childhood death. Patients suffer from different infections and experience crisis of HLH. HSCT remains the sole curative treatment for GS-2. We prospectively analyzed the outcomes of transplantation with RIC regimen in five patients. The median age at transplantation was 21.6 months (range: 12-30). All of the patients underwent HSCT from HLA-matched related donors. Currently, four patients are cured, and symptoms of recurrent infections and HLH crisis are not seen in them. The only patient who died had undergone HSCT in the accelerated phase of HLH. One patient who developed acute GvHD had a favorable response to therapy. No chronic GvHD occurred in patients. It seems that the use of RIC regimen as a method of transplant preparation is effective and tolerable in this group of patients with various comorbidities. It is recommended to carry out HSCT in these patients at lower ages, before presentations of different infections and HLH crisis.
1906	Novel mutations in the UNC13D gene carried by a Chinese neonate with hemophagocytic lymphohistiocytosis.	23709445	Hemophagocytic lymphohistiocytosis (HLH) in different ethnicities has been described in the literature, but few cases in patients of Chinese descent have been reported. Here, we describe the case of a Chinese neonate presenting with HLH carrying novel, compound heterozygous mutations of the UNC13D gene, including [c.2295_2298delGCAG, p.Glu765Aspfs*27] in exon 23, c.-250C>T, c.1+30G>A, c.279C>T, c.888G>C, c.18+36A>G, c.20-48T>C, c.1977C>T, c.2296C>T, c.24-46C>T, c.26-9_26-8insC, c.2599A>G, c.28+48C>T and c.3198A>G, some of which have not been reported in the literature. Cytokine profile analyses were performed in this patient, and the results were consistent with our previous findings in HLH patients. Cytokine profile monitoring may be helpful in differentiating among various clinical phases of HLH.
1907	Lymphoma-associated hemophagocytic lymphohistiocytosis: experience in adults from a single institution.	23700280	Lymphoma-associated hemophagocytic lymphohistiocytosis (HLH) is a rare but fatal disease. Differences between B cell and T cell lymphoma-associated HLH remain unclear, specifically clinical characteristics and survival. We retrospectively analyzed 30 lymphoma-associated HLH patients from July 2004 to October 2012. Patients were divided into B cell (n = 13) and T cell (n = 17) lymphoma groups. Patients' age, performance status, presence of Epstein-Barr virus infection, international prognostic index, presence of disseminated intravascular coagulopathy, serum triglyceride, fibrinogen, and lactate dehydrogenase levels were not significantly different between B cell and T cell lymphoma groups. HLH was an indicator for treatment resistance in patients with B cell (p = 0.048), but not T cell (p = 0.217), lymphoma. Patients in the T cell lymphoma group, however, had higher serum ferritin levels than patients in the B cell lymphoma group (11,525.6 versus 3,790.6 ng/mL; p = 0.043). The median survival time for patients in the B cell and T cell lymphoma groups was 330 and 96 days, respectively. Although the difference was not statistically significant (p = 0.273), our results suggested a trend toward a better overall survival time in patients with B cell lymphoma. This survival advantage could be at least partially due to use of rituximab (p = 0.045) for the treatment of patients with B cell lymphoma. Our results also suggested that allogeneic hematopoietic stem cell transplantation could possibly provide survival benefits to T cell lymphoma-associated HLH by graft-versus-lymphoma effect.
1908	Analysis of fatal cases of pandemic influenza A (H1N1) virus infections in pediatric patients with leukemia.	23697998	Pandemic influenza A/H1N1/2009 virus usually causes mild illness in healthy children. Chronic medical conditions are recognized as increasing the risk for complications of influenza virus infection. Although most studies including children with acute leukemia and H1N1 virus have reported no deaths, some anectodal reports with low patient numbers have reported mortality rates as high as 28.5%. Here, we report patients with leukemia and H1N1 virus and review the literature.Medical records of all children with leukemia and H1N1 virus in our institution were reviewed for demographic, clinical, and laboratory data. We also carried out a systematic review of the English-language literature. Among the 24 articles found, only patients with leukemia and pandemic H1N1 infections were reviewed by two independent reviewers.Eight of 98 children who received chemotherapy for leukemia were diagnosed with pandemic H1N1 infection. One developed pneumonia and acute respiratory distress syndrome (ARDS) and died. Another one developed hemophagocytic lymphohistiocytosis (HLH) and died due to secondary infection during the 6th week of treatment for HLH. In our study, 2 of 8 patients had a fatal course (25%), compared with an overall mortality of 2.5% in the studies retrieved from PubMed (6/232).Pandemic H1N1 influenza virus caused mortality in patients with ARDS or HLH; an unexpected finding for pandemic H1N1 (2009) influenza virus. Thus, for children with leukemia and infected with H1N1 virus, short- and long-term complications should be kept in mind during evaluation.
1909	Hematopoietic stem cell transplantation for children with primary immunodeficiency diseases: single center experience in Jordan.	23692601	HSCT can be curative for many PID. Little is known about the outcome of HSCT for patients with PID in the developing countries. We retrospectively reviewed all children with PID who received HSCT at KHCC in Jordan between August 2003 and October 2011. Twenty-eight patients were identified. The median age was 16 months (3 months-17 yr). Patients' diagnoses were SCID (n = 16), CHS (n = 3), HLH (n = 3), WAS (n = 2), Griscelli syndrome (n = 1), ALPS (n = 1), Omenn's syndrome (n = 1), and DiGeorge syndrome (n = 1). Seventeen patients received HLA-matched related HSCT, eight received maternal un-manipulated haploidentical HSCT, and three received unrelated cord blood transplantation. Nine patients (32%) developed BCGosis secondary to reactivation of pretransplant vaccination. Three died while still receiving anti-tuberculosis drugs, one still on treatment, and all others have recovered. Six patients had graft failure; four of them received no conditioning regimens. At a median follow up of 32 months (range 1-67), 21 patients are alive, with overall survival of 72%. We conclude that HSCT for PID patients can be performed with a good outcome in developing countries; however, delayed diagnosis or referral and BCG reactivation are unique challenges.
1910	Treatment of Epstein Barr virus-induced haemophagocytic lymphohistiocytosis with rituximab-containing chemo-immunotherapeutic regimens.	23692048	Haemophagocytic lymphohistiocytosis (HLH) is a life threatening complication of Epstein-Barr virus (EBV) infection. The anti-CD20 antibody rituximab depletes B cells, leading to improved outcomes for patients with EBV-associated B-lymphoproliferative disorders. To gather data on the use of rituximab in EBV-HLH, we performed a retrospective investigation involving 42 EBV-HLH patients who had received treatment with rituximab-containing regimens. On average, patients received 3 rituximab infusions (range 1-10) at a median dose of 375 mg/m(2) . In all patients, rituximab was administered with other HLH-directed medications, including steroids, etoposide and/or ciclosporin. Rituximab-containing regimens appeared well tolerated and improved clinical status in 43% of patients. Examination of laboratory data obtained prior to and within 2-4 weeks after the first rituximab dose revealed significant reductions in EBV load (median load pre-rituximab: 114,200 copies/ml, median post-rituximab: 225 copies/ml, P = 0.0001) and serum ferritin levels (median ferritin pre-rituximab: 4260 μg/l, median post-rituximab: 1149 μg/l, P = 0.001). Thus, when combined with conventional HLH-directed therapies, rituximab improves symptoms, reduces viral load and diminishes inflammation. These data support the incorporation of rituximab into future prospective clinical trials for patients with EBV-HLH.
1911	Hemophagocytic lymphohistiocytosis: an update on diagnosis and pathogenesis.	23690113	Hemophagocytic lymphohistiocytosis (HLH) is a frequently fatal and likely underdiagnosed disease involving a final common pathway of hypercytokinemia, which can result in end-organ damage and death. Although an early diagnosis is crucial to decrease mortality, the definitive diagnosis is often challenging because of the lack of specificity of currently accepted diagnostic criteria and the absence of confirmatory gold standards. Because of the wide range of laboratory assays involved in the diagnosis of HLH, practicing pathologists from a broad spectrum of clinical specialties need to be aware of the disease so that they may appropriately flag results and convey them to their clinical counterparts. Our article summarizes these new advances in the diagnosis of HLH and includes a review of clinical findings, updated understanding of the pathogenesis, and promising new testing methods.
1912	Sudden unexpected death due to hemophagocytic lymphohistiocytosis syndrome.	23682740	The hemophagocytic lymphohistiocytosis (HLH) syndrome is a hyperimmune disorder characterized by lymphohistiocytic infiltrations, elevated cytokine levels in the blood, macrophage activation, and hemophagocytosis, frequently presenting with a febrile septic picture. This unusual disease is more common in infancy and childhood than adulthood. It is classified as primary or familial when a genetic defect is identified and secondary or acquired when triggered by certain infections, autoimmune disorders, or malignancies. If or when such patients expire, they typically do so within a hospital or under a physician's care and so such cases rarely come to the attention of forensic pathologists. We report on the unexpected deaths of two hospitalized adult cases of HLH brought to autopsy without a premortem diagnosis. Postmortem examination demonstrated marked hepatosplenomegaly and lymphadenopathy in association with hemophagocytosis. Although very uncommon HLH must be considered in infants, children, or adults who die unexpectedly with an undiagnosed septic presentation.
1913	Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy.	23678006	Blinatumomab is a CD19/CD3-bispecific T-cell receptor-engaging (BiTE) antibody with efficacy in refractory B-precursor acute lymphoblastic leukemia. Some patients treated with blinatumomab and other T cell-activating therapies develop cytokine release syndrome (CRS). We hypothesized that patients with more severe toxicity may experience abnormal macrophage activation triggered by the release of cytokines by T-cell receptor-activated cytotoxic T cells engaged by BiTE antibodies and leading to hemophagocytic lymphohistiocytosis (HLH). We prospectively monitored a patient during blinatumomab treatment and observed that he developed HLH. He became ill 36 hours into the infusion with fever, respiratory failure, and circulatory collapse. He developed hyperferritinemia, cytopenias, hypofibrinogenemia, and a cytokine profile diagnostic for HLH. The HLH continued to progress after discontinuation of blinatumomab; however, he had rapid improvement after IL-6 receptor-directed therapy with tocilizumab. Patients treated with T cell-activating therapies, including blinatumomab, should be monitored for HLH, and cytokine-directed therapy may be considered in cases of life-threatening CRS. This trial was registered at www.clinicaltrials.gov as #NCT00103285.
1914	Familial hemophagocytic lymphohistiocytosis type 3 diagnosed at school age: a case report.	23669735	Familial hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening condition characterized by immune hyperactivation and clinical signs of extreme inflammation. We describe a 7-year-old male who presented with fever resistant to antibiotic therapy, pancytopenia, splenomegaly, hypertriglyceridemia, and hyperferritinemia. Bone marrow aspirate showed hemophagocytosis. Epstein-Barr virus genome was positive in blood. Functional screening showed reduced capacity of cytotoxic degranulation. Mutation analysis of the FHL-related genes revealed compound heterozygous for UNC13D mutations: c. 753+1G>T, and the novel c.544C>T (p.P182S). Patients with a clinical presentation of HLH, even if older than typically seen, should be screened for familial HLH by mutation analysis.
1915	EBV-Positive T/NK-Cell Lymphoproliferative Disease of Childhood.	23667373	Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH), EBV-positive systemic T-cell lymphoproliferative disease (STLPD) of childhood, and chronic active EBV (CAEBV) infection may develop after primary EBV infection. This study reviewed the clinicopathological spectrum of EBV-associated T- and natural killer (NK)-cell LPD, including STLPD and CAEBV infection, with an analysis of T-cell clonality.Clinicopathological features of seven patients with EBV-associated HLH or STLPD and 12 patients with CAEBV infection were reviewed. Immunohistochemical staining and a T-cell receptor (TCR) gene rearrangement study were performed.STLPD and EBV-positive HLH showed significantly overlapping clinicopathological findings. One patient with STLPD and one patient with EBV-positive HLH demonstrated moderate to severe atypia of the infiltrating lymphocytes, whereas the remaining patients lacked significant atypia. Twelve patients had CAEBV infection, four of whom suffered mosquito-bite hypersensitivity, five showed NK lymphocytosis, and one suffered hydroa vacciniforme. Infiltrating lymphocytes were predominantly small and devoid of atypia. Hemophagocytic histiocytosis was found in seven of 11 patients. Monoclonality was detected in three (50%) of the six patients with successful TCR gene analysis.EBV-positive HLH and STLPD share similar clinicopathological findings and may constitute a continuous spectrum of acute EBV-associated T- or NK-cell proliferative disorders. The distinction of EBV-positive T-cell LPD from EBV-positive HLH may be difficult during routine diagnoses because of the technical limitations of clonality assessment.
1916	Molecular characterization, expression patterns, and association analysis with carcass traits of porcine USF1 gene.	23666615	The upstream stimulatory factor 1 (USF1) is a transcription factor controlling expression of several genes involved in lipid and glucose homeostasis. In this study, two isoforms of the porcine USF1 gene were detected by reverse transcription polymerase chain reaction (RT-PCR), termed USF1 wild-type (wt) and USF1/CD, both of them contain a helix-loop-helix leucine zipper (HLH-LZ) conserved domain. Tissue distribution analysis showed that the two transcripts of porcine USF1 gene were ubiquitously expressed in all tested tissues, except for heart. Moreover, we found that a single nucleotide polymorphism (SNP, C/T) in intron 10 was significantly associated with ratio of lean to fat (P < 0.05), dress percentage (P < 0.05), average backfat thickness (P < 0.05), loin eye width (P < 0.05), lean meat percentage (P < 0.01), loin eye height (P < 0.01), and loin eye area (P < 0.01). This result suggests that porcine USF1 gene may be a candidate gene of meat production trait.
1917	[Refractory acute myeloid leukemia developed malignancy-associated hemophagocytic lymphohistiocytosis during treatment of invasive fungal infection].	23666221	We here report a 2-year-old female with relapsed acute myeloid leukemia (AML) with MLL gene rearrangement in the bone marrow and central nervous system. The 3'-RACE (Rapid Amplification of cDNA Ends) method identified the MLLT10 gene as a fusion partner of the MLL gene. The patient was complicated with hemophagocytic lymphohistiocytosis (HLH) and invasive aspergillosis (IPA) after re-induction treatment with FLAG-IDA following etoposide, cytarabine, and mitoxantrone. Although treatment with systemic anti-fungal drugs was effective for IPA, HLH did not improve. We considered tumor-associated HLH to be initiated from leukemic stem cells (LSCs) in the bone marrow niche because reverse transcription-polymerase chain reaction (RT-PCR) analysis of a bone marrow biopsy sample was positive for MLL-MLLT10. Gemtuzumab ozogamicin and sorafenib had no major effect on acquiring complete remission, and the patient died of progressive AML with an exacerbation of HLH and aspergillosis. LSCs are known to be resistant to conventional chemotherapy due to their quiescence in the cell cycle. Novel therapeutic concepts are important to eradicate LSCs in order to cure AML patients.
1918	[Transfusion-related acute lung injury during the treatment of EBV-associated hemophagocytic lymphohistiocytosis].	23666220	Transfusion-related acute lung injury (TRALI) is a severe pulmonary complication following blood transfusions. We experienced a case of possible TRALI during the course of EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). A 19-year-old woman was admitted to our hospital suffering from fever and abdominal pain. Her laboratory data revealed pancytopenia, liver damage, coagulopathy, and a high titer of EBV-DNA. Computed tomography showed hepatosplenomegaly and bone marrow aspiration revealed hemophagocytosis and the proliferation of atypical lymphocytes. A diagnosis of EBV-HLH was made and plasma exchange was performed. Severe hypoxia due to pulmonary edema developed two hours after starting the plasma transfusion. Methylprednisolone pulse therapy and non-invasive positive pressure ventilation ameliorated her respiratory condition. Anti-HLA class I and II antibodies were detected in donor sera and a cross-match test between patient lymphocytes and donor plasma was positive. To the best of our knowledge, this is the first case report of TRALI complicated with EBV-HLH. It is possible that hypercytokinemia accompanied by HLH was associated with the onset of TRALI.
1919	Haemophagocytic lymphohistiocytosis in a Ghanaian child.	23661845	We report a case of a previously well nine-month-old infant who presented with prolonged fever, hepatosplenomegaly and pancytopenia. A diagnosis of haemophagocytic lymphohistiocytosis (HLH) was made during the course of hospital admission. There was good initial response to dexamethasone but the patient died less than two months after diagnosis. This is the first report of HLH from Ghana. The disease has a significant mortality rate if untreated and a high index of suspicion is required in all severely ill children.
1920	Perforin deficiency impairs a critical immunoregulatory loop involving murine CD8(+) T cells and dendritic cells.	23660960	Humans and mice with impaired perforin-dependent cytotoxic function may develop excessive T-cell activation and the fatal disorder hemophagocytic lymphohistiocytosis (HLH) after infection. Though cytotoxic lymphocytes can kill antigen-presenting cells, the physiological mechanism of perforin-mediated immune regulation has never been demonstrated in a disease-relevant context. We used a murine model of HLH to examine how perforin controls immune activation, and we have defined a feedback loop that is critical for immune homeostasis. This endogenous feedback loop involves perforin-dependent elimination of rare, antigen-presenting dendritic cells (DCs) by CD8(+) T cells and has a dominant influence on the magnitude of T-cell activation after viral infection. Antigen presentation by a minor fraction of DCs persisted in T-cell- or perforin-deficient animals and continued to drive T-cell activation well beyond initial priming in the latter animals. Depletion of DCs or transfer of perforin-sufficient T cells dampened endogenous DC antigen presentation and T-cell activation, demonstrating a reciprocal relationship between perforin in CD8(+) T cells and DC function. Thus, selective cytotoxic "pruning" of DC populations by CD8(+) T cells limits T-cell activation and protects against the development of HLH and potentially other immunopathological conditions.
1921	[EB virus-associated lymphoproliferative disorders and cytokine storm].	23628063	Epstein-barr virus (EBV)-associated lymphoproliferative disorders (EBV-LPD) include a series of diseases from chronic to aggressive EBV-positive T-cells, NK cells, T/NK cells or B-cell LPD. The occurrence and development of EBV-LPD are closely associated with the cytokine storm, the clinical manifestations are very complex. Studies found that the different types of EBV-LPD express different cytokine secretion patterns, which help to further understand the progression of diseases, thus the EBV-LPD can be diagnosed early and treated early. In this article, different cytokine secretion patterns and their roles in EBV-LPD progression are reviewed. The main problems discussed above are the classification of EBV-LPD, T/NK-LPD and cytokine, B-LPD and cytokine, HLH and cytokine, and so on.
1922	[Detection of serum neopterin in patients with hemophagocytic lymphohistiocytosis and its significance].	23628055	This study was aimed to detect the peripheral blood serum neopterin (Npt) level in the patients with hemophagocytic lymphohistiocytosis (HLH) and to explore its significance in HLH. The enzyme-linked immunosorbent assay (ELISA) was applied to detect the serum Npt level and sCD25 level in 20 HLH patients before and after treatment and 15 healthy controls. The results indicated that the serum Npt and sCD25 levels in HLH patients were significantly higher than those in healthy controls (P < 0.0001). The serum Npt and sCD25 levels in the HLH group decreased significantly after treatment, respectively (P < 0.0001). The correlation analysis of Npt with sCD25 before and after treatment showed that they had significant correlation (r = 0.81, P < 0.05 before treatment; r = 0.65, P < 0.05 after treatment). Meanwhile, the level of serum Npt and ferritin had a significant correlation in newly diagnosed HLH patients (r = 0.55, P < 0.05). It is concluded that the serum Npt may play an important role in the HLH pathogenesis, the enhancement of Npt levels has an important significance for diagnosis and evaluation for HLH.
1923	[Clinical analysis of Epstein-Barr virus-associated hemophagocytic syndrome in children].	23628054	The primary infection of Epstein-Barr virus (EBV) may results in hemophagocytic syndrome, known as EBV-associated hemophagocytic syndrome (EBV-AHS), but the clinical risk factors complicating this fatal disease in children with infectious mononucleosis (IM) are unknown. The aim of this study was to identify clinical features of EBV-AHS and to evaluate the curative effect of HLH-2004 protocol. The clinical and laboratory data of 644 IM children including 27 children developed into EBV-AHS and 43 HPS children associated with other diseases were retrospectively analyzed and logistic regression was used to identify the clinical risk factors complicating EBV-AHS. The results showed as follows: (1) the prevalence of EBV-AHS in IM children was 4.2% (27/644), and the prevalence in group aged younger than 3 years was higher than in other age groups. The incidence age of EBV-AHS was significantly younger than that of other HPS patients; (2) Liver function damage of group aged older than 7 years was much more severe in HPS patients. (3) Compared with other HPS patients, male patients were more common and liver function damage was severe in EBV-AHS patients, especially in the patients aged at 2 years or younger. (4) The fatality rate in the EBV-AHS patients was 37.0% (10/27). (5)After treatment with HLH-2004 protocol, the fatality rate in patients with EBV-AHS decreased from 50.0% to 18.2%, the overall survival (OS) of 3 years significantly increased (P = 0.032). It is concluded that IM is a benign self-limited disease, of which only about 4.2% patients will develop into EBV-AHS. Clinical risk factors identified in this study may be helpful for early diagnosis of IM children with complicated EBV-ASH, the HLH-2004 protocol can obviously improve prognosis of EBV-HPS.
1924	Neurological expression of genetic immunodeficiencies and of opportunistic infections.	23622332	Immunodeficiencies may result from genetic defects or may be acquired after viral infection or therapeutic immunosuppression. In either case, neurological symptoms are frequent. Hemophagocytic lymphohistiocytosis (HLH), previously designated as macrophage activation syndrome (MAS), results usually from genetic defects impairing the cytotoxicity of CD8 T lymphocytes and natural killer (NK) cells. Neurological symptoms may be the first and only manifestation of the disease. The neurological and neuroradiological aspects of MAS, if isolated, may closely resemble those of other central nervous system (CNS) diseases such as acute disseminated encephalomyelitis (ADEM) or encephalitis. An early treatment including allogeneic hematopoietic stem cell transplantation can prevent further brain lesions. The main infections of the CNS observed in conjunction with therapeutic immunosuppression are described. It is important to know which phase of immunosuppression the child is in at the time of suspected CNS infection and always to consider that a relapse of the primary tumor may mimic a CNS infection.
1925	A child with Epstein-Barr Virus-associated hemophagocytic lymphohistiocytosis complicated by coronary artery lesion mimicking Kawasaki disease.	23619112	There is considerable overlap between hemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD) in terms of aberrant immune response though the etiology of KD remains unknown. We present a case fulfilling the criteria of both HLH and KD complicated by coronary artery dilatation: HLH was confirmed to be triggered by Epstein-Barr virus. This case alarms us the possibility that even patients with HLH may be complicated by coronary artery lesion, which is one of the hallmarks of KD. We would like to draw attention that if features of KD become apparent in patients with HLH, echocardiographic examinations should be performed not to miss coronary artery lesion.
1926	MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability.	2360431623728462	Fat is stored or mobilized according to food availability. Malfunction of the mechanisms that ensure this coordination underlie metabolic diseases in humans. In mammals, lysosomal and autophagic function is required for normal fat storage and mobilization in the presence or absence of food. Autophagy is tightly linked to nutrients. However, if and how lysosomal lipolysis is coupled to nutritional status remains to be determined. Here we identify MXL-3 and HLH-30 (TFEB orthologue) [corrected] as transcriptional switches coupling lysosomal lipolysis and autophagy to nutrient availability and controlling fat storage and ageing in Caenorhabditis elegans. Transcriptional coupling of lysosomal lipolysis and autophagy to nutrients is also observed in mammals. Thus, MXL-3 and HLH-30 orchestrate an adaptive and conserved cellular response to nutritional status and regulate lifespan.
1927	Report of a Fatal Pediatric Case of Hemophagocytic Lymphohistiocytosis Associated with Pandemic Influenza A (H1N1) Infection in 2009.	23602384	An acute viral infection may lead to death, as seen in the recent novel 2009 influenza A (H1N1) virus pandemic that rapidly spread worldwide. Hemophagocytic lymphohistiocytosis (HLH) is a systemic disorder with high mortality rates and can be triggered by various etiological agents, including viral infections. H1N1-associated HLH is a very rare condition that may cause extremely severe complications leading to multiple organ failure (MOF) and death. We report an unusual case of a rapidly progressive and fatal H1N1 infection that was complicated with HLH and MOF in a previously healthy 8-year-old Asian female who initially presented with fever and abdominal pain. To enable early recognition and proper treatment, physicians should be aware of the possibility of this fatal complication, which may present with unusual initial symptoms.
1928	Reaching the threshold: a multilayer pathogenesis of macrophage activation syndrome.	23588947	Macrophage activation syndrome (MAS) is a potentially fatal complication of rheumatic diseases. The condition is considered part of secondary hemophagocytic lymphohistiocytoses (HLH). There are similarities in genetic background, pathogenesis, and clinical and laboratory features with primary HLH (p-HLH). We describe findings in mouse models of secondary HLH, comparing them with models of p-HLH and the cellular and molecular mechanisms involved, and relate them to recent findings in patients with secondary HLH. A multilayer model is presented in which background inflammation, infections, and genetics all contribute in different proportions and in several ways. Once the "threshold" has been reached, inflammatory cytokines are the final effectors, independent of the interplay between different upstream pathogenic factors.
1929	Advances in understanding the pathogenesis of HLH.	23577835	Haemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder resulting from immune dysfunction reflecting either primary immune deficiency or acquired failure of normal immune homeostasis. Familial HLH includes autosomal recessive and X-linked disorders characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines, secondary to defects in genes encoding proteins involved in granule-dependent cytolytic pathways. In older children and adults, HLH is associated more often with infections, malignancies, autoimmune diseases, and acquired immune deficiencies. HLH, macrophage activation syndrome, sepsis, and systemic inflammatory response syndrome are different clinical entities that probably represent a common immunopathological state, termed cytokine storm. These conditions may be clinically indistinguishable; all include massive inflammatory response, elevated serum cytokine levels, multi-organ involvement, haemophagocytic macrophages, and often death. Tissues of haematopoietic and lymphoid function are directly involved; other organs are secondarily damaged by circulating cytokines and chemokines. Haemophagocytic disorders are now increasingly diagnosed in the context of severe inflammatory reactions to viruses, malignancies and systemic connective tissue diseases. Many of these cases may reflect underlying genetic predispositions to HLH. The detection of gene defects has contributed considerably to our understanding of HLH, but the mechanisms leading to acquired HLH have yet to be fully determined.
1930	TEN-like eruption in setting of EBV positive T-cell lymphoproliferative disease with HLH, in a child.	23573996	Haemophagocytic lymphohistiocytosis complicating Epstein-Barr virus positive T-cell lymphoproliferative disease of childhood is a rare and life-threatening entity. We report a child with this condition presenting with a toxic epidermal necrolysis-like eruption.
1931	Severe hemophagocytic lymphohistiocytosis in adults-experience from an intensive care unit from North India.	23559726	Hemophagocytic lymphohistiocytosis (HLH) has been reported to complicate fulminant tropical infections but data on severe HLH with multi-organ dysfunction (MODS) are scant.Retrospective review of medical electronic records of our intensive care unit (ICU) over a 2-year period.We describe 10 adult patients with HLH and MODS. Patients had short symptom duration prior to presentation and had rapid deterioration during their hospitalization course. Fever, organomegaly, neurologic abnormalities, hepatic abnormalities, and cutaneous signs were very common. No patient had diagnosed HLH at ICU admission (median 4 criteria [Inter Quartile Range 2-4.25]). All patients required mechanical ventilation and 80% required vasopressors. Infection-associated HLH (IAHS) was the most common etiology (80%). Seventy percent (7/10) of patients were treated with steroids and 20% received intravenous immunoglobulin. Etoposide and/or cyclosporine were administered in 20% (2/10). Nosocomial infections occurred in 40% and the ICU mortality was 70%.Severe HLH with MODS has a very high mortality. Data on adult cohorts with IAHS in the tropics with defined treatment protocols are urgently needed.
1932	E47 and Id1 interplay in epithelial-mesenchymal transition.	23555842	E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.
1933	Interferon-γ mediates anemia but is dispensable for fulminant toll-like receptor 9-induced macrophage activation syndrome and hemophagocytosis in mice.	23553372	Macrophage activation syndrome (MAS) is a devastating cytokine storm syndrome complicating many inflammatory diseases and characterized by fever, pancytopenia, and systemic inflammation. It is clinically similar to hemophagocytic lymphohistiocytosis (HLH), which is caused by viral infection of a host with impaired cellular cytotoxicity. Murine models of MAS and HLH illustrate that interferon-γ (IFNγ) is the driving stimulus for hemophagocytosis and immunopathology. This study was undertaken to investigate the inflammatory contributors to a murine model of Toll-like receptor 9 (TLR-9)-induced fulminant MAS.Wild-type, transgenic, and cytokine-inhibited mice were treated with an IL-10 receptor blocking antibody and a TLR-9 agonist, and parameters of MAS were evaluated.Fulminant MAS was characterized by dramatic elevations in IFNγ, IL-12, and IL-6 levels. Increased serum IFNγ levels were associated with enhanced IFNγ production within some hepatic cell populations but also with decreased numbers of IFNγ-positive cells. Surprisingly, IFNγ-knockout mice developed immunopathology and hemophagocytosis comparable to that seen in wild-type mice. However, IFNγ-knockout mice did not become anemic and had greater numbers of splenic erythroid precursors. IL-12 neutralization phenocopied disease in IFNγ-knockout mice. Interestingly, type I IFNs contributed to the severity of hypercytokinemia and weight loss, but their absence did not otherwise affect MAS manifestations.These data demonstrate that both fulminant MAS and hemophagocytosis can arise independently of IFNγ, IL-12, or type I IFNs. They also suggest that IFNγ-mediated dyserythropoiesis, not hemophagocytosis, is the dominant cause of anemia in fulminant TLR-9-induced MAS. Thus, our data establish a novel mechanism for the acute anemia of inflammation, but suggest that a variety of triggers can result in hemophagocytic disease.
1934	[Characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2010].	23547457	To analyze the characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2012.Polymerase chain reaction (PCR) was used to amplify the EBNA-3C, EBNA1 and LMP1 genes. The amplified products were sequenced directly and the sequences were analyzed by BioEdit 7. 0. 9 and MEGA 4. 0. 2.Type A EBV was detected in 98% samples. Nucleotide sequence analysis of the carboxy-terminal region of EBNA1 showed that Vvvl was deteted in 98% samples. DNA sequence analysis of LMP1 C-terminus indicated that China 1 was 90% in this study. There were no significant differences in the frequency of Vvv1 and China 1 between the IM and HLH samples (P = 1.00). Linkage analysis of EBV types, EBNA1 and LMP1 variants indicated that 90% of EBV type A was associated with EBNA1-Vvv1 variant and LMP1-China 1 variant in 40 cases. Full length of LMP1 gene was successfully amplified in 35 cases. Four Chinese groups (CG1-4) were identified. The percentage of CG1-CG4 were 85%, 6%, 6% and 3%, respectively.EBV type A is predominant in primary EBV infection in children in Beijing Area. EBNA1-Vvv1 and LMP1-China 1 variants were predominant genotypes in this area. There is a high linkage between EBNA1-Vvv1 variant and LMP1-China 1 variant. Four Chinese groups (CG1-4) were identified according to the full length of LMP1 gene and CG1 was the most prevalent.
1935	Extramacrochaetae imposes order on the Drosophila eye by refining the activity of the Hedgehog signaling gradient.	23536565	The compound eye of Drosophila melanogaster is configured by a differentiating wave, the morphogenetic furrow, that sweeps across the eye imaginal disc and transforms thousands of undifferentiated cells into a precisely ordered repetitive array of 800 ommatidia. The initiation of the furrow at the posterior margin of the epithelium and its subsequent movement across the eye field is controlled by the activity of the Hedgehog (Hh) signaling pathway. Differentiating photoreceptors that lie behind the furrow produce and secrete the Hh morphogen, which is captured by cells within the furrow itself. This leads to the stabilization of the full-length form of the zinc-finger transcription factor Cubitus interruptus (Ci(155)), the main effector of Hh signaling. Ci(155) functions as a transcriptional activator of a number of downstream targets, including decapentaplegic (dpp), a TGFβ homolog. In this report, we describe a mechanism that is in place within the fly retina to limit Hh pathway activity within and ahead of the furrow. We demonstrate that the helix-loop-helix (HLH) protein Extramacrochaetae (Emc) regulates Ci(155) levels. Loss of emc leads to an increase in Ci(155) levels, nuclear migration, apical cell constriction and an acceleration of the furrow. We find that these roles are distinct from the bHLH protein Hairy (H), which we show restricts atonal (ato) expression ahead of the furrow. Secondary furrow initiation along the dorsal and ventral margins is blocked by the activity of the Wingless (Wg) pathway. We also show that Emc regulates and cooperates with Wg signaling to inhibit lateral furrow initiation.
1936	Genome-wide microarrray analysis reveals roles for the REF-1 family member HLH-29 in ferritin synthesis and peroxide stress response.	23533643	In Caenorhabditis elegans, the six proteins that make up the REF-1 family have been identified as functional homologs of the Hairy/Enhancer of Split (HES) proteins. These transcription factors act in both Notch dependent and Notch-independent pathways to regulate embryonic events during development; however, their post-embryonic functions are not well defined. As a first step toward understanding how the REF-1 family works together to coordinate post-embryonic events, we used gene expression microarray analysis to identify transcriptional targets of HLH-29 in L4/young adult stage animals. Here we show that HLH-29 targets are genes needed for the regulation of growth and lifespan, including genes required for oxidative stress response and fatty acid metabolism, and the ferritin genes, ftn-1 and ftn-2. We show that HLH-29 regulates ftn-1 expression via promoter sequences upstream of the iron-dependent element that is recognized by the hypoxia inducible factor, HIF-1. Additionally, hlh-29 mutants are more resistant to peroxide stress than wild-type animals and ftn-1(RNAi) animals, even in the presence of excess iron. Finally we show that HLH-29 acts parallel to DAF-16 but upstream of the microphthalmia transcription factor ortholog, HLH-30, to regulate ftn-1 expression under normal growth conditions.
1937	Elevated Granzyme B in Cytotoxic Lymphocytes is a Signature of Immune Activation in Hemophagocytic Lymphohistiocytosis.	23524976	Patients with hemophagocytic lymphohistiocytosis (HLH) exhibit immune hyper-activation as a consequence of genetic defects in secretory granule proteins of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Murine models of HLH demonstrate significant activation of CTL as central to the disease pathogenesis, but evaluation of CTL and NK activation in children with HLH or inflammatory conditions is not well described. CD8 T cells only express granzyme B (GrB) following stimulation and differentiation into CTL; therefore, we reasoned that GrB expression may serve as a signature of CTL activation. It is unknown whether human NK cells are similarly activated in vivo, as marked by increased granule proteins. Perforin and GrB levels are measured in both CTL and NK cells by flow cytometry to diagnose perforin deficiency. We retrospectively compared GrB expression in blood samples from 130 children with clinically suspected and/or genetically defined HLH to age-matched controls. As predicted, CD8 expressing GrB cells were increased in HLH, regardless of genetic etiology. Remarkably, the GrB protein content also increased in NK cells in patients with HLH and decreased following immunosuppressive therapy. This suggests that in vivo activation of NK cells occurs in hyper-inflammatory conditions. We conclude that increased detection of GrB in CTL and NK are an immune signature for lymphocyte activation in HLH, irrespective of genetic subtype and may also be a useful measure of immune activation in other related conditions.
1938	The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues.	23523789	Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.
1939	Leishmania in HLH: a rare finding with significant treatment implications.	23511497	Hemophagocytic lymphohistiocytosis (HLH) associated with visceral leishmaniasis (VL) is a very rare phenomenon. We report the first known North American case in a 21 month old boy. He was initially diagnosed with Epstein Barr virus (EBV) triggered HLH and treated with the international treatment protocol, HLH-2004. Stem cell transplant was planned due to repeated reactivations of disease, but his pretransplant bone marrow revealed an unexpected protozoan-Leishmania donovani. Treatment with liposomal amphotericin B led to resolution of all manifestations of HLH. We discuss how the clinical and laboratory features of both entities can closely mimic each other and are extremely difficult to differentiate. This case also raises the question of whether to screen all children with suspected HLH for Leishmania in a non endemic area.
1940	Positions of the glycans in molluscan hemocyanin, determined by fluorescence spectroscopy.	23494164	Molluscan hemocyanins are glycoproteins with different quaternary and carbohydrate structures. It was suggested that the carbohydrate chains of some Hcs are involved in their antiviral and antitumor effect, as well in the organization of the quaternary structure of the molecules. Using a well-known complex for saccharide sensing, positions and access to the carbohydrate chains in the native hemocyanins from Rapana venosa (RvH) and Helix lucorum (HlH) and also their structural subunits (RvH1, RvH2 and βcHlH) and functional units (FUs) were analysed by fluorescence spectroscopy and circular dichroism. Almost no effect was observed in the fluorescence emission after titration of the complex with native RvH and HlH due to lack of free hydroxyl groups which are buried in the didecameric form of the molecules. Titration with the structural subunits βcHlH and RvH2, increasing of the emission indicates the presence of free hydroxyl groups compared to the native molecules. Complex titration with the structural subunit βc-HlH of H. lucorum Hcs leads to a 2.5 fold increase in fluorescence intensity. However, the highest emission was measured after titration of the complex with FU βcHlH-g. The result was explained by the structural model of βcHlH-g showing the putative position of the glycans on the surface of the molecule. The results of the fluorescent measurements are in good correlation with those of the circular dichroism data, applied to analyse the effect of titration on the secondary structure of the native molecules and functional units. The results also support our previously made suggestion that the N-linked oligosaccharide trees are involved in the quaternary organization of molluscan Hcs.
1941	Computational modelling to optimize the hybrid configuration for hypoplastic left heart syndrome.	23487529	Hybrid palliation for hypoplastic left heart syndrome (HLHS) is associated with mortality and late ventricular dysfunction. Increased ventricular workload and coronary perfusion limitation may be the important factors. Using mathematical modelling, this study investigated the effects of differing hybrid configurations on the demands on this single ventricle circulation.A multicompartmental Windkessel model of hybrid HLH-aortic atresia circulation was adopted, with a time-varying elastance representing ventricular functionality. The effects of diameter increases in bilateral pulmonary artery bandings (PABs) (+0.5, 2.5-4 mm) and ductal stent (+1, 4-10 mm) on cardiovascular haemodynamics, systemic oxygenation and ventricular energetics were assessed.Simulations showed that an increase in PAB diameter of 2.5-4 mm resulted in an increased Q (0.61-2.66), and diastolic stent backflow (-0.2 to -0.78 l/min) with reduced systemic perfusion (0.82-0.77 l/min) and diastolic pressures (48.3-41.2 mmHg). Arterial and venous saturations increased, SaO2 (%) was 62-88 and SvO(2) 41-65. To maintain mean systemic pressures, substantial increases in cardiac output (1.3-2.8 l/min) and ventricular stroke work (576-1360 mmHg ml) were required. A decrease in the ductal stent diameter over the range 10-7 mm had a negligible haemodynamic effect: reduced systemic systolic pressure (77-72 mmHg) and increase in ventricular stroke work (781-790 mmHg ml). When the ductal diameter was restricted to <7 mm, it resulted in a significant reduced systemic flow and increased stroke work. Optimal hybrid configuration was defined at PAB 3 mm and ductal stent ≥7 mm.In this model, increasing the PAB diameter, or a stent diameter <7 mm, substantially increased single ventricle workload and reduced systemic perfusion and diastolic pressure. This may compromise myocardial oxygen demand-supply, particularly in the setting of retrograde-dependent coronary perfusion.
1942	Risk perception of prescription drugs: results of a survey among experts in the European regulatory network.	23478076	Experts are perceived to be veridical and to focus only on objective data when evaluating risk. Only a few research studies have attempted to characterize the subjectivity in risk evaluation among experts.The hypothesis of this study is that expert evaluation of a pharmaceutical drug can be partly explained by dimensions that describe the drug and by individual characteristics.Seventy-five medical assessors in 9 EU countries evaluated a list of 28 pharmaceutical drugs using 4 scales: risk, benefit, seriousness of harm, and patients' knowledge of the risk. They were also given a mock "clinical dossier" and asked to rate it on 8 dimensions: risk, benefit, worry, magnitude of the exposure, scientific knowledge of the risk, familiarity of the risk, ethical concerns, and risk acceptability.Female assessors perceived significantly higher benefits than men for a large number of the 28 drugs. Principal component analysis of the ratings for the clinical dossiers revealed 2 underlying components: seriousness of harm and scientific evidence. A regression model predicting the risk perception of the drug showed that the variables seriousness of harm (benefit, worry, magnitude of exposure, ethical concerns, and risk acceptability), years of regulatory experience, gender, and type of drug explained 54% of the variability among assessors.Assessors' view of the risks associated with pharmaceutical drugs is influenced by worry for patient safety, magnitude of patient exposure, and ethical concerns. These dimensions may influence their perceptions of benefit and risk acceptability. Senior assessors are more risk averse than junior assessors, and female assessors seem to be sensitive to the promise of benefit from medicines and consequently may be less risk averse than male assessors.
1943	Gene expression profiles of cumulus cells obtained from women treated with recombinant human luteinizing hormone + recombinant human follicle-stimulating hormone or highly purified human menopausal gonadotropin versus recombinant human follicle-stimulating hormone alone.	23472943	To evaluate cumulus cell (CC) expression profile modulation after different stimulation protocols.CCs transcriptome variations were evaluated by microarray in patients undergoing different treatments for ovarian stimulation, namely, r-hLH + r-hFSH and hp-hMG, compared with a control group treated with r-hFSH.Healthy patients undergoing assisted reproduction protocols.Sixteen healthy women with regular cycles and tubal disease or unexplained infertility.Four patients received hp-hMG, four received r-hFSH + r-hLH, and eight received r-hFSH daily. Aspiration of the oocytes was performed 36 hours after hCG administration. Only samples derived from cumulus-oocyte complexes containing mature oocytes showing polar body were processed.Comparison of genes differentially expressed in both treatment groups with the use of a hierarchic clustering analysis.Data clustering analysis allowed detection of four clusters containing genes differentially expressed in both treatment groups compared with control. Functional analysis of the affected transcripts revealed genes involved in oocyte development and maturation.r-hLH and hCG, though acting on the same receptor, produce a differential activation of intracellular pathways. It can be hypothesized that this effect depends on their different structures and specific binding affinity for the receptor.
1944	Two acute myeloblastic leukemia cases concomitant with hemophagocytic lymphohistiocytosis and review of the literature.	23462611	We present two patients with acute myeloblastic leukemia (FAB AML-M7 and AML-M2) complicated by hemophagocytic lymphohistiocytosis (HLH) and relevant literature review. To our knowledge, our first case is the first and youngest patient having AML-M7 associated with HLH reported in the literature. Our cases and cases in the literature highlight the high mortality rate of leukemia associated with HLH and the need for further investigation for the most appropriate therapy.
1945	A cell-penetrating peptide suppresses the hypoxia inducible factor-1 function by binding to the helix-loop-helix domain of the aryl hydrocarbon receptor nuclear translocator.	23454269	The heterodimeric hypoxia inducible factor-1 (HIF-1) complex is composed of the hypoxia inducible factor-1 alpha (HIF-1α) and the aryl hydrocarbon receptor nuclear translocator (ARNT). Activation of the HIF-1 function is essential for tumor growth and metastasis. We previously showed that transfection of a plasmid containing an ARNT-interacting peptide (Ainp1) cDNA suppresses the HIF-1 signaling in Hep3B cells. Here we generated TAT fusion of the Ainp1 peptide (6His-TAT-Ainp1) to determine whether and how the Ainp1 peptide suppresses the HIF-1 function. The bacterially expressed 6His-TAT-Ainp1 was purified under denatured condition and then refolded by limited dialysis. The refolded 6His-TAT-Ainp1 interacts with the helix-loop-helix (HLH) domain of ARNT in a similar fashion as the native 6His-Ainp1. 6His-TAT-Ainp1 colocalizes with ARNT in the nucleus of HeLa and Hep3B cells after protein transduction. The transduced protein reaches the maximum intracellular levels within 2 h while remains detectable up to 96 h in HeLa cells. At 2 μM concentration, 6His-TAT-Ainp1 is not cytotoxic in HeLa cells but suppresses the cobalt chloride-activated, hypoxia responsive enhancer-driven luciferase expression in a dose-dependent manner. In addition, it decreases the cobalt chloride-dependent induction of the HIF-1 target genes at both the message (vascular endothelial growth factor and aldolase C) and protein (carbonic anhydrase IX and glucose transporter 1) levels. The protein levels of HIF-1α and ARNT are not altered in the presence of 6His-TAT-Ainp1. In summary, we provided evidence to support that the Ainp1 peptide directly suppresses the HIF-1 function by interacting with the ARNT HLH domain, and in turn interfering with the heterodimerization of HIF-1α and ARNT.
1946	The risk of hemophagocytic lymphohistiocytosis in Hermansky-Pudlak syndrome type 2.	23403622	Genetic disorders of lymphocyte cytotoxicity predispose patients to hemophagocytic lymphohistiocytosis (HLH). Reduced lymphocyte cytotoxicity has been demonstrated in Hermansky-Pudlak syndrome type 2 (HPS2), but only a single patient was reported who developed HLH. Because that patient also carried a potentially contributing heterozygous RAB27A mutation, the risk for HLH in HPS2 remains unclear. We analyzed susceptibility to HLH in the pearl mouse model of HPS2. After infection with lymphocytic choriomeningitis virus, pearl mice developed all key features of HLH, linked to impaired virus control caused by a moderate defect in CTL cytotoxicity in vivo. However, in contrast to perforin-deficient mice, the disease was transient, and all mice fully recovered and controlled the infection. An additional heterozygous Rab27a mutation did not aggravate the cytotoxicity defect or disease parameters. In the largest survey of 22 HPS2 patients covering 234 patient years, we identified only 1 additional patient with HLH and 2 with incomplete transient HLH-like episodes, although cytotoxicity or degranulation was impaired in all 16 patients tested. HPS2 confers a risk for HLH that is lower than in Griscelli or Chediak-Higashi syndrome, probably because of a milder defect in cytotoxicity. Preemptive hematopoietic stem cell transplantation does not appear justified in HPS2.
1947	Fulminant hemophagocytic lymphohistiocytosis secondary to a reactivated EBV infection: a case report.	2339823723682633	Hemophagocytic lymphohistiocytosis (HLH) is an aggressive inflammatory syndrome that results from inappropriate activation of the immune system. HLH has a high mortality if not treated. We describe a case of a fulminant HLH, associated with a reactivation of an EBV infection. The patient responded well to steroid treatment.
1948	Expression pattern of id proteins in medulloblastoma.	23397264	Inhibitor of DNA binding or inhibitor of differentiation (Id) proteins are up regulated in a variety of neoplasms, particularly in association with high-grade, poorly differentiated tumors, while differentiated tissues show little or no Id expression. The four Id genes are members of the helix-loop-helix (HLH) family of transcription factors and act as negative regulators of transcription by binding to and sequestering HLH complexes. We tested the hypothesis that Id proteins are overexpressed in medulloblastoma by performing immunohistochemistry using a medulloblastoma tissue microarray with 45 unique medulloblastoma and 11 normal control cerebella, and antibodies specific for Id1, Id2, Id3, and Id4. A semi-quantitative staining score that took staining intensity and the proportion of immunoreactive cells into account was used. Id1 was not detected in normal cerebella or in medulloblastoma cells, but 78 % of tumors showed strong Id1 expression in endothelial nuclei of tumor vessels. Id2 expression was scant in normal cerebella and increased in medulloblastoma (median staining score: 4). Id3 expression was noted in some neurons of the developing cerebellar cortex, but it was markedly up regulated in medulloblastoma (median staining score: 12) and in tumor endothelial cells. Id4 was not expressed in normal cerebella or in tumor cells. Id2 or Id3 overexpression drove proliferation in medulloblastoma cell lines by altering the expression of critical cell cycle regulatory proteins in favor of cell proliferation. This study shows that Id1 expression in endothelial cells may contribute to angiogenic processes and that increased expression of Id2 and Id3 in medulloblastoma is potentially involved in tumor cell proliferation and survival.
1949	Cytokine profiles in children with primary Epstein-Barr virus infection.	23382108	Primary Epstein-Barr virus (EBV) infection causes infectious mononucleosis and hemophagocytic lymphohistiocytosis (HLH) in children, where EBV infects B and CD8(+) T cells, respectively. We measured pro-inflammatory and anti-inflammatory cytokines in both diseases. Significantly higher concentrations of various mediators, including interferon-γ, neopterin, interleukin (IL)-6, IL-10, IL-18, and heme oxygenase-1, were observed in EBV-HLH. Because of their similarity to the profile of familial HLH, this profile was likely a consequence of HLH, but not ectopic infection. TNF-α levels were elevated in both diseases. Elevation of those mediators may contribute to the disease pathogenesis of EBV-HLH by activating and inhibiting host immune responses.
1950	GDF15-mediated upregulation of ferroportin plays a key role in the development of hyperferritinemia in children with hemophagocytic lymphohistiocytosis.	23335088	Growth differentiation factor 15 (GDF15), a divergent TGFβ superfamily, has recently been implicated in the modulation of iron homeostasis, acting as an upstream negative regulator of hepcidin, the key iron regulatory hormone produced primarily by hepatocytes. However, little is known about possible roles that GDF15 might play in the regulation of iron homeostasis and development of hyperferritinemia in children with hemophagocytic lymphohistiocytosis (HLH).We compared serum GDF15 level and mRNA expressions of GDF15 and key molecules of iron metabolism, and made correlations between their expressions in children with HLH and control children.Serum GDF15 level was remarkably higher in HLH group than that in controls, with median serum concentration of 1,700 and 260 pg/ml, respectively (P < 0.001). In addition, GDF15 mRNA was significantly upregulated but independent of hypoxia-inducible factor-mediated oxygen signaling pathway. More importantly, GDF15 induction was positively correlated to upregulation of ferroportin, the only cellular iron exporter, and to upregulation of ferritin heavy chain.Our study suggests that GDF15 induction helps suppress further activation of macrophages in stressful physiologic states as HLH, and is intimately implicated in the development of hyperferritinemia by modulating the hepcidin-ferroportin axis, resulting in enhanced ferroportin-mediated iron efflux.
1951	ATBS1 INTERACTING FACTORs negatively regulate Arabidopsis cell elongation in the triantagonistic bHLH system.	23333962	We recently demonstrated that cell elongation in plants is regulated by a triantagonistic bHLH system, in which three bHLH proteins, Activator of Cell Elongation 1 (ACE1), Arabidopsis ILI1 binding BHLH 1 (AtIBH1) and Paclobutrazol Resistance 1 (PRE1), competitively regulate the expression of genes for cell elongation. Here we show that ATBS1 Interacting Factor 2 (AIF2), AIF3 and AIF4 interact with PRE1 and ACE1, similar to AtIBH1, and also negatively regulate cell elongation in the triantagonistic bHLH system. The expression of each AIF is constitutive or induced by light, but AtIBH1 expression is dependent on BR signaling and developmental phase. These results indicate that AIFs and AtIBH1 may play different roles in cell elongation in different signaling pathways.
1952	Differentiating macrophage activation syndrome in systemic juvenile idiopathic arthritis from other forms of hemophagocytic lymphohistiocytosis.	23333131	To identify measures distinguishing macrophage activation syndrome (MAS) in systemic juvenile idiopathic arthritis (sJIA) from familial hemophagocytic lymphohistiocytosis (FHL) and virus-associated hemophagocytic lymphohistiocytosis (VA-HLH) and to define appropriate cutoff values. To evaluate suggested dynamic measures differentiating MAS in patients with sJIA from sJIA flares.In a cohort of patients referred for evaluation of hemophagocytic lymphohistiocytosis, we identified 27 patients with sJIA and MAS (MAS/sJIA) fulfilling the criteria of the proposed preliminary diagnostic guideline for the diagnosis of MAS in sJIA. Ten measures at diagnosis were compared between the MAS/sJIA group and 90 patients with FHL and 42 patients with VA-HLH, and cutoff values were determined. In addition, 5 measures were analyzed for significant change from before MAS until MAS diagnosis.Neutrophil count and C-reactive protein were significantly higher in patients with MAS/sJIA compared with patients with FHL and patients with VA-HLH, with 1.8×10(9)/L neutrophils (sensitivity 85%, specificity 83%) and 90 mg/L C-reactive protein (74%, 89%) as cutoff values. Soluble CD25<7900 U/L (79%, 76%) indicated MAS/sJIA rather than FHL/VA-HLH. Platelet (-59%) and white blood cell count (-46%) displayed a significant decrease, and neutrophil count (-35%) and fibrinogen (-28%) showed a trend during the development of MAS. However, a substantial portion of patients had values at diagnosis of MAS within or above the normal range for white blood cells (84%), neutrophils (77%), platelets (26%), and fibrinogen (71%).Readily available measures can rapidly differentiate between MAS/sJIA and FHL/VA-HLH. The findings substantiate that a decline of measures may facilitate the distinction of MAS from flares of sJIA.
1953	Diagnosing and treating hemophagocytic lymphohistiocytosis in the tropics: systematic review from the Indian subcontinent.	23331391	Hemophagocytic lymphohistiocytosis (HLH) is a catastrophic syndrome of unrestrained immune activation. Evaluation and management of HLH in the tropics is challenging.To examine the reported etiologies and management of HLH reported from the sub-continent.Systematic review of all published cases from the Indian sub-continent.We found only 156 published cases of HLH from the sub-continent. HLH was reported from the immediate perinatal period to 46 years of age. Infection-associated HLH (IAHS) constituted 46.8% of all cases of HLH (44% and 51% in children and adults respectively). In adults, tropical infections triggered 51% of these cases of IAHS. Steroids were used in 47% of children and 10% of adults. Etoposide and/or cyclosporine were used in 8% children and 8% of adults only. Intravenous immunoglobulin was used in another 30% of children and 4% of the adults. HLH-related mortality occurred in 31.8% and 28% of children and adults respectively.HLH is under-reported in the sub-continent and has high mortality. Cyclosporine and etoposide are seldom administered early despite diagnosis of HLH. Larger cohorts with IAHS triggered by tropical infections are urgently needed to understand its natural history and implications of this differing prescription pattern on mortality.
1954	Increased complications and morbidity in children with hemophagocytic lymphohistiocytosis undergoing hematopoietic stem cell transplantation.	23331022	Hematopoietic stem cell transplantation (HSCT) is the only curative option for patients with primary hemophagocytic lymphohistiocytosis (HLH) and for patients with secondary HLH who fail to respond to therapy.Retrospective study of HSCT for HLH with focus on complications and outcome.Eighteen children (10 males), with a median age of 1.2 yr (5 months-16 yr), received HSCT for HLH. Fourteen children had primary HLH. Four children underwent transplant while not in remission. Sixteen received myeloablative and two received reduced intensity conditioning regimen. A high incidence of complications was found: 13 (72%) children had 22 episodes of culture-proven infections; seven (38%) had hepatic veno-occlusive disease; nine (50%) developed respiratory complications; and nine (50%) required intensive care unit admission. Eight children had acute graft-versus-host disease (GVHD), and three developed chronic GVHD. Three patients died from multi-organ failure before day +100, and another patient died from pulmonary hemorrhage after day 100. Three patients failed to engraft (two developed recurrent HLH and died from complications after a second HSCT). Three of four children not in remission at the time of transplantation died. Actuarial survival at three yr was 61%.HSCT for HLH carries significant risks with high infection, organ dysfunction, and ICU admissions rates.
1955	Central nervous system (CNS) involvement is a critical prognostic factor for hemophagocytic lymphohistiocytosis.	23320006	Hemophagocytic lymphohistiocytosis (HLH) is a rare multisystem disorder that frequently involves the central nervous system (CNS). We compared the clinical characteristics, treatment, and prognosis of patients with HLH according to the degree of CNS involvement.The clinical manifestations, initial laboratory data, treatment, and outcomes for 50 patients diagnosed with HLH and treated at Asan Medical Center between January 1995 and August 2011 were retrospectively reviewed and analyzed. CNS involvement was defined as the presence of neurological symptoms or an elevated white blood cell (WBC) count in the cerebrospinal fluid (CSF).Among these 50 patients, 23 (46%) developed CNS disease. Among patients with CNS disease, 19 had neurological symptoms, including seizures, altered consciousness, facial palsy, dysarthria, and dysphagia. Four patients had elevated CSF WBC counts without neurological symptoms. Twelve patients had abnormal brain imaging results, including high signal intensity lesions on T2-weighted magnetic resonance imaging (MRI) findings, ventriculomegaly, hemorrhage, atrophy, and leptomeningeal enhancement. Patients with CNS disease had lower ferritin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels as well as reduced 5-year survival as compared to those without CNS disease.CNS involvement is common among patients with HLH. Overall, patients with CNS disease achieve poorer outcomes than patients without CNS involvement. To improve outcomes, physicians must carefully monitor the neurological manifestations in patients with HLH and administer the appropriate course of intensified chemotherapy to patients with CNS disease.
1956	Transcriptional dynamics elicited by a short pulse of notch activation involves feed-forward regulation by E(spl)/Hes genes.	23300480	Dynamic activity of signaling pathways, such as Notch, is vital to achieve correct development and homeostasis. However, most studies assess output many hours or days after initiation of signaling, once the outcome has been consolidated. Here we analyze genome-wide changes in transcript levels, binding of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and RNA Polymerase II (Pol II) immediately following a short pulse of Notch stimulation. A total of 154 genes showed significant differential expression (DE) over time, and their expression profiles stratified into 14 clusters based on the timing, magnitude, and direction of DE. E(spl) genes were the most rapidly upregulated, with Su(H), Pol II, and transcript levels increasing within 5-10 minutes. Other genes had a more delayed response, the timing of which was largely unaffected by more prolonged Notch activation. Neither Su(H) binding nor poised Pol II could fully explain the differences between profiles. Instead, our data indicate that regulatory interactions, driven by the early-responding E(spl)bHLH genes, are required. Proposed cross-regulatory relationships were validated in vivo and in cell culture, supporting the view that feed-forward repression by E(spl)bHLH/Hes shapes the response of late-responding genes. Based on these data, we propose a model in which Hes genes are responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining the critical functions these key regulators play in many developmental and disease contexts.
1957	Recurrent macrophage activation syndrome associated with heterozygous perforin W374X gene mutation in a child with systemic juvenile idiopathic arthritis.	23274377	Recurrent macrophage activation syndrome (MAS) is rarely reported.To describe recurrent MAS in a 2.5-year-old girl with systemic juvenile idiopathic arthritis and heterozygous perforin mutation, which may have a role in the patient's first recurrence despite use of the HLH-2004 treatment protocol.In the presented case, MAS was initially controlled after the addition of etoposide to the treatment regimen. However, recurrence occurred 6.5 months after cessation of the HLH-2004 protocol. Subsequent recurrences may have occurred because of the family's noncompliance with treatment.The patient's extremely high serum ferritin level (267,054 ng/mL) and the recurrent course of MAS may have been because of the coexistence of juvenile idiopathic arthritis and heterozygous perforin W374X mutation. We suggest to search for mutations in HLH genes in recurrent MAS cases.
1958	[Secondary hemophagocytic syndromes].	25775810	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening extreme whole body inflammatory state. It results from the pathological hyperactivation of the immune system, because of congenital or acquired abnormalities of cytotoxicity and NK or T cells. Uncontrolled stimulation of lymphocytes and macrophages lead to hypercytokinemia, organ infiltration by these cells and multiple organ failure. There are genetic HLH and secondary HLH, associated with infections, autoimmune disorders, malignancies. The frequency of the secondary form is difficult to estimate because of the wrong and difficult diagosis. The clinical course is often insidious and nonspecific. Symptoms are varied. The most important are: unremitting fever, hepatosplenomegaly. Generalized edema, rash, lymphadenopathy may occur. Liver failure, respiratory, circulatory and multiple organ failure could develop in a very short time. Most common abnormalities in additional tests are: cytopenias, hypofibrinogenaemia, hypertriglyceridaemia, hyperferritinaemia, hypertransaminasaemia, elevated parameters of inflammation (excepting lowering erythrocyte sedimentaion rate). Criteria for diagnosis and therapeutic protocols referto the genetic forms of HLH. Currently, there are no guidelines for secondary HLH. Diagnostic and therapeutic difficulties also arise from clinical picture, similar as in the systemic inflammatory response syndrome, sepsis, multiple organ dysfunction syndrome. We present the clinical presentation, diagnostic pitfalls and treatment of secondary HLH, based on a review of the current literature and our own observations.
1959	Acute liver dysfunction as a presentation of haemophagocytic lymphohistiocytosis.	24670591	Haemophagocytic lymphohistiocytosis (HLH) is an uncommon disorder of histiocyte function which presents with fever, cytopaenias and end-organ dysfunction. We report the case of a 62-year-old man with acute liver dysfunction as an initial presentation of HLH.
1960	Secondary hemophagocytic lymphohistiocytosis in adults: an update on diagnosis and therapy.	23271259	Hemophagocytic lymphohistiocytosis (HLH), also known as hemophagocytic syndrome (HPS), is a rare, life-threatening, hematologic disorder manifested by clinical findings of extreme inflammation and unregulated immune activation. In both its congenital (primary) and adult (secondary) forms, it is most often characterized by fevers, hepatomegaly or splenomegaly, and bi- or trilineage cytopenias. In addition, elevated liver enzymes, hyperferritinemia, hypertriglyceridemia, and hypofibrinogenemia are commonly seen in HLH patients. A high index of suspicion is necessary for early diagnosis. Furthermore, a thorough diagnostic evaluation is necessary, and prompt treatment of the underlying causes is key in order to prevent irreversible tissue damage. Here we discuss the clinical signs, diagnosis, and treatments associated with this rare and potentially lethal disorder as manifested in adults.
1961	Fetal diagnosis of hypoplastic left heart, associations and outcomes in the current era.	23250856	Hypoplastic left heart (HLH) is one of the most common forms of cardiac abnormality detectable during gestation by fetal echocardiography. Antenatal diagnosis allows for appropriate counseling and time to consider treatment options. We report the actual outcome data after fetal diagnosis of HLH.Retrospective analysis of the outcome in all cases with HLH from 1994 - 2011 presenting in fetal life at two tertiary referral centers for prenatal diagnosis and pediatric cardiology.105 cases were included and the overall survival is 40.9 % (43/105) after prenatal diagnosis. There was an 81.1 % survival rate in infants undergoing surgery and a 64.1 % survival rate from an intention-to-treat position. Two neonates died due to tamponade and cardiac arrest following balloon septostomy and one neonate from sepsis before surgery. Extracardiac anomalies occurred in three fetuses, and karyotype anomalies in seven fetuses (18.9 %). In 4 of 5 babies born with additional extracradiac or karyotype anomalies, parents opted for compassionate care. The first had trisomy 13, the second had trisomy 18, the third neonate presented with spina bifida, and the fourth presented with hydronephrosis and pulmonary atresia. Termination of pregnancy took place in 17 cases (16.1 %).Thorough antenatal evaluation should include karyotyping, detailed extracardiac and intracardiac assessment to accurately predict the risks of surgery. Prenatal counseling might be modified after the exclusion of additional anomalies. These data provide up-to-date information for parental counseling.ZIEL:: Das hypoplastische Linksherz (HLH) ist eine der häufigsten angeborenen Herzfehlbildungen, die durch fetale Echokardiografie in der Schwangerschaft diagnostiziert werden können. Die pränatale Diagnose erlaubt eine detaillierte Beratung der Eltern. In der vorliegenden Arbeit werden aktuelle Outcome-Daten nach pränataler Diagnose eines HLH vorgelegt.Retrospektive Analyse aller Fälle mit einer pränatalen Diagnose eines HLH zwischen 1994 – 2011 an 2 tertiären Zentren für pränatale Diagnostik und pädiatrischer Kardiologie.105 Fälle wurden eingeschlossen. Die Gesamtüberlebensrate nach pränataler Diagnose ist 40.9 % (43/105). Das Überleben nach operativer Therapie ist 81,1 % und 64,1 % in den Fällen mit beabsichtigter Therapie. Vor der operativen Therapie verstarben 2 Neugeborene an einer Herzbeuteltamponade mit Herzstillstand nach einer Ballon-Septostomie und ein Neugeborenes verstarb an einer Sepsis. Extrakardiale Anomalien wurden in 3 Fällen, chromosomale Anomalien in 7 Fällen (18.9 %) diagnostiziert. In 4 von 5 Neugeborenen mit extrakardialen und chromosomalen Anomalien entschieden sich die Eltern für eine Compassionate Care, in einem Fall mit Trisomie 13 und in einem weiteren Fall mit Trisomie 18, in einem weiteren Fall mit Spina bifida und ein weiteres Neugeborenes mit Hydronephrose und Pulmonalklappenatresie. Ein Schwangerschaftsabbruch wurde in 17 Fällen (16,1 %) registriert.Die exakte pränatale Untersuchung bei Vorliegen eines HLH sollte eine Karyotypisierung und die Analyse extrakardialer und intrakardialer assoziierter Anomalien beinhalten, um das Risiko des operativen Eingriffs besser einschätzen zu können. Mit Fortgang der Schwangerschaft und Ausschluss zusätzlicher Anomalien kann die Beratung angepasst werden. Die vorgestellten Daten können als Grundlage der elterlichen Beratung herangezogen werden.
1962	A clinicopathological analysis of 26 patients with infection-associated haemophagocytic lymphohistiocytosis and the importance of bone marrow phagocytosis for the early initiation of immunomodulatory treatment.	23243150	To analyse the clinicopathological presentation, outcome and importance of bone marrow haemophagocytosis in patients with infection-associated haemophagocytic lymphohistiocytosis (IA-HLH) in a tertiary care hospital in Northern India.Between January 2007 and December 2009, 26 consecutive patients meeting the diagnostic criteria for IA-HLH, based on the HLH2004 protocol of the Histiocyte Society, were followed up for between 12 and 34 months (median 20 months).IA-HLH was diagnosed in three of the five patients who died 5-6 weeks after the onset of the illness, whereas diagnosis in the remaining group was made a median of 2 weeks after the onset of the illness. The predominant presenting features were fever (100%), hepatomegaly (69%), splenomegaly (58%) and anaemia (96%). All patients showed >3% haemophagocytosis on bone marrow studies-in four cases after serial aspiration/biopsies. Twenty-one (80.8%) cases were non-fatal and five (19.2%) patients died. The non-fatal cases included eight (38.1%) cases of viral infection, seven (33.3%) bacterial infections, two (9.6%) fungal and four (19.0%) protozoal infections; whereas four (80%) bacterial infections and one (20%) viral infection were associated with the fatal cases. The mean of the nadir blood counts of white blood cells, absolute neutrophil counts and platelets; the mean of all the peak biochemical parameters of liver function tests, lactate dehydrogenase and ferritin and the lowest fibrinogen values before treatment, differed significantly (p<0.05) between the non-fatal and the fatal group, being worse in the latter.IA-HLH is important because it can obscure the typical clinical features of the underlying primary disease, thus delaying the diagnosis and having a negative effect on the outcome. Although bone marrow haemophagocytosis is not a mandatory diagnostic criterion, we found it to be a useful tool together with biochemical parameters for early recognition of HLH, especially in developing countries lacking molecular and flow laboratories. The severity of pancytopenia and derangement in biochemical markers were significantly higher in the patients who died.
1963	A competitive peptide inhibitor KIDARI negatively regulates HFR1 by forming nonfunctional heterodimers in Arabidopsis photomorphogenesis.	23224238	Dynamic dimer formation is an elaborate means of modulating transcription factor activities in diverse cellular processes. The basic helix-loop-helix (bHLH) transcription factor LONG HYPOCOTYL IN FAR-RED 1 (HFR1), for example, plays a role in plant photomorphogenesis by forming non-DNA binding heterodimers with PHYTOCHROMEINTERACTING FACTORS (PIFs). Recent studies have shown that a small HLH protein KIDARI (KDR) negatively regulates the HFR1 activity in the process. However, molecular mechanisms underlying the KDR control of the HFR1 activity are unknown. Here, we demonstrate that KDR attenuates the HFR1 activity by competitively forming nonfunctional heterodimers, causing liberation of PIF4 from the transcriptionally inactive HFR1-PIF4 complex. Accordingly, the photomorphogenic hypocotyl growth of the HFR1-overexpressing plants can be suppressed by KDR coexpression, as observed in the HFR1-deficient hfr1-201 mutant. These results indicate that the PIF4 activity is modulated through a double layer of competitive inhibition by HFR1 and KDR, which could in turn ensure fine-tuning of the PIF4 activity under fluctuating light conditions.
1964	A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis.	2322159823221594	Environmental and endogenous signals, including light, temperature, brassinosteroid (BR), and gibberellin (GA), regulate cell elongation largely by influencing the expression of the paclobutrazol-resistant (PRE) family helix-loop-helix (HLH) factors, which promote cell elongation by interacting antagonistically with another HLH factor, IBH1. However, the molecular mechanism by which PREs and IBH1 regulate gene expression has remained unknown. Here, we show that IBH1 interacts with and inhibits a DNA binding basic helix-loop-helix (bHLH) protein, HBI1, in Arabidopsis thaliana. Overexpression of HBI1 increased hypocotyl and petiole elongation, whereas dominant inactivation of HBI1 and its homologs caused a dwarf phenotype, indicating that HBI1 is a positive regulator of cell elongation. In vitro and in vivo experiments showed that HBI1 directly bound to the promoters and activated two EXPANSIN genes encoding cell wall-loosening enzymes; HBI1's DNA binding and transcriptional activities were inhibited by IBH1, but the inhibitory effects of IBH1 were abolished by PRE1. The results indicate that PREs activate the DNA binding bHLH factor HBI1 by sequestering its inhibitor IBH1. Altering each of the three factors affected plant sensitivities to BR, GA, temperature, and light. Our study demonstrates that PREs, IBH1, and HBI1 form a chain of antagonistic switches that regulates cell elongation downstream of multiple external and endogenous signals.
1965	[Smog chamber simulation of ozone formation from atmospheric photooxidation of propane].	23213871	Atmospheric photochemical reactions of propane and NO, were simulated with a self-made smog chamber. The effects of relative humidity (RH) and [C3H8]0/[NOx]0 ratio on ozone formation were studied. The results showed that both the maximum ozone concentration and the maximum value of incremental reactivity (IRmax) of propane decreased linearly with increasing RH. Under lower RH conditions, the occurrence time of peak ozone concentration was about 22 h after the beginning of reaction, and IRmax varied from 0.0231 to 0.0391, while under higher RH conditions the occurrence time of peak ozone concentration was 16 h, and IRmax ranged from 0.0172 to 0.0320. During the 20 h of reaction, within the first 12 h RH did not significantly affect the yield of acetone, whereas after 12 h the lower RH condition could lead to relatively greater amount of acetone. During the first 4-20 h of experiments, acetone concentrations ranged from 153 x 10(-9) to 364 x 10(-9) at 17% RH and from 167 x 10(-9) to 302 x 10(-9) at 62% RH, respectively. Maximum ozone concentrations decreased with increasing [C3H8]0/[NOx]0 ratio and a better negative linear relationship between them was obtained under the lower RH conditions. The smog chamber data and the results from simulation of the C3H8-NOx reactions using the sub-mechanism of MCM were compared, and a significant deviation was found between these two results.
1966	HLH-29 regulates ovulation in C. elegans by targeting genes in the inositol triphosphate signaling pathway.	23213416	The reproductive cycle in the nematode Caenorhabditis elegans depends in part on the ability of the mature oocyte to ovulate into the spermatheca, fuse with the sperm during fertilization, and then exit the spermatheca as a fertilized egg. This cycle requires the integration of signals between the germ cells and the somatic gonad and relies heavily on the precise control of inositol 1,4,5 triphosphate (IP(3))levels. The HLH-29 protein, one of five Hairy/Enhancer of Split (HES) homologs in C. elegans, was previously shown to affect development of the somatic gonad. Here we show that HLH-29 expression in the adult spermatheca is strongly localized to the distal spermatheca valve and to the spermatheca-uterine valve, and that loss of hlh-29 activity interferes with oocyte entry into and egg exit from the spermatheca. We show that HLH-29 can regulate the transcriptional activity of the IP(3) signaling pathway genes ppk-1, ipp-5, and plc-1 and provide evidence that hlh-29 acts in a genetic pathway with each of these genes. We propose that the HES-like protein HLH-29 acts in the spermatheca of larval and adult animals to effectively increase IP(3) levels during the reproductive cycle.
1967	Diagnostic evaluation of patients with suspected haemophagocytic lymphohistiocytosis.	23206255	Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome characterized by severely disturbed immune homeostasis. It can affect all age groups. Diagnostic evaluation of the patient with suspected HLH has to address three main questions: (i) does the patient have HLH? There is no simple diagnostic test, but a number of clinical and laboratory criteria define this clinical syndrome. (ii) Can a trigger be identified? A variety of infections, malignant or autoimmune diseases can contribute to the disturbed immune homeostasis with important consequences for treatment. (iii) Does the patient suffer from a genetic disease predisposing to HLH? Recent advances in the understanding of the genetic and pathophysiological basis of HLH have enabled a better and more rapid answer to this question, which is relevant for prognosis and the decision to perform haematopoietic stem cell transplantation. This review summarizes the current diagnostic approach to the patient with HLH.
1968	Pulmonary hypertension following haematopoietic stem cell transplantation for primary haemophagocytic lymphohistiocytosis.	23192896	We report two children who developed severe, fatal pulmonary hypertension (PHT) after mismatched unrelated donor cord blood transplantation using reduced intensity conditioning for HLH. PHT was diagnosed on post mortem lung biopsies with no evidence of HLH, pulmonary veno-occlusive disease, infection or of idiopathic pulmonary hypertension. PHT may be an association with HLH and physicians treating HLH should be aware of this potential association.
1969	Hemophagocytic lymphohistiocytosis in syntaxin-11-deficient mice: T-cell exhaustion limits fatal disease.	2319053123349371	Syntaxin-11 (Stx11), an atypical member of the SNARE protein family, is part of the cytolytic machinery of T and NK cells and involved in the fusion of lytic granules with the plasmamembrane. Functional loss of syntaxin-11 in humans causes defective degranulation and impaired cytolytic activity of T and NK cells. Furthermore, patients with STX11 deficiency develop familial hemophagocytic lymphohistiocytosis type 4 (FHL4), a life-threatening disease of severe hyperinflammation. We established Stx11-deficient mice as an animal model for FHL4. Stx11-deficient mice exhibited severely reduced degranulation and cytolytic activity of CTL and NK cells and developed all clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) after infection with lymphocytic choriomeningitis virus (LCMV). The HLH phenotype was further characterized by hyperactive CD8 T cells and continuous IFN-γ production. However, in contrast to perforin-deficient mice, which represent a model for FHL2, progression of HLH was not fatal. Survival of Stx11-deficient mice was determined by exhaustion of antigen-specific T cells, characterized by expression of inhibitory receptors, sequential loss of effector functions, and finally T-cell deletion. Blockade of inhibitory receptors on T cells in Stx11-deficient mice converted nonfatal disease course into fatal HLH, identifying T-cell exhaustion as an important factor for determination of disease severity in HLH.
1970	Founder effects in two predominant intronic mutations of UNC13D, c.118-308C>T and c.754-1G>C underlie the unusual predominance of type 3 familial hemophagocytic lymphohistiocytosis (FHL3) in Korea.	23180437	Familial hemophagocytic lymphohistiocytosis (familial HLH or FHL) is a potentially fatal autosomal recessive disorder. Our previous study demonstrated that UNC13D mutations (FHL3) account for ~90 % of FHL in Korea with recurrent splicing mutation c.754-1G>C (IVS9-1G>C). Notably, half of the FHL3 patients had a monoallelic mutation of UNC13D. Deep intronic mutations in UNC13D were recently reported in patients of European descent. In this study, we performed targeted mutation analyses for deep intronic mutations and investigated on the founder effect in FHL3 in Korean patients. The study patients were 72 children with HLH including those with FHL3 previously reported to have a monoallelic UNC13D mutation. All patients were recruited from the Korean Registry of Hemophagocytic Lymphohistiocytosis. In addition to conventional sequencing of FHL2-4, targeted tests for c.118-308C>T and large intronic rearrangement mutations of UNC13D were performed. Haplotype analysis was performed for founder effects using polymorphic markers in the FHL3 locus. FHL mutations were detected in 20 patients (28 %). Seventeen patients had UNC13D mutations (FHL3, 85 %) and three had PRF1 mutations (FHL2, 15 %). UNC13D:c.118-308C>T was detected in ten patients, accounting for 38 % of all mutant alleles of UNC13D, followed by c.754-1G>C (26 %). Haplotype analyses revealed significantly shared haplotypes in both c.118-308C>T and c.754-1G>C, indicating the presence of founder effects. The deep intronic mutation UNC13D:c.118-308C>T accounts for the majority of previously missing mutations and is the most frequent mutation in FHL3 in Korea. Founder effects of two recurrent intronic mutations of UNC13D explain the unusual predominance of FHL3 in Korea.
1971	Hemophagocytic lymphohistiocytosis--a diagnostic dilemma: two cases and review.	23172902	Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory disorder characterized by activation and proliferation of lymphocytes and histiocytes with cytokine release and uncontrolled hemophagocytosis, especially late in the course of the disease. Clinical features include relapsing fevers, hepatosplenomegaly, cytopenias, lymphadenopathy, and coagulopathy. The diagnosis can be challenging, as the early signs and symptoms are nonspecific and no specific laboratory tests exist. This syndrome is frequently not recognized and has a significant mortality rate. Typical scenarios in which HLH should be considered include mononucleosis (fever, hepatosplenomegaly, and lymphadenopathy) in an infant or young child, aseptic meningitis associated with cytopenias, or a viral syndrome-like illness with cytopenias and lymphadenopathy or splenomegaly, for example. Our approach includes measuring a ferritin level as a screening tool early in the course of such an illness. Two cases of HLH are reviewed, illustrating the frequent complexity of these cases and potential pitfalls to making a prompt diagnosis.
1972	Hemophagocytic lymphohistiocytosis in pregnancy: a case report and review of treatment options.	23168071	Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening inflammatory disorder characterized by uncontrolled proliferation and activation of histiocytes with phagocytosis of normal hematopoietic cells. A 41-year-old woman, 19 weeks pregnant with twins, and a history of Still's disease, presented with rash, fever, and headache. Laboratory studies revealed transaminitis, hyperbilirubinemia, and eventually severe neutropenia as well as elevations in ferritin, lactate dehydrogenase, and C-reactive protein. A bone marrow biopsy confirmed HLH. She declined standard HLH-treatment but responded well to high-dose corticosteroids. Her blood counts remained stable following corticosteroid taper, and she delivered healthy twin girls at 30-week gestation. Few cases of HLH during pregnancy have been reported. In some cases, the condition has proved fatal. Therefore recognizing signs and symptoms of HLH is essential to avoid treatment delay. In our case, high-dose corticosteroids alone were a safe and effective therapy for the mother and fetuses resulting in long-term disease control.
1973	Distinct severity of HLH in both human and murine mutants with complete loss of cytotoxic effector PRF1, RAB27A, and STX11.	2316046423349371	Inherited defects of granule-dependent cytotoxicity led to the life-threatening immune disorder hemophagocytic lymphohistiocytosis (HLH), characterized by uncontrolled CD8 T-cell and macrophage activation. In a cohort of HLH patients with genetic abnormalities expected to result in the complete absence of perforin, Rab27a, or syntaxin-11, we found that disease severity as determined by age at HLH onset differed significantly, with a severity gradient from perforin (early onset) > Rab27a > syntaxin-11 (late onset). In parallel, we have generated a syntaxin-11-deficient (Stx11(-/-)) murine model that faithfully reproduced the manifestations of HLH after lymphocytic choriomeningitis virus (LCMV) infection. Stx11(-/-) murine lymphocytes exhibited a degranulation defect that could be rescued by expression of human syntaxin-11 but not expression of a C-terminal-truncated mutant. Comparison of the characteristics of LCMV infection-induced HLH in the murine counterparts of the 3 human conditions revealed a similar gradient in the phenotypic severity of HLH manifestations. Strikingly, the severity of HLH was not correlated with the LCMV load and not fully with differences in the intensity of cytotoxic activity. The capacity of antigen presentation differed in vivo between Rab27a- and Syntaxin-11-deficient mutants. Our data indicate that cytotoxic effectors may have other immune-regulatory roles in addition to their role in controlling viral replication.
1974	Hemophagocytic [corrected] lymphohistiocytosis associated with nephrotic syndrome and multi-organ failure.	23146987	Hemophagocytic lymphohistiocytosis (HLH) is still an important elusive and misdiagnosed condition despite of improved knowledge. Nephrotic syndrome associated with HLH is not a common feature and has been rarely reported in hemophagocytic syndrome. We report a 27-year-old man with HLH who progressed to multi-organ failure as well as nephrotic-range proteinuria, generalized edema, and hypoalbuminemia.
1975	Cloning, purification and preliminary X-ray data analysis of the human ID2 homodimer.	23143248	The ID proteins are named for their role as inhibitors of DNA binding and differentiation. They contain a helix-loop-helix (HLH) domain but lack a basic DNA-binding domain. In complex with basic HLH (bHLH) transcription factors, gene expression is regulated by DNA-binding inactivation. Although the HLH domain is highly conserved and shares a similar topology, the IDs preferentially bind class I bHLH-group members such as E47 (TCF3) but not the class III bHLH member Myc. A structure of an ID protein could potentially shed light on its mechanism. Owing to their short half-lives in vivo and reported in vitro instability, this paper describes the strategies that went into expressing sufficient soluble and stable ID2 to finally obtain diffraction-quality crystals. A 2.1 Å resolution data set was collected from a crystal belonging to space group P3(1)21 with unit-cell parameters a=b=51.622, c=111.474 Å, α=β=90, γ=120° that was obtained by hanging-drop vapour diffusion in a precipitant solution consisting of 0.1 M MES pH 6.5, 2.0 M potassium acetate. The solvent content was consistent with the presence of one or two molecules in the asymmetric unit.
1976	Allogeneic hematopoietic cell transplantation for XIAP deficiency: an international survey reveals poor outcomes.	23131490	There have been no studies on patient outcome after allogeneic hematopoietic cell transplantation (HCT) in patients with X-linked inhibitor of apoptosis (XIAP) deficiency. To estimate the success of HCT, we conducted an international survey of transplantation outcomes. Data were reported for 19 patients. Seven patients received busulfan-containing myeloablative conditioning (MAC) regimens. Eleven patients underwent reduced intensity conditioning (RIC) regimens predominantly consisting of alemtuzumab, fludarabine, and melphalan. One patient received an intermediate-intensity regimen. Survival was poor in the MAC group, with only 1 patient surviving (14%). Most deaths were from transplantation-related toxicities, including venoocclusive disease and pulmonary hemorrhage. Of the 11 patients who received RIC, 6 are currently surviving at a median of 570 days after HCT (55%). Preparative regimen and HLH activity affected outcomes, and of RIC patients reported to be in remission from HLH, survival is 86% (P = .03). We conclude that MAC regimens should not be used for patients with XIAP deficiency. It is possible that the loss of XIAP and its antiapoptotic functions contributes to the high incidence of toxicities observed with MAC regimens. RIC regimens should be pursued with caution and, if possible, efforts should be made to ensure HLH remission before HCT in these patients.
1977	Composite peripheral T-cell lymphoma not otherwise specified, and B-cell small lymphocytic lymphoma presenting with hemophagocytic lymphohistiocytosis.	23129836	We report a case of a 68-year-old female patient who developed hemophagocytic lymphohistiocytosis (HLH) secondary to peripheral T-cell lymphoma (PTCL) not otherwise specified (NOS) that developed in the setting of treatment-resistant B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). The patient's B-cell lymphoma had a good initial response to chemotherapy for 4 years, after which it became less responsive and was thought to have undergone transition to a higher-grade lymphoma. Different regimens of chemoradiotherapy were then tried with modest response until the patient presented 3 years later with signs and symptoms of HLH. The patient died 1 month later, and an autopsy was performed. Significant para-aortic lymphadenopathy and splenomegaly were found. Microscopic, immunohistochemical and molecular evaluations confirmed the presence of composite B-cell and T-cell lymphoma in the para-aortic enlarged lymph nodes. Bone marrow examination showed hemophagocytosis, and the liver demonstrated infiltration by activated macrophages with hepatocellular necrosis. This report highlights the importance of searching for a possible underlying T-cell lymphoma in light of HLH. Different theories have been proposed to explain the rare occurrence of concurrent B- and T-cell lymphomas, but the development of HLH in this patient highlights the importance of immune dysregulation as a proposed mechanism to explain some cases of composite lymphomas. A review of the literature and discussion of the relative merits of these hypotheses are presented in the context of this case.
1978	Lymphocyte-depleted Hodgkin lymphoma complicating hemophagocytic lymphohistiocytosis as an initial manifestation: a case report and review of the literature.	23124152	Hemophagocytic lymphohistocytosis (HLH) is commonly associated with infectious diseases or T/NK cell-lymphoma; however that with Hodgkin lymphoma (HL) was rarely reported. Herein, we describe a young male diagnosed with lymphocyte-depleted HL (LD-HL) complicated by HLH as an initial manifestation. He was given high-dose steroid therapy plus recombinant thrombomodulin, and subsequent ABVd (doxorubicin, bleomycin, vinblastine, dacarbazine) treatment. In spite of the achievement of a partial remission treated with one cycle of ABVd, he relapsed after 3 cycles. To our knowledge, the present case is very rare, and more intensive treatment might be needed for the long-term control of HLH-complicated HL.
1979	A divalent ion is crucial in the structure and dominant-negative function of ID proteins, a class of helix-loop-helix transcription regulators.	23119064	Inhibitors of DNA binding and differentiation (ID) proteins, a dominant-negative group of helix-loop-helix (HLH) transcription regulators, are well-characterized key players in cellular fate determination during development in mammals as well as Drosophila. Although not oncogenes themselves, their upregulation by various oncogenic proteins (such as Ras, Myc) and their inhibitory effects on cell cycle proteins (such as pRb) hint at their possible roles in tumorigenesis. Furthermore, their potency as inhibitors of cellular differentiation, through their heterodimerization with subsequent inactivation of the ubiquitous E proteins, suggest possible novel roles in engineering induced pluripotent stem cells (iPSCs). We present the high-resolution 2.1Å crystal structure of ID2 (HLH domain), coupled with novel biochemical insights in the presence of a divalent ion, possibly calcium (Ca2+), in the loop of ID proteins, which appear to be crucial for the structure and activity of ID proteins. These new insights will pave the way for new rational drug designs, in addition to current synthetic peptide options, against this potent player in tumorigenesis as well as more efficient ways for stem cells reprogramming.
1980	The role of the bHLH protein hairy in morphogenetic furrow progression in the developing Drosophila eye.	23118874	In Drosophila eye development, a wave of differentiation follows a morphogenetic furrow progressing across the eye imaginal disc. This is subject to negative regulation attributed to the HLH repressor proteins Hairy and Extramacrochaete. Recent studies identify negative feedback on the bHLH gene daughterless as one of the main functions of extramacrochaete. Here the role of hairy was assessed in relation to daughterless and other HLH genes. Hairy was not found to regulate the expression of Daughterless, Extramacrochaete or Atonal, and Hairy expression was largely unregulated by these other genes. Null alleles of hairy did not alter the rate or pattern of differentiation, either alone or in the absence of Extramacrochaete. These findings question whether hairy is an important regulator of the progression of retinal differentiation in Drosophila, alone or redundantly with extramacrochaete.
1981	Midkine-A functions upstream of Id2a to regulate cell cycle kinetics in the developing vertebrate retina.	23111152	Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina's stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish.The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype.These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins.
1982	Fetal pulmonary venous flow and restrictive foramen ovale in hypoplastic left heart.	23108924	Hypoplastic left heart (HLH) with intact or restrictive interatrial communication (HLH-IAS/RAS) is associated with high mortality rates. The object was to correlate pulmonary venous (PV) Doppler spectra and direct foramen ovale (FO) assessment with the neonatal need for early atrial septostomy (EAS) and neonatal outcome.We reviewed all prenatal echocardiograms and outcomes of 51 fetuses with HLH and information about the interatrial communication between 1994 - 2011.  IAS/RAS was defined as a small/absent interatrial shunt on 2-dimensional imaging. Three PV Doppler spectra were observed: type A: continuous forward flow with a small a wave reversal; type B: continuous forward flow with increased a-wave reversal; type C: brief to-and-fro flow.Three of 51 neonates with type C pulmonary venous flow pattern and suspicion of IAS/RAS on 2-dimensional (2-DE) evaluation required EAS.  In one fetus pulmonary venous flow changed from type B to type C spectra throughout gestation. Fetuses with type C spectra showed a 71.4 % survival after 30 days compared to 92.3 % in fetuses with type A spectra. Short term survival after EAS was 33 %.Prenatal PV flow pattern and 2-DE of the FO size help in identifying the fetus at risk for neonatal EAS and patient selection for fetal cardiac intervention. Most late second trimester values will not change over time.ZIEL:: Das hypoplastische Linksherz (HLH) mit intakter oder restriktiver interatrialer Kommunikation (HLH-IAS/RAS) ist mit einer hohen Mortalitätsrate assoziiert. Mit dieser Arbeit sollte die Bestimmung von Pulmonalvenen(PV)-Doppler-Spektren, die direkte Messung des Foramen ovale, die Notwendigkeit einer frühen atrialen Septostomie (EAS) und das neonatale Outcome auf eine Korrelation hin untersucht werden.Wir untersuchten alle pränatalen Echokardiogramme und Outcomes von 51 Feten mit der Diagnose eines HLH und der Information über die interatriale Kommunikation zwischen 1994 und 2011. IAS/RAS wurde als schmaler/nicht darstellbarer Shunt im 2-dimensionalen B-Bild definiert. 3 verschiedene PV-Doppler-Spektren konnten nachgewiesen werden: Typ A: kontinuierlicher Vorwärtsfluss mit einem geringen Rückwärtsfluss während der a-Welle; Typ B: kontinuierlicher Vorwärtsfluss mit gesteigertem Rückwärtsfluss während der a-Welle; Typ C: Pendelfluss.Bei 3 der 51 Neugeborenen mit einem PV Spektrum vom Typ C und dem Verdacht auf IAS/RAS in der 2-dimensionalen Auswertung (2-DE) war eine EAS notwendig. In einem Fall kam es im Laufe der Schwangerschaft zu einem Wechsel von Typ B zu Typ C. Feten mit Typ-C-Spektrum wiesen nach 30 Tagen eine Überlebensrate von 71,4 % auf, bei Feten mit Typ-A-Spektrum lag diese dagegen bei 92,3 %. Das Kurzzeit-Überleben nach EAS betrug 33 %.Mithilfe der pränatalen Bestimmung der PV-Flussmuster und der 2-DE der FO-Größe können Feten mit einem Risiko für eine neonatale EAS und Patienten für eine fetale kardiale Intervention selektiert werden. Im späten zweiten Trimester erhobene Messwerte ändern sich im weiteren Verlauf der Schwangerschaft meist nicht mehr.
1983	Haemophagocytic syndrome in rheumatic patients. A systematic review.	23104659	Hemophagocytic lymphohistiocytosis (HLH), is a potentially fatal hyperinflammatory syndrome characterized fever, hepatosplenomegaly, and cytopenias. HLH can be either primary, with a genetic aetiology, or secondary, associated with malignancies, autoimmune diseases, or infections. Among rheumatic disorders, HLH occurs most frequently in systemic juvenile idiopathic arthritis.To draw attention on this severe syndrome that may often go undiagnosed in patient with rheumatic diseases.PubMed search was performed by combining the terms (haemophagocytic, haemophagocytosis, hemophagocytosis, hemophagocytic, erythrophagocytosis, macrophage activation syndrome) and (rheumatic, rheumatologic, arthritis, lupus, Sjögren's syndrome, scleroderma, polymyositis, dermatomyositis, polymyalgia rheumatic, mixed connective tissue disease, polychondritis, sarcoidosis, polyarteritis nodosa, Henoch-Schönlein, serum sickness, wegener's granulomatosis, giant cell arteritis, temporal arteritis, Takayasu's arteritis, Behçet's syndrome, Kawasaki, Buerger's).117 papers describing 421 patients were considered. HLH was described in systemic lupus erythematosus in 94 patients, in Still's disease in 37 patients, in rheumatoid arthritis in 13 patients, in systemic juvenile arthritis in 219 patients, in dermatomyositis in 7 patients, in Kawasaki disease in 25 patients, in systemic sclerosis in 5 patients, in Behcet disease in one patient, in polyarteritis nodosa in 6 patients, in ankylosing spondylitis in 2 patients, in mixed connective tissue disease in one patient, in sarcoidosis in 5 patients, in Sjögren's syndrome in 3 patients, in Wegener's granulomatosis in one patient, and in unclassifiable disorders in two patients.HLH occurring in the course of rheumatic diseases is an important and often underdiagnosed clinical entity, which can affect prognosis.
1984	Secondary hemophagocytic lymphohistiocytosis in zoonoses. A systematic review.	23104648	Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome that is often fatal despite treatment. It is caused by a dysregulation in natural killer T-cell function, resulting in activation and proliferation of histiocytes with uncontrolled hemophagocytosis and cytokines overproduction. The syndrome is characterized by fever, hepatosplenomegaly, cytopenias, liver dysfunction, and hyperferritinemia. HLH can be either primary, with a genetic aetiology, or secondary, associated with malignancies, autoimmune diseases, or infections.To focus on secondary HLH complicating zoonotic diseases.PubMed search of human cases of HLH occurring during zoonotic diseases was performed combining the terms (haemophagocytic or haemophagocytosis or hemophagocytosis or hemophagocytic or erythrophagocytosis or macrophage activation syndrome) with each one of the etiological agents of zoonoses.Among bacterial diseases, most papers reported cases occurring during brucellosis, rickettsial diseases and Q fever. Regarding viral diseases, most of the cases were reported in patients with avian influenza A subtype H5N1. Among the protozoan zoonoses, most of the cases were reported in patients with visceral leishmaniasis. Regarding zoonotic fungi, most of the cases were reported in AIDS patient with histoplasmosis. No cases of secondary HLH were reported in patient with zoonotic helminthes.Zoonotic diseases are an important cause of HLH. Secondary HLH can delay the correct diagnosis of the zoonotic disease, and can contribute to an adverse outcome.
1985	Removal of Polycomb repressive complex 2 makes C. elegans germ cells susceptible to direct conversion into specific somatic cell types.	23103163	How specific cell types can be directly converted into other distinct cell types is a matter of intense investigation with wide-ranging basic and biomedical implications. Here, we show that removal of the histone 3 lysine 27 (H3K27) methyltransferase Polycomb repressor complex 2 (PRC2) permits ectopically expressed, neuron-type-specific transcription factors ("terminal selectors") to convert Caenorhabditis elegans germ cells directly into specific neuron types. Terminal-selector-induced germ-cell-to-neuron conversion can be observed not only upon genome-wide loss of H3K27 methylation in PRC2(-) animals but also upon genome-wide redistribution of H3K27 methylation patterns in animals that lack the H3K36 methyltransferase MES-4. Manipulation of the H3K27 methylation status not only permits conversion of germ cells into neurons but also permits hlh-1/MyoD-dependent conversion of germ cells into muscle cells, indicating that PRC2 protects the germline from the aberrant execution of multiple distinct somatic differentiation programs. Taken together, our findings demonstrate that the normally multistep process of development from a germ cell via a zygote to a terminally differentiated somatic cell type can be short-cut by providing an appropriate terminal selector transcription factor and manipulating histone methylation patterns.
1986	Efficacy of different gonadotropin combinations to support ovulation induction in WHO type I anovulation infertility: clinical evidences of human recombinant FSH/human recombinant LH in a 2:1 ratio and highly purified human menopausal gonadotropin stimulation protocols.	23095369	The World Helath Organization (WHO) Group I anovulation, or hypogonadotropic hypogonadism (HH), is characterized by reduced hypothalamic/pituitary activity which results in abnormally low serum FSH and LH levels and negligible estrogen activity.To compare the efficacy of human recombinant FSH (r-hFSH) plus human recombinant LH (r-hLH) in a 2:1 ratio with highly purified human menopausal gonadotropin (hMG-HP) urinary extract, containing LH-like activity, in women with HH.This two-arm randomized open-label study included 35 HH women (aged 25-36 yr) attending our Center. Eighteen patients received 150 IU hMG-HP (150 IU FSH + 150 IU LH-like activity) and seventeen received 150IU r-hFSH/75IU rhLH daily for a maximum of 16 days. Ovulation was induced by a single administration of hCG on the day after the last hMG-HP or r-hFSH/r-hLH.The primary efficacy endpoint was ovulation induction as measured by follicle ≥17 mm, pre-ovulatory estradiol (E 2 ) ≥400 pmol/l and mid-luteal phase progesterone (P 4 ) ≥25 nmol/l. Secondary efficacy endpoints included E 2 levels/follicle at mid-cycle, number of follicles at mid-cycle and pregnancy rate (PR). Following a total of 70 cycles, 70% of r-hFSH/r-hLH treated patients met the primary endpoint vs 88% in hMG-HP group (p=0.11). However, PR in r-hFSH/r-hLH group was 55.6% compared to 23.3% in hMG-HP group (p=0.01).The primary endpoint achievement did not correlate with PR. This study has shown the superiority of LH compared to hCG in supporting FSH-induced follicular development in HH women.
1987	Remission and relapse of hemophagocytic lymphohistiocytosis in a patient harboring a PRF1 homozygous mutation: a case report.	23073044	The aim of this paper was to describe a case of familial hemophagocytic lymphohistiocytosis (HLH) in a pediatric patient with a PRF1 homozygous mutation. An 8-year-old boy diagnosed with HLH was in remission after undergoing nonspecific treatment; however, merely 2 months later, he was presented at our hospital with a relapse of HLH. His genetic analysis showed that he had a homozygous mutation c.1066C>T in the PRF1 gene. Timely distinction of primary HLH from secondary HLH is critical.
1988	Association of nonimmune hydrops fetalis with familial hemophagocytic lymphohistiocytosis in identical twin neonates with perforin His222Arg (c665A>G) mutation.	23073042	This is the first study demonstrating that nonimmune hydrops fetalis (NIHF) in identical twin neonates is associated with biallelic gene defect causing familial hemophagocytic lymphohistiocytosis.Preterm male twins (31(4/7) wk) with NIHF and hepatosplenomegaly gradually developed pancytopenia, hyperferritinemia, hyponatremia, hypoalbuminemia, and elevated alanine aminotransferase, aspartate aminotransferase, bilirubin, and lactate dehydrogenase levels. Suspected sepsis led to antibiotic therapy. Upon detection of hemophagocytosis in bone marrow, multiorgan failure and pulmonary bleeding led to death. Homozygous His222Arg (c665A>G) mutation was identified in Perforin.Familial hemophagocytic lymphohistiocytosis should be considered in first days of NIHF cases to have chance for HLH-2004 therapy. Missense mutations of Perforin codon His222 may lead to intrauterine presentation.
1989	LH and hCG action on the same receptor results in quantitatively and qualitatively different intracellular signalling.	23071612	Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED(50): 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.
1990	Haemophagocytic lymphohistiocytosis: a cause for rare but fatal outcome in tuberculosis.	23035171	Haemophagocytic lymphohistiocytosis (HLH), also called haemophagocytic syndrome (HPS) is characterised by a dysregulated activation and proliferation of macrophages, leading to uncontrolled phagocytosis of platelets, erythrocytes, lymphocytes and their haematopoietic precursors throughout the reticuloendothelial system. Mycobacterium tuberculosis-associated HPS is a rare and underdiagnosed association. We report a 34-year-old male patient diagnosed with tubercular pleural effusion responding poorly to antitubercular treatment. Patient later developed generalised lymphadenopathy, pancytopaenia and liver dysfunction and was eventually diagnosed as HLH. Despite being treated as per HLH protocol 2004 he could not be saved.
1991	c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells.	230212162302121123037452	The c-Myc HLH-bZIP protein has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and differentiation at the cellular, tissue, or organismal levels via regulation of numerous target genes. No principle yet unifies Myc action due partly to an incomplete inventory and functional accounting of Myc's targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications were compared during lymphocyte activation and in ES cells as well. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action explains the vast majority of Myc biology observed in literature.
1992	The treatment of crimean-congo hemorrhagic fever with high-dose methylprednisolone, intravenous immunoglobulin, and fresh frozen plasma.	23018575	Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne disease caused by Nairovirus, and it is sometimes characterized by reactive hemophagocytic histiocytosis (HLH). The reasons for reactive HLH are macrophage-activating syndrome and disseminated intravascular coagulation due to cytokine storm, liver dysfunction, and endothelial damage by the virus. In this study, the effectiveness of high-dose methylprednisolone (HDMP) (5 to 30 mg/kg/d), fresh frozen plasma (FFP), and intravenous immunoglobulin (IVIG) was investigated in patients with CCHF associated with reactive HLH. Twelve patients with CCHF in association with reactive HLH were included in the study. The patients were successfully treated with HDMP to suppress the macrophage activation, FFP to treat disseminated intravascular coagulation, and IVIG to treat severe thrombocytopenia. No patients received ribavirin. Fever reduced in 1.6 ± 0.8 days, WBC count increased above 4.500/µL in 4.0 ± 2.4 days, platelet count increased above 150.000/µL in 8.5 ± 2.5 days, and D-dimer level decreased under 1 mcg/dL in 5.8 ± 3.6 days. Consequently, HDMP, FFP, and IVIG may be effective in patients with CCHF associated with reactive HLH during hemorrhagic period of the disease.
1993	An unusual cause of fever.	23005894	A 5-month old infant presented with a short history of fever of unknown origin. He initially appeared well although there was a resting tachycardia during periods of normal temperature, and pancytopenia. He remained febrile in spite of antibiotic therapy and by 48 h he had developed marked hepatosplenomegaly and coagulopathy. At 72 h a bone marrow aspirate and trephine biopsy showed haemophagocytosis. By this time the infant was encephalopathic and haemodynamically unstable with multi-organ dysfunction. Treatment with high-dose methylprednisolone and cyclosporin was commenced with an initial good response. Unfortunately the patient ultimately died of infective complications of treatment and reactivation of the underlying disease process. Haemophagocytic lymphohistiocytosis (HLH) is a rare disorder of the mononuclear phagocyte system characterised by proliferation of morphologically benign histiocytes resulting in hypercytokinaemia and uncontrolled activation of immune cells. The diagnosis of familial HLH should be considered in any infant with fever, splenomegaly and cytopenia of at least two cell lines. HLH may be cured by immunosuppressive therapy and eventual stem cell transplantation but rapid disease progression to multi-organ failure results in a high mortality.
1994	Engulfment of hematopoietic stem cells caused by down-regulation of CD47 is critical in the pathogenesis of hemophagocytic lymphohistiocytosis.	22990013	Hemophagocytic lymphohistiocytosis (HLH) is characterized by deregulated engulfment of hematopoietic stem cells (HSCs) by BM macrophages, which are activated presumably by systemic inflammatory hypercytokinemia. In the present study, we show that the pathogenesis of HLH involves impairment of the antiphagocytic system operated by an interaction between surface CD47 and signal regulatory protein α (SIRPA). In HLH patients, changes in expression levels and HLH-specific polymorphism of SIRPA were not found. In contrast, the expression of surface CD47 was down-regulated specifically in HSCs in association with exacerbation of HLH, but not in healthy subjects. The number of BM HSCs in HLH patients was reduced to approximately 20% of that of healthy controls and macrophages from normal donors aggressively engulfed HSCs purified from HLH patients, but not those from healthy controls in vitro. Furthermore, in response to inflammatory cytokines, normal HSCs, but not progenitors or mature blood cells, down-regulated CD47 sufficiently to be engulfed by macrophages. The expression of prophagocytic calreticulin was kept suppressed at the HSC stage in both HLH patients and healthy controls, even in the presence of inflammatory cytokines. These data suggest that the CD47-SIRPA antiphagocytic system plays a key role in the maintenance of HSCs and that its disruption by HSC-specific CD47 down-regulation might be critical for HLH development.
1995	Id2a functions to limit Notch pathway activity and thereby influence the transition from proliferation to differentiation of retinoblasts during zebrafish retinogenesis.	22981606	During vertebrate retinogenesis, the precise balance between retinoblast proliferation and differentiation is spatially and temporally regulated through a number of intrinsic factors and extrinsic signaling pathways. Moreover, there are complex gene regulatory network interactions between these intrinsic factors and extrinsic pathways, which ultimately function to determine when retinoblasts exit the cell cycle and terminally differentiate. We recently uncovered a cell non-autonomous role for the intrinsic HLH factor, Id2a, in regulating retinoblast proliferation and differentiation, with Id2a-deficient retinae containing an abundance of proliferative retinoblasts and an absence of terminally differentiated retinal neurons and glia. Here, we report that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through a novel RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, we identify a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis.
1996	Familial hemophagocytic lymphohistiocytosis may present during adulthood: clinical and genetic features of a small series.	22970278	Familial Hemophagocytic lymphohistiocytosis (FHL) is a rare immune deficiency with defective cytotoxic function. The age at onset is usually young and the natural course is rapidly fatal if untreated. A later onset of the disease has been sporadically reported even in adolescents and adults. We report the results of our retrospective data collection of all cases diagnosed with FHL at an age of 18 years or older and enrolled in the Italian Registry of HLH. All cases were diagnosed with FHL based on evidence of genetic defect in one FHL-related gene. A total of 11 patients were diagnosed with FHL. They were 9 males and 2 females, from 10 unrelated families; their age ranged between 18 and 43 years (median, 23 years). Family history was unremarkable in eight families at the time of the diagnosis. Their genetic diagnoses are: FHL2 (n = 6), FHL3 (n = 2), FHL5 (n = 1), XLP1 (n = 2). Clinical, molecular and functional data are described. These data confirm that FHL may present beyond the pediatric age and up to the fifth decade. FHL2 due to perforin defect is the most frequently reported subtype. Adult specialists should consider FHL in the differential diagnosis of patients with cytopenia and liver or central nervous system disorders, especially when a lymphoproliferative disease is suspected but eventually not confirmed. FHL may turn to be fatal within a short time course even in adults. This risk, together with the continuous improvement in the transplant technique, especially in the area of transplant from matched unrelated donor, resulting in reduced treatment related mortality, might suggest a wider use of SCT in this population. Current diagnostic approach allows prompt identification of patients by flow-cytometry screening, then confirmed by the genetic study, and treatment with chemo-immunotherapy followed by stem cell transplantation.
1997	Demonstration of type II latency in T lymphocytes of Epstein-Barr Virus-associated hemophagocytic lymphohistiocytosis.	22961815	Epstein-Barr virus (EBV) is the most common infectious cause of non-genetic hemophagocytic lymphohistiocytosis (HLH). To investigate EBV-infected lymphocytes and immune dysfunction in EBV-associated HLH, blood samples from a 6-year-old boy were longitudinally analyzed using molecular techniques. EBV-positive lymphocytes were detected as CD5(+) , CD8(+) , and/or HLA DR(+) lymphocytes on Day 25 of the disease, mostly disappearing thereafter. CD8(+) cells specific for lytic antigen BRLF1 were detected, but cells specific for latent antigens EBNA3 and LMP2 were not. EBV genes EBNA1, LMP1, LMP2, EBER1, BARTs were detected, suggesting a latency type II gene expression pattern in this case.
1998	Essential roles of Da transactivation domains in neurogenesis and in E(spl)-mediated repression.	22949507	E proteins are a special class of basic helix-loop-helix (bHLH) proteins that heterodimerize with many bHLH activators to regulate developmental decisions, such as myogenesis and neurogenesis. Daughterless (Da) is the sole E protein in Drosophila and is ubiquitously expressed. We have characterized two transcription activation domains (TADs) in Da, called activation domain 1 (AD1) and loop-helix (LH), and have evaluated their roles in promoting peripheral neurogenesis. In this context, Da heterodimerizes with proneural proteins, such as Scute (Sc), which is dynamically expressed and also contributes a TAD. We found that either one of the Da TADs in the Da/Sc complex is sufficient to promote neurogenesis, whereas the Sc TAD is incapable of doing so. Besides its transcriptional activation role, the Da AD1 domain serves as an interaction platform for E(spl) proteins, bHLH-Orange family repressors which antagonize Da/Sc function. We show that the E(spl) Orange domain is needed for this interaction and strongly contributes to the antiproneural activity of E(spl) proteins. We present a mechanistic model on the interplay of these bHLH factors in the context of neural fate assignment.
1999	Drosophila Hox and sex-determination genes control segment elimination through EGFR and extramacrochetae activity.	22912593	The formation or suppression of particular structures is a major change occurring in development and evolution. One example of such change is the absence of the seventh abdominal segment (A7) in Drosophila males. We show here that there is a down-regulation of EGFR activity and fewer histoblasts in the male A7 in early pupae. If this activity is elevated, cell number increases and a small segment develops in the adult. At later pupal stages, the remaining precursors of the A7 are extruded under the epithelium. This extrusion requires the up-regulation of the HLH protein Extramacrochetae and correlates with high levels of spaghetti-squash, the gene encoding the regulatory light chain of the non-muscle myosin II. The Hox gene Abdominal-B controls both the down-regulation of spitz, a ligand of the EGFR pathway, and the up-regulation of extramacrochetae, and also regulates the transcription of the sex-determining gene doublesex. The male Doublesex protein, in turn, controls extramacrochetae and spaghetti-squash expression. In females, the EGFR pathway is also down-regulated in the A7 but extramacrochetae and spaghetti-squash are not up-regulated and extrusion of precursor cells is almost absent. Our results show the complex orchestration of cellular and genetic events that lead to this important sexually dimorphic character change.
2000	[Prognostic factors for hemophagocytic lymphohistiocytosis in children].	22898280	To study the main factors influencing prognosis of hemophagocytic lymphohistiocytosis (HLH) in children by summarizing the clinical features of HLH and investigating the relationship between relevant factors and prognosis.The medical data of 63 children with HLH were retrospectively reviewed. Kaplan-Meier method was employed to draw survival curves. Factors influencing prognosis were assessed with Cox univariate analysis, and Cox multivariate analysis was done on statistically significant factors.The 3-year and 5-year survival rates were both 62.9%. The survival rate decreased from 98.4% at 1 day after definite diagnosis to 73.2% at 4 months. Univariate analysis demonstrated only one factor, which was that the condition of platelet recovery after treatment of 2 to 3 weeks was significantly related to prognosis (P=0.002). In children receiving etoposide therapy, temperature recovery after one day of treatment was significantly related to prognosis (P=0.016).Children with HLH have a satisfactory prognosis, but the survival rate reduces rapidly in the first 4 months after definite diagnosis. Platelet recovery after treatment of 2 to 3 weeks and temperature recovery after one day of treatment are factors influencing prognosis of HLH in children.
2001	Hypoalbuminaemia is an independent predictor for hemophagocytic lymphohistiocytosis in childhood Epstein-Barr virus-associated infectious mononucleosis.	22897479	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal condition in children with Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). This study aimed to identify commonly available clinical and laboratory predictors that might help clinicians decide to perform the bone marrow and immunological tests for HLH in paediatric EBV-associated IM.A retrospective case-control study of patients aged <18 yr diagnosed with EBV-associated IM and HLH from 1991 to 2010 in a tertiary medical centre was conducted. A diagnosis of HLH was defined as fulfilling the criteria of the guidelines of the HLH-2004 protocol of the Histiocyte Society and consisted of at least evidence of hemophagocytosis in a bone marrow biopsy.A total of 177 IM and 27 HLH patients were enrolled. The mean age was 5.3 yr with a female-to-male ratio of 1.06. The most common characteristics (>70% of patients) were fever, lymphadenopathy and hepatomegaly. In addition to the diagnostic criteria of HLH including fever, splenomegaly, cytopenia, hyperferritinaemia, hypertriglyceridemia and/or hypofibrinogenaemia, children with HLH had a significantly higher rate of prolonged fever >10 d, hepatomegaly, jaundice, general malaise, elevated aspartate aminotransferase, lactate dehydrogenase, C-reactive protein and hypoalbuminaemia compared to those with IM (all P < 0.01). Multiple logistic regression confirmed that hypoalbuminaemia (OR = 23.1, P = 0.01) was an independent predictor of paediatric HLH, with a high sensitivity (96%) and a good negative likelihood ratio (0.06) in patients with EBV-associated IM.Hypoalbuminaemia is a unique characteristic and potentially a valuable predictor for HLH in paediatric EBV-associated IM.
2002	Protection from inflammatory organ damage in a murine model of hemophagocytic lymphohistiocytosis using treatment with IL-18 binding protein.	22891066	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition due to the association of an infectious agent with lymphocyte cytotoxicity defects, either of congenital genetic origin in children or presumably acquired in adults. In HLH patients, an excess of lymphocyte or macrophage cytokines, such as IFN-γ and TNFα is present in serum. In animal models of the disease, IFN-γ and TNF-α have been shown to play a central pathogenic role. In humans, unusually high concentrations of IL-18, an inducer of IFN-γ, and TNF-α have been reported, and are associated with an imbalance between IL-18 and its natural inhibitor IL-18 binding protein (IL-18BP) resulting in an excess of free IL-18. Here we studied whether IL-18BP could reduce disease severity in an animal model of HLH. Mouse cytomegalovirus infection in perforin-1 knock-out mice induced a lethal condition similar to human HLH characterized by cytopenia with marked inflammatory lesions in the liver and spleen as well as the presence of hemophagocytosis in bone marrow. IL-18BP treatment decreased hemophagocytosis and reversed liver as well as spleen damage. IL-18BP treatment also reduced both IFN-γ and TNF-α production by CD8(+) T and NK cells, as well as Fas ligand expression on NK cell surface. These data suggest that IL-18BP is beneficial in an animal model of HLH and in combination with anti-infectious therapy may be a promising strategy to treat HLH patients.
2003	Genomic occupancy of HLH, AP1 and Runx2 motifs within a nuclease sensitive site of the Runx2 gene.	22886425	Epigenetic mechanisms mediating expression of the Runt-related transcription factor Runx2 are critical for controlling its osteogenic activity during skeletal development. Here, we characterized bona fide regulatory elements within 120 kbp of the endogenous bone-related Runx2 promoter (P1) in osteoblasts by genomic DNase I footprinting and chromatin immuno-precipitations (ChIPs). We identified a ~10 kbp genomic domain spanning the P1 promoter that interacts with acetylated histones H3 and H4 reflecting an open chromatin conformation in MC3T3 osteoblasts. This large chromatin domain contains a single major DNaseI hypersensitive (DHS) region that defines a 0.4 kbp "basal core" promoter. This region encompasses two endogenous genomic protein/DNA interaction sites (i.e., footprints at Activating Protein 1 [AP1], E-box and Runx motifs). Helix-Loop-Helix (HLH)/E-box occupancy and presence of the DHS region persists in several mesenchymal cell types, but AP1 site occupancy occurs only during S phase when Runx2 expression is minimal. Point-mutation of the HLH/E box dramatically reduces basal promoter activity. Our results indicate that the Runx2 P1 promoter utilizes two stable principal protein/DNA interaction domains associated with AP1 and HLH factors. These sites function together with dynamic and developmentally responsive sites in a major DHS region to support epigenetic control of bone-specific transcription when osteoblasts transition into a quiescent or differentiated state.
2004	[Screening for cytotoxic defects with flow cytometric detection of CD107α on natural killer cells and cytotoxic lymphocyte cells].	22883044	To establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 (LAMP-1, CD107α) on the cell surface of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) and evaluate the screening value of this assay for cytotoxic defects-related diseases such as familial hemophagocytic lymphopro-liferative (FHL) syndrome.Three suspected Chediak-Higashi Syndrome (CHS) patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin (PHA) and CD8+T cells by anti-CD3, the CD107α expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the pathogenic genes via DNA-PCR/RT-PCR and direct sequencing.The CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [(0.18 ± 0.07)% vs. (4.47 ± 2.36)%, P < 0.05] and NK cells after activation by PHA [(0.27 ± 0.07)% vs. (5.80 ± 2.83)%, P < 0.05]. The frequency of CD107α-expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0.5%, 0.6% vs. (5.80 ± 2.83)%] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL cells also showed no significant difference [0.3%, 0.9%, 0.2% vs. (4.47 ± 2.36)%] in three patients. All of their mean fluorescence intensity (MFI) showed the same trend. Patient 1 and 3 were identified to have LYST mutations (Patient 1: c.5411-5414 del TTTC, L1741fsX1758 and c.7975 C > T, R2596X; Patient 3: c.4863G > A, R1563H and c.5392-5393delAA, E1739fsX1756). There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STX11 were identified.We developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.
2005	Septo-optic dysplasia and hemophagocytic lymphohistiocytosis in an infant.	22876565	Macrophage activation in hemophagocytic lymphohistiocytosis (HLH) leads to severe inflammation resulting in cytopenias and multi-organ dysfunction. Septo-optic dysplasia (SOD) is an as-yet unaffiliated disorder that manifests with optic, hepatic, endocrine and/or constitutional defects. We detail the first reported occurrence of both HLH and SOD in one patient. This two-month old patient presented with acute hepatitis, direct hyperbilirubinemia, anemia and thrombocytopenia. Treatment followed standard of care practices for SOD and HLH. The patient subsequently underwent an allogeneic bone marrow transplant within eight months of diagnosis and remained in full remission at day +90. We suggest considering a diagnosis of HLH in patients with SOD who present with severe liver failure refractory to standard therapy.
2006	P6981, an arylstibonic acid, is a novel low nanomolar inhibitor of cAMP response element-binding protein binding to DNA.	22851716	Several basic leucine zipper (B-ZIP) transcription factors have been implicated in cancer, substance abuse, and other pathological conditions. We previously identified arylstibonic acids that bind to B-ZIP proteins and inhibit their interaction with DNA. In this study, we used electrophoretic mobility shift assay to analyze 46 arylstibonic acids for their activity to disrupt the DNA binding of three B-ZIP [CCAAT/enhancer-binding protein α, cyclic AMP-response element-binding protein (CREB), and vitellogenin gene-binding protein (VBP)] and two basic helix-loop-helix leucine zipper (B-HLH-ZIP) [USF (upstream stimulating factor) and Mitf] proteins. Twenty-five arylstibonic acids showed activity at micromolar concentrations. The most active compound, P6981 [2-(3-stibonophenyl)malonic acid], had half-maximal inhibition at ~5 nM for CREB. Circular dichroism thermal denaturation studies indicated that P6981 binds both the B-ZIP domain and the leucine zipper. The crystal structure of an arylstibonic acid, NSC13778, bound to the VBP leucine zipper identified electrostatic interactions between both the stibonic and carboxylic acid groups of NSC13778 [(E)-3-(3-stibonophenyl)acrylic acid] and arginine side chains of VBP, which is also involved in interhelical salt bridges in the leucine zipper. P6981 induced GFP-B-ZIP chimeric proteins to partially localize to the cytoplasm, demonstrating that it is active in cells. P6981 inhibited the growth of a patient-derived clear cell sarcoma cell line whose oncogenic potential is driven by a chimeric protein EWS-ATF1 (Ewing's sarcoma protein-activating transcription factor 1), which contains the DNA binding domain of ATF1, a B-ZIP protein. NSC13778 inhibited the growth of xenografted clear cell sarcoma, and no toxicity was observed. These experiments suggest that antimony containing arylstibonic acids are promising leads for suppression of DNA binding activities of B-ZIP and B-HLH-ZIP transcription factors.
2007	Quantification of Epstein-Barr virus DNA is helpful for evaluation of chronic active Epstein-Barr virus infection.	22850617	Chronic active Epstein-Barr virus infection (CAEBV) presents with chronic or recurrent infectious mononucleosis-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Immunological methods are useful for the diagnosis of viral infections. However, CAEBV patients do not necessarily have high titers of Epstein-Barr virus (EBV)-specific antibodies. Hosts that are immunocompromised after hematopoietic stem cell transplantations sometimes suffer from systemic EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV-positive lymphoma. Patients with EBV-associated diseases are often diagnosed by analyses of bone marrow. Cytomegalovirus (CMV) can cause serious pneumonia or retinitis in immunocompromised hosts. In order to noninvasively understand the clinical status of patients with EBV-associated diseases, we conducted real-time polymerase chain reaction (PCR) methods in their peripheral blood in order to quantify EBV and CMV DNA levels, which reflect viral activity. Here, we describe a 30-year-old Japanese female patient with CAEBV. The patient had repeated fever, fatigue, and liver dysfunction. The histopathological results of liver biopsies were positive for EBV-encoded RNA-1. Acute hepatitis was associated with the EBV infection. The whole-blood EBV DNA levels were high and above 1.0 × 10⁷ copies/mL. After immunosuppressive and antiviral therapies, EBV DNA levels lowered. However, she had to receive bone marrow transplantation because of her EBV-HLH. As the number of lymphocytes increased in the post-transplantation period, EBV DNA levels gradually increased again. The simultaneous detection of CMV DNA was more sensitive than the CMV antigenemia test that is often used to diagnose CMV infections. Unfortunately, the patient died due to a fungal infection. Observing EBV DNA levels closely with real-time quantitative PCR methods is helpful for evaluating the changes in the clinical course.
2008	Early growth response 2 negatively modulates osteoclast differentiation through upregulation of Id helix-loop-helix proteins.	22842221	Early growth response 2 (Egr2) is a zinc finger transcription factor that acts as an important modulator of various physiological processes. In this study, we show that Egr2 negatively regulates receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. The overexpression of Egr2 in bone marrow-derived macrophages (BMMs) suppresses the formation of multinuclear osteoclasts and the expression of osteoclastogenic markers, including nuclear factor of activated T cells c1 (NFATc1). On the other hand, Egr2 overexpression does not impact the phagocytic activity of osteoclast precursors or the expression of macrophage-specific markers in the presence of the osteoclastogenic stimuli, RANKL and M-CSF. We further demonstrate that Egr2 induces the expression of the inhibitors of differentiation/DNA binding (Ids) helix-loop-helix (HLH) transcription factors, which are important repressors in RANKL-mediated osteoclastogenesis. Egr2 transactivates the Id2 promoter and increases its recruitment to the Id2 promoter region. In addition, Egr2-dependent induction of Id2 promoter activity, and its binding to the Id2 promoter is abrogated by the overexpression of the Egr2 repressor, NGFI-A binding protein 2 (Nab2). Accordingly, coexpression with Nab2 restores Egr2-mediated suppression of osteoclast differentiation. Furthermore, knockdown of Egr2 using shRNA enhances osteoclastogenesis and decreases Id2 gene expression. Ectopic expression of Id2 reverses the phenotype mediated by Egr2 silencing. Taken together, our results identify Egr2 as an important modulator of RANKL-induced osteoclast differentiation and provide the link between RANKL, Egr2 and Id proteins in osteoclast-lineage cells.
2009	Drug repurposing in pediatrics and pediatric hematology oncology.	22835502	Drug 'repurposing', that is, using old drugs for new indications, has been proposed as a more efficient strategy for drug development than the current standard of beginning with novel agents. In this review, we explore the scope of drug repurposing in pediatric hematology oncology and in pediatrics in general. Drugs commonly used in children were identified using the Harriet Lane Handbook (HLH) and searched in PubMed for different uses. Additional drugs were identified by searching PubMed and Google.com for 'drug repurposing' or 'drug repositioning'. Almost 10% of drugs with primary uses in pediatrics have been repurposed in pediatric hematology oncology or pediatrics. The observant clinician, pharmacologist and translational bioinformatician, as well as structural targeting, will have a role in discovering new repurposing opportunities.
2010	Neuropsychological assessment in a case of adult-onset hemophagocytic lymphohistiocytosis (HLH).	22784400	We present a case of an individual diagnosed with hemophagocytic lymphohistiocytosis (HLH), an extremely rare and commonly fatal disorder characterized by rapid dysregulation of immune system processes. Typical age of onset is in childhood, with adult-onset occurring less frequently. The pathophysiology of this condition is characterized by a hyperinflammatory response with infiltration of visceral organs, lymph nodes, bone marrow, and the central nervous system. The clinical presentation has been documented in the extant medical literature. However, there appear to be no published reports of neuropsychological functioning in HLH patients. The patient we present here is a 28-year-old woman with 16 years of education who developed HLH subsequent to systemic lupus erythematosus flare-up. She was initially comatose for 3 weeks. Acute MRI reported multiple subcortical abnormalities, including the brainstem. The patient underwent chemotherapy, immunosuppresant, and steroid treatments. She underwent a neuropsychological evaluation at 2.5 and 7 months post initial presentation. Preliminary neuropsychological evaluation found impairments in motor abilities and aspects of executive functions. Subsequent evaluation showed improved executive function and relative sparing of higher-order cognitive abilities, but continued impairment on motor tests. To our knowledge this is the first study to report neuropsychological data for an adult diagnosed with HLH.
2011	[A case report of adult onset of primary hemophagocytic syndrome with literature review].	22781720	To introduce the clinical manifestations and laboratory tests of adult onset of primary hemophagocytic syndrome (HPS), and to investigate the essentials of diagnosis and the genotype characteristics in adult onset patient.The definite diagnosis of HPS was made according to HLH-2004. Exons of PRF1, STX11, UNC13D, SH2D1A and RAB27A genes coding region were amplified using polymerase chain reaction.A 48-year-old man was admitted to our hospital because of recurrent fever, pancytopenia and lymph node enlargement. His laboratory test revealed bone marrow hemophagocytosis, elevated ferritin level (2000 µg/L), reduced level of NK cell activity (20.13%) and elevated soluble CD25 level (12277 U/ml). Based on the HLH-2004 diagnostic criteria, the patient was diagnosed as HPS. The patient had viral infection, and no other primary disease was identified that would cause HPS. The patient responded poorly to anti-viral therapy. DNA sequencing was used to confirm that perforin gene mutations might be one of the causes of the patient suffered from primary HPS.Although primary HPS usually affects infants and young children, it also occurred in teens and adults. It is essential to perform genetic screenings to patient whose illnesses recur with unknown causes. In addition, detection of molecular genetic alterations can be used to distinguish primary HPS from acquired HPS.
2012	Assessment of immunochemotherapy and stem cell transplantation on EBV-associated hemophagocytic lymphohistiocytosis in children: a systematic review and meta analysis.	22774410	Although immunochemotherapy had been reported to be effective initial treatment for patients with Epstein Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH), and stem cell transplantation (SCT) was employed for patients with refractory disease, the long-term outcome of these patients underwent such treatment remained uncertain. The main purpose of this study was to make a primary system review on the outcome of EBV-HLH patients treated with immunochemotherapy and/or SCT.A system review and meta analysis was conducted on studies which collected from published PubMed and China Knowledge Resource Integrated Database (CNKI). The analysis was based on clinical characteristics and follow-up. Search strategy and selection criteria were identified by relevant articles, the period was defined from January 1990 to October 2010. Search terms included all relevant terms. English and Chinese language papers were reviewed.A total of 11 articles include 342 EBV-HLH patients that were identified with our search terms fulfilled the eligibility criteria. Overall 104/342 patients (30.4%) died at the end of respective study. In 288 patients who did not receive SCT, 93/288 patients (32.3%) patients died. While in 54 patients who underwent SCT, 11/54 patients (20.4%) died at the end of respective study. Four articles had the contents both of immunochemotherapy and SCT. When using a meta analysis compared the mortality between immunochemotherapy and SCT groups, there was no statistical significance could be found, the Odds Ratio is 1.10 (0.43-2.84), (p = 0.84). When compared the mortality between SCT group and total EBV-HLH patients, there was still no statistical significance could be found, the Odds Ratio is 0.99 (0.39-2.53), (p = 0.98).Etoposide-containing immunochemotherapy and SCT both decreased the mortality in EBV-HLH patients in the past decade. There was not enough evidence to suggest that SCT is better than immunochemotherapy in children with EBV-HLH. And such result may justify further research.
2013	Structure, dynamics and domain organization of the repeat protein Cin1 from the apple scab fungus.	22771296	Venturia inaequalis is a hemi-biotrophic fungus that causes scab disease of apple. A recently-identified gene from this fungus, cin1 (cellophane-induced 1), is up-regulated over 1000-fold in planta and considerably on cellophane membranes, and encodes a cysteine-rich secreted protein of 523 residues with eight imperfect tandem repeats of ~60 amino acids. The Cin1 sequence has no homology to known proteins and appears to be genus-specific; however, Cin1 repeats and other repeat domains may be structurally similar. An NMR-derived structure of the first two repeat domains of Cin1 (Cin1-D1D2) and a low-resolution model of the full-length protein (Cin1-FL) using SAXS data were determined. The structure of Cin1-D1D2 reveals that each domain comprises a core helix-loop-helix (HLH) motif as part of a three-helix bundle, and is stabilized by two intra-domain disulfide bonds. Cin1-D1D2 adopts a unique protein fold as DALI and PDBeFOLD analysis identified no structural homology. A (15)N backbone NMR dynamic analysis of Cin1-D1D2 showed that a short stretch of the inter-domain linker has large amplitude motions that give rise to reciprocal domain-domain mobility. This observation was supported by SAXS data modeling, where the scattering length density envelope remains thick at the domain-domain boundary, indicative of inter-domain dynamics. Cin1-FL SAXS data models a loosely-packed arrangement of domains, rather than the canonical parallel packing of adjacent HLH repeats observed in α-solenoid repeat proteins. Together, these data suggest that the repeat domains of Cin1 display a "beads-on-a-string" organization with inherent inter-domain flexibility that is likely to facilitate interactions with target ligands.
2014	Hemophagocytic lymphohistiocytosis associated with H1N1 virus infection and visceral leishmaniasis in a 4.5-month-old infant.	22760147	We present a case of a 4.5-month-old boy from Turkey with hemophagocytic lymphohistiocytosis (HLH) associated with H1N1 virus and Leishmania spp. coinfection. Because visceral leishmaniasis can mimic hematologic disorders like HLH, it is important to rule out this clinical condition before starting immunosuppressive therapy. In our case, treatment with liposomal amphotericin B resulted in a dramatic resolution of clinical and laboratory abnormalities.
2015	Transcriptional regulation of HLH-6-independent and subtype-specific genes expressed in the Caenorhabditis elegans pharyngeal glands.	22759833	The Caenorhabditis elegans pharyngeal glands represent one of five cell types in the pharynx. We have previously shown that the bHLH transcription factor, HLH-6, is required for gland development and for expression of many, but not all, gland genes (Smit et al., 2008). Here, we have identified additional gland-expressed genes and find that transcriptional regulatory inputs other than HLH-6 are necessary for their regulation. We demonstrate that at least two hlh-6 independent gland genes, nas-12 and Y8A9A.2, require a cis-acting motif (HRL3- Hlh-6 Regulatory eLement 3), previously described based on its requirement for hlh-6 expression (Ghai and Gaudet, 2008). We also show that expression of the gland-expressed genes, ZK596.1, scl-3, wrt-3, and Y76B12C.3, rely on cis-elements and trans-acting factor(s) other than HLH-6 and HRL3. In addition, we show that negative regulatory mechanisms are employed to refine the spatial expression of some genes, resulting in expression in only a subset of the five gland cells. We show that one of these genes, Y8A9A.2, is negatively regulated by the NHR transcription factor encoded by nhr-48, which represses Y8A9A.2 expression in the g1A cells. We also show that another gene expressed in the reciprocal subset of gland cells, phat-5, is negatively regulated in the g1P and g2 cells by an unknown factor acting through a conserved cis-element in the phat-5 promoter. Overall, this work reveals levels of regulation of gene expression in a single cell type beyond that previously known, and suggests mechanisms by which the different gland sub-types are distinguished.
2016	Hemophagocytic lymphohistiocytosis of indeterminate cause: a fatal adult case.	31528566	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon life-threatening disorder characterized by wide spread non-neoplastic proliferation and inappropriate activation of mature macrophages resulting in hypercytokinemia. This uncontrollable and ineffective systemic immune response causes fever, hepatosplenomegaly, cytopenias and subsequently multiorgan failure. The authors report a case of a 41-year-old male patient with a 30-day history of weight loss, fever, icterus, hepatomegaly, and cytopenias. The diagnostic workup disclosed hypertriglyceridemia, hypofibrinogenemia, and elevated ferritin. Bone marrow examination and clinical course raised the suspicion of HLH and treatment was started with high-dose corticosteroids and immune globulin. The patient underwent multi-organ failure and expired after 58 days of hospitalization. The autopsy finding included massive bone marrow infiltration by non-neoplastic histiocytes, many of them showing hemophagocytosis, which immunohistochemical study revealed diffuse CD68-positive histiocytes, which were negative for S100 protein. Hemophagocytosis was also observed in the lungs, lymph nodes and liver. The immediate cause of death was attributed to a massive intestinal bleeding due to extensive ischemic necrosis at the duodenum/jejunal transition area.
2017	Severe transient left ventricular pseudohypertrophy during treatment of hemophagocytic lymphohistiocytosis: a case report.	22735880	An association between hemophagocytic lymphohistiocytosis (HLH) and severe transient left ventricular (LV) hypertrophy has not been described to date. Possible explanations, including etoposide toxicity, are discussed.A 2-month-old male infant with HLH was treated according to the HLH-2004 protocol. Initial cardiac evaluation was within normal limits. During the second month of therapy, a heart murmur was discovered; electrocardiogram demonstrated signs of LV hypertrophy, and echocardiogram confirmed the presence of thickness of LV walls. This complication was transient: clinical findings, echocardiogram, and electrocardiogram recorded 6 months afterward were all within normal limits.The case suggests the need for close echocardiographic monitoring during HLH treatment.
2018	Life-threatening scrub typhus with hemophagocytosis and acute respiratory distress syndrome in an infant.	22735791	Scrub typhus is a rickettsial disease, caused by Orientia tsutsugamushi, which is transmitted via the bite of a chigger. This disease is one of the most important infectious diseases in the Asia-Pacific area; however, a severe infant case has not yet been reported. Here, we present the case of an 8-month-old boy with scrub typhus accompanied by hemophagocytic lymphohistiocytosis (HLH). His rapid course was complicated by acute respiratory distress syndrome (ARDS), status epilepticus and disseminated intravascular coagulation (DIC). He recovered after clarithromycin therapy and intensive supportive care. Although being extremely rare, scrub typhus can be life-threatening in an infant; therefore, physicians in endemic countries should be aware of the necessity for early recognition and prompt treatment of suspected cases.
2019	New structural determinants for c-Myc specific heterodimerization with Max and development of a novel homodimeric c-Myc b-HLH-LZ.	22733550	c-Myc must heterodimerize with Max to accomplish its functions as a transcription factor. This specific heterodimerization occurs through the b-HLH-LZ (basic region, helix 1-loop-helix 2-leucine zipper) domains. In fact, many studies have shown that the c-Myc b-HLH-LZ (c-Myc'SH) preferentially forms a heterodimer with the Max b-HLH-LZ (Max'SH). The primary mechanism underlying the specific heterodimerization lies on the destabilization of both homodimers and the formation of a more stable heterodimer. In this regard, it has been widely reported that c-Myc'SH has low solubility and homodimerizes poorly and that repulsions within the LZ domain account for the homodimer instability. Here, we show that replacing one residue in the basic region and one residue in Helix 1 (H(1)) of c-Myc'SH with corresponding residues conserved in b-HLH proteins confers to c-Myc'SH a higher propensity to form a stable homodimer in solution. In stark contrast to the wild-type protein, this double mutant (L362R, R367L) of the c-Myc b-HLH-LZ (c-Myc'RL) shows limited heterodimerization with Max'SH in vitro. In addition, c-Myc'RL forms highly stable and soluble complexes with canonical as well as non-canonical E-box probes. Altogether, our results demonstrate for the first time that structural determinants driving the specific heterodimerization of c-Myc and Max are embedded in the basic region and H(1) of c-Myc and that these can be exploited to engineer a novel homodimeric c-Myc b-HLH-LZ with the ability of binding the E-box sequence autonomously and with high affinity.
2020	Are all mutant SNARES equal?	2273052022451424	In this issue of Blood, Pagel et al carefully deliniate some fascinating phenotype/genotype correlations in a larger cohort than in their earlier reports of familial hemophagocytic lymphohistiocytosis (HLH).
2021	Transcription factor redundancy and tissue-specific regulation: evidence from functional and physical network connectivity.	22730465	Two major transcriptional regulators of Caenorhabditis elegans bodywall muscle (BWM) differentiation, hlh-1 and unc-120, are expressed in muscle where they are known to bind and regulate several well-studied muscle-specific genes. Simultaneously mutating both factors profoundly inhibits formation of contractile BWM. These observations were consistent with a simple network model in which the muscle regulatory factors drive tissue-specific transcription by binding selectively near muscle-specific targets to activate them. We tested this model by measuring the number, identity, and tissue-specificity of functional regulatory targets for each factor. Some joint regulatory targets (218) are BWM-specific and enriched for nearby HLH-1 binding. However, contrary to the simple model, the majority of genes regulated by one or both muscle factors are also expressed significantly in non-BWM tissues. We also mapped global factor occupancy by HLH-1, and created a genetic interaction map that identifies hlh-1 collaborating transcription factors. HLH-1 binding did not predict proximate regulatory action overall, despite enrichment for binding among BWM-specific positive regulatory targets of hlh-1. We conclude that these tissue-specific factors contribute much more broadly to the transcriptional output of muscle tissue than previously thought, offering a partial explanation for widespread HLH-1 occupancy. We also identify a novel regulatory connection between the BWM-specific hlh-1 network and the hlh-8/twist nonstriated muscle network. Finally, our results suggest a molecular basis for synthetic lethality in which hlh-1 and unc-120 mutant phenotypes are mutually buffered by joint additive regulation of essential target genes, with additional buffering suggested via newly identified hlh-1 interacting factors.
2022	PET/CT images of a patient with haemophagocytic lymphohistiocytosis.	22729341	Haemophagocytic lymphohistiocytosis (HLH) is a rare immune disorder that predominantly affects macrophages and T lymphocytes and leads to multiple organ disease and death. The characteristic pathological finding in the bone marrow and the other affected tissues is haemophagocytosis of macrophages (macrophages digesting erythrocyte). Primary (hereditary) and secondary (acquired) forms of the disease are present. A patient with documented HLH disease revealed by positron emission tomography/CT is reported in this paper.
2023	Hyperferritinemia in the critically ill child with secondary hemophagocytic lymphohistiocytosis/sepsis/multiple organ dysfunction syndrome/macrophage activation syndrome: what is the treatment?	22715953	Hyperferritinemia is associated with increased mortality in pediatric sepsis, multiple organ dysfunction syndrome (MODS), and critical illness. The International Histiocyte Society has recommended that children with hyperferritinemia and secondary hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) should be treated with the same immunosuppressant/cytotoxic therapies used to treat primary HLH. We hypothesized that patients with hyperferritinemia associated secondary HLH/sepsis/MODS/MAS can be successfully treated with a less immunosuppressant approach than is recommended for primary HLH.We conducted a multi-center cohort study of children in Turkish Pediatric Intensive Care units with hyperferritinemia associated secondary HLH/sepsis/MODS/MAS treated with less immunosuppression (plasma exchange and intravenous immunoglobulin or methyl prednisolone) or with the primary HLH protocol (plasma exchange and dexamethasone or cyclosporine A and/or etoposide). The primary outcome assessed was hospital survival.Twenty-three children with hyperferritinemia and secondary HLH/sepsis/MODS/MAS were enrolled (median ferritin = 6341 μg/dL, median number of organ failures = 5). Univariate and multivariate analyses demonstrated that use of plasma exchange and methyl prednisolone or intravenous immunoglobulin (n = 17, survival 100%) was associated with improved survival compared to plasma exchange and dexamethasone and/or cyclosporine and/or etoposide (n = 6, survival 50%) (P = 0.002).Children with hyperferritinemia and secondary HLH/sepsis/MODS/MAS can be successfully treated with plasma exchange, intravenous immunoglobulin, and methylprednisone. Randomized trials are required to evaluate if the HLH-94 protocol is helpful or harmful compared to this less immune suppressive and cytotoxic approach in this specific population.
2024	Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells.	22701468	Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.
2025	[Current and future directions of pathologic analysis in hemophagocytic syndrome].	22690617	Hemophagocytic syndrome is a rare disorder with dysfunction of cytotoxic T lymphocyte (CTL) or NK cell activity, leading to excessive production of inflammatory cytokines and various clinical symptoms. HLH can be classified as either primary or secondary form; primary HLH includes familial hemophagocytic lymphohistiocytosis (FHL) and several immune deficiencies. All affected genes are involved in the transport and membrane fusion, or exocytosis of perforin/granzyme in lytic granules. Making a rapid screening of FHL with flow cytometry followed by genetic analysis is mandatory for the appropriate treatment of this fatal disease. Whereas, pathogenesis of secondary HLH is still unknown; several genetic backgrounds to affect on the pathway of T-cell activity will be associated with secondary HLH. With perforin- or Munc-deficient mouse model that develop HLH-like symptoms after virus infection, CD8+ T cells and interferon-gamma have been proven to be necessary for the HLH development. These data will provide new targets for specific therapeutic intervention of HLH in the future.
2026	Recent advances in the diagnosis and treatment of hemophagocytic lymphohistiocytosis.	22682420	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease of severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages secreting high amounts of inflammatory cytokines. It is a frequent manifestation in patients with predisposing genetic defects, but can occur secondary to various infectious, malignant, and autoimmune triggers in patients without a known genetic predisposition. Clinical hallmarks are prolonged fever, cytopenias, hepatosplenomegaly, and neurological symptoms, but atypical variants presenting with signs of chronic immunodeficiency are increasingly recognized. Impaired secretion of perforin is a key feature in several genetic forms of the disease, but not required for disease pathogenesis. Despite progress in diagnostics and therapy, mortality of patients with severe HLH is still above 40%. Reference treatment is an etoposide-based protocol, but new approaches are currently explored. Key for a favorable prognosis is the rapid identification of an underlying genetic cause, which has been facilitated by recent immunological and genetic advances. In patients with predisposing genetic disease, hematopoietic stem cell transplantation is performed increasingly with reduced intensity conditioning regimes. Current research aims at a better understanding of disease pathogenesis and evaluation of more targeted approaches to therapy, including anti-cytokine antibodies and gene therapy.
2027	SAP and XIAP deficiency in hemophagocytic lymphohistiocytosis.	2267219423279032	Hemophagocytic lymphohistiocytosis (HLH) is a multisystem inflammatory disorder due to cytokine overproduction from excessively activated lymphocytes and macrophages. HLH has been divided into two subgroups: primary HLH and secondary HLH. Primary HLH includes PRF1, UNC13D, STX11, STXBP2, RAB27A, LYST, SH2D1A and XIAP gene mutations; and secondary HLH is associated with infections, malignancies and autoimmune diseases. Among primary HLH-related genes, SH2D1A and XIAP are genetically responsible for X-linked lymphoproliferative syndrome (XLP) due to signaling-lymphocytic-activation-molecule-associated protein (SAP) and XIAP deficiencies, respectively. XLP is characterized by extreme vulnerability to Epstein-Barr virus infection. The major clinical manifestations of XLP consist of HLH (60%), lymphoproliferative disorder (30%) and dysgammaglobulinemia (30%). Analysis of clinical phenotypes of XLP patients suggests that XLP predominantly shows familial HLH phenotypes, whereas some XLP patients present sporadic HLH. For many decades, clinicians and investigators have been concerned with possible XLP in young boys presenting with Epstein-Barr-virus-associated HLH. This review aims to describe the new knowledge about XLP and to draw the attention of the pediatrician to XLP, which should be differentiated from other forms of HLH.
2028	[Clinical characteristics and disease course in children with haemophagocytic lymphohistiocytosis treated at the University Children's Hospital in Belgrade].	22650106	Haemophagocytic lymphohistiocytosis (HLH) is a disorder characterised by long-standing fever, splenomegaly and bicytopoenia or pancytopoenia. Lymphadenopathy, jaundice and neurological symptoms mayalsooccur. HLH may ensue in various forms of innate or acquired immunodeficiency with impaired cytotoxic lymphocyte function resulting in excessive macrophage activation.To describe and analyse clinical characteristics of patients treated for HLH at the University Children's Hospital of Belgrade from August 2000 to August 2010.Retrospective analysis of medical records.Diagnosis of HLH was established in 13 children (five boys and eight girls) aged from one month to 14 years. In six children HLH was secondary (to visceral leishmaniasis in two, Ebstein-Barr virus infection in one, Langerhans' cell histiocytosis in one and systemic juvenile rheumatoid arthritis in two). Of the remaining seven patients, genes for perforin and syntaxin 11 were examined in two and no mutations were found. Of the remaining seven patients, genes encoding perforin and syntaxin 11 were analyzed in two, but no mutations were found. All children had fever, splenomegaly, cytopoenias, hyperferritinaemia and hypertriglyceridaemia, but haemophagocytosis was seen in only six (46.1%). Six children were cured (four with secondary HLH and two with primary HLH).Two children are undergoing treatment, while five succumbed (three before treatment could be administered and two due to complications). In four of the six cured children, HLH arose in the first year of life. Cure rate in those who underwent haematopoietic stem cell transplantation was 2/3.Results underscore the importance of timely diagnosis and treatment of HLH, warranting that in all children with fever, splenomegaly and/or cytopoenias, with or without haemophagocytosis, HLH be actively sought.
2029	Fulminant Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	22648663	Hemophagocytic lymphohistiocytosis (HLH) is a rare, severe, and often fatal disorder. Its hereditary and sporadic form can present as a significant diagnostic challenge to the otolaryngologist. This report describes two fatal cases of adult patients with HLH initially presented as infectious mononucleosis to an otorhinolaryngologist. The clinical presentation, serological and histological features, and management are discussed.
2030	[Fatal haemophagocytic lymphohistiocytosis and mononucleosis in a patient with colitis ulcerosa].	22640789	We report a case of Epstein-Barr virus primo infection with the development of lethal haemophagocytic lymphohistiocytosis (HLH) in a 22 year-old man, who was being treated with azathioprin for colitis ulcerosa. HLH is a rare, life-threatening disease, which is caused by an inappropriate activation of the immune system with haemophagocytosis and multiorgan system dysfunction. Early initiation of treatment significantly improves survival.
2031	Scrub typhus-associated severe hemophagocytic lymphohistiocytosis with encephalomyelitis leading to permanent sequelae: a case report and review of the literature.	22627574	Hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially fatal disorder. There have been a few reports on HLH secondary to scrub typhus in adults. Here, we describe the case of a 9-year-old Korean girl who presented with the typical findings of HLH. Despite adequate antirickettsial and HLH treatment, the neurological impairment worsened and remained. This is the first case report of severe neurological impairment resulting from the very rare association of HLH with scrub typhus. Therefore, in endemic areas, a high index of suspicion for scrub typhus is warranted in patients presenting with HLH.
2032	Frequency and outcomes of cardiac operations and catheter interventions in Turner syndrome.	22621800	Cardiac malformations occur commonly in Turner syndrome (TS), but the outcomes of cardiac operations and catheter-based procedures are unknown. The Pediatric Cardiac Care Consortium database was queried for individuals with TS and other female subjects without genetic abnormalities or syndromes (non-TS [NTS]). Procedures for left-sided heart lesions represented most TS procedures (95.2%). Three hundred ninety-eight patients with TS who underwent 637 of these procedures of interest were compared with 25,913 female NTS subjects who underwent 56,625 procedures. The numbers of procedures per admission (1.47 vs 1.61, p = 0.01) and per patient (1.85 vs 2.16, p <0.0001) were significantly lower in patients with TS. Procedures for cyanotic heart disease other than hypoplastic left heart (HLH) were performed 4.5-fold less frequently in patients with TS. Patients with TS and NTS subjects had equivalent hospital lengths of stay, except for patients with TS who underwent hypoplastic aortic arch operations, patent ductus arteriosus ligation, pulmonary artery balloon dilation, balloon atrial septostomy, and catheter closure of atrial septal defects. There were 34 deaths among patients with TS and 1,795 among NTS subjects (8.6% vs 7.2%, p = 0.30). When HLH was excluded, mortality was lower in the TS group (3.9% vs 6.5%, p = 0.05). Operations for partial anomalous pulmonary venous connection (14.3% vs 1.9%, p = 0.03) and HLH (90.4% vs 70.5%, p = 0.08) were more likely to result in death in patients with TS. In conclusion, given generally comparable lengths of stay and numbers of procedures as well as uniformly excellent results, these data suggest that the diagnosis of TS does not increase the utilization of limited health care resources. Operations for HLH and partial anomalous pulmonary vein connection carry additional risk for those with TS. These results will permit risk stratification, prognostication, and counseling of individuals with TS and their families.
2033	Clinical analysis of macrophage activation syndrome in pediatric patients with autoimmune diseases.	22615046	Macrophage activation syndrome (MAS) belongs to secondary hemophagocytic lymphohistiocytosis (HLH) syndrome. It is usually associated with rheumatic diseases. We retrospectively reviewed our hospital's medical records of 102 HLH/MAS patients from the past 20 years. Demographics, clinical data, treatment, and outcomes were analyzed. Among 102 patients, eight patients with underlying juvenile systemic lupus erythematous (two patients), mixed connective tissue disease (one patient), primary anti-phospholipid syndrome (one patient), and systemic type juvenile rheumatoid arthritis (sJRA; four patients) with 13 episodes of MAS were studied. Clinical manifestations of MAS included fever (100 %), hepatosplenomegaly (77 %), lymphadenopathy (38 %), skin rash (62 %), and neurological involvement (31 %). Laboratory features included leukopenia (54 %), anemia (46 %), thrombocytopenia (77 %), jaundice (27 %), hypofibrinogenemia (40 %), decreased erythrocyte sedimentation rate (67 %), and elevated liver enzymes (77 %), lactate dehydrogenase (100 %), ferritin (88 %), triglycerides (91 %), C-reactive protein (85 %), plasma D-dimer (50 %), and hemophagocytosis in bone marrow (83 %). The Epstein-Barr virus and adenovirus infection triggered MAS in two patients with sJRA. Methylprednisolone pulse therapy was effective in two out of three patients, and high-dose intravenous immunoglobulin (IVIG) was effective in two out of six patients. Patients with sJRA responded well to corticosteroids and cyclosporine. Complications included opportunistic infection with Pneumocystis jiroveci, multiple organ failure, and intensive care unit myopathy. The mortality rate was one out of eight (12.5 %). Our results showed that MAS could be fatal and complicate various pediatric autoimmune diseases. It generally has a good response to corticosteroids and IVIG. Prompt recognition and timely treatment can result in good outcomes.
2034	Hemophagocytic syndrome in a 4-month-old infant with biotinidase deficiency.	22605457	Hemophagocytic syndromes such as hemophagocytic lymphohistiocytosis (HLH) are life-threatening hyperinflammatory conditions caused by inherited or acquired immune disorders. Awareness of the clinical symptoms and diagnostic criteria for hemophagocytic syndromes is crucial to start timely life-saving therapy. We present a case of a 4-month-old boy presenting with HLH. However, the patient was subsequently diagnosed with biotinidase deficiency and was successfully treated with biotin-replacement therapy, upon which the hemophagocytic syndrome ceased. Subsequent laboratory evaluations revealed normal lymphocyte cytotoxicity and no mutations in genes associated with familial HLH were found. Biotinidase deficiency should be considered as a differential diagnosis of patients fulfilling HLH criteria.
2035	Physical and functional interaction between the ID1 and p65 for activation of NF-κB.	22592405	Inhibitor of differentiation or DNA binding-1 (ID1) is an important helix-loop-helix (HLH) transcription factor involved in diverse biological functions including cell differentiation, proliferation, apoptosis, and senescence. Recently, it was reported that ID1 can activate the NF-κB signaling pathway in a variety of cancer cells and a T cell line, but the mechanisms involved in ID1-mediated transactivation of NF-κB are not clear. In this study, we demonstrate by both in vitro pull-down assays and a cell-based in vivo two-hybrid system that ID1-mediated NF-κB activation is due to its physical interaction with p65. We have identified that the transcriptional activation domain (TAD) in p65 and the HLH domain in ID1 are vital for their interaction. Interestingly, a single site mutation (Leu76) in the HLH domain of ID1 protein drastically decreased its ability to bind with p65. Using a dual-luciferase assay, we demonstrated that the interaction between ID1 and p65 modulates activation of the NF-κB signaling pathway in vivo. In addition, we demonstrated that, by affecting the nuclear translocation of p65, ID1 is essential in regulating TNF-α-induced p65 recruitment to its downstream target, the cellular inhibitor of apoptosis protein 2 (cIAP2) promoter.
2036	ETV6 fusion genes in hematological malignancies: a review.	22578774	Translocations involving band 12p13 are one of the most commonly observed chromosomal abnormalities in human leukemia and myelodysplastic syndrome. Their frequently result in rearrangements of the ETV6 gene. At present, 48 chromosomal bands have been identified to be involved in ETV6 translocations, insertions or inversions and 30 ETV6 partner genes have been molecularly characterized. The ETV6 protein contains two major domains, the HLH (helix-loop-helix) domain, encoded by exons 3 and 4, and the ETS domain, encoded by exons 6 through 8, with in between the internal domain encoded by exon 5. ETV6 is a strong transcriptional repressor, acting through its HLH and internal domains. Five potential mechanisms of ETV6-mediated leukemogenesis have been identified: constitutive activation of the kinase activity of the partner protein, modification of the original functions of a transcription factor, loss of function of the fusion gene, affecting ETV6 and the partner gene, activation of a proto-oncogene in the vicinity of a chromosomal translocation and dominant negative effect of the fusion protein over transcriptional repression mediated by wild-type ETV6. It is likely that ETV6 is frequently involved in leukemogenesis because of the large number of partners with which it can rearrange and the several pathogenic mechanisms by which it can lead to cell transformation.
2037	Hemophagocytic lymphohistiocytosis complicated by central nervous system lesions in a patient with dermatomyositis: a case presentation and literature review.	22576792	We report a case of dermatomyositis (DM) and hemophagocytic lymphohistiocytosis (HLH) complicated by central nervous system (CNS) lesions and review eight literature cases of DM and HLH. A 17-year-old woman, admitted to our hospital because of severe muscle weakness and high fever, was diagnosed with DM based on elevated serum levels of muscle enzymes and a typical skin rash. Pancytopenia, high serum ferritin and soluble interleukin (IL)-2 receptor, and hepatosplenomegaly were also noted. Bone-marrow examination was negative for hemophagocytosis. Steroid therapy combined with immunoglobulin i.v. was ineffective against the DM, pancytopenia, hepatic dysfunction, and hyperferritinemia. On the 27th hospital day, seizures and acute respiratory failure occurred. In the course of improving muscle enzyme levels after starting adjunctive treatment with cyclosporine, the patient suffered disturbed consciousness, dyskinesia, and tremor. Brain magnetic resonance imaging (MRI) revealed T2 hyperintense lesions in the pons. Additional cyclophosphamide pulse therapy successfully decreased serum ferritin. Unfortunately, the diffuse alveolar damage (DAD) confirmed by biopsy progressed and the patient died. Autopsy findings revealed DAD throughout both lungs, HLH liver lesions, and a hemorrhagic necrotic lesion of the pons in the brain. Even when pathological examination yields no findings of hemophagocytosis, it is important to comprehensively and rapidly diagnose HLH based on the clinical picture. Because DM complicated by HLH may be associated with abnormal production of cytokines and systemic autoimmune responses, it may be necessary to immediately administer additional immunosuppressive therapy. We describe and discuss the extraordinary, severe form of DM in our patient, along with cases in the literature.
2038	Human parechovirus-3 infection in nine neonates and infants presenting symptoms of hemophagocytic lymphohistiocytosis.	22569793	Human parechovirus-3 (HPeV-3) has been reported to cause a sepsis-like illness in neonates and young infants. We experienced the occurrence of HPeV-3 infection in nine neonates and young infants (eight boys, one girl; aged 14-52 days, median 31 days). They were admitted to our hospital with the chief complaints of fever persisting for 3-5 days (median 4 days) and lethargy. Five infants presented with abdominal distension and six had a rash (including acral reddening), as was previously reported with this viral infection. Abdominal distension with navel protrusion and acral reddening during the course were characteristic. Laboratory data were characterized by elevated values for serum AST, LDH, FDP, D-dimer, ferritin, soluble IL-2 receptor, triglyceride, choline esterase, and urinary β(2)-microglobulin. Two of our nine patients presented with a hemophagocytic lymphohistiocytosis (HLH)-like illness and required specific therapy. These data suggest that HPeV-3 is an important virus that can cause hypercytokinemia, which sometimes leads to HLH, and systemic inflammatory response syndrome in neonates and young infants.
2039	[Significance of soluble interleukin-2 receptor and NK cell activity in patients with hemophagocytic lymphohistiocytosis].	22541108	This study was aimed to detect the level of soluble interleukin-2 receptor (sCD25) and cytotoxic activity of NK lymphocytes in patients with hemophagocytic lymphohistiocytosis (HLH), and to explore their clinical significance in HLH. The enzyme-linked immunosorbent assay was used to detect the sCD25 level in serum of 20 patients with HLH, 15 healthy controls, 20 cases of acute myeloid leukemia and 20 cases of systemic lupus erythematosus. The NK cell cytotoxicity in peripheral blood of patients with HLH and controls were detected by flow cytometry with CD107a antibody labeling and LDH release assay. The results indicated that the level of sCD25 in HLH patients was significantly higher than that in healthy controls and disease groups (P < 0.001). The NK cell cytotoxicity in peripheral blood detected by both methods in patients with HLH were lower than that in healthy controls (P < 0.05), and the results detected by flow cytometry correlated significantly with those by LDH release assay (r = 0.73, P < 0.05). It is concluded that detection of sCD25 levels and NK cell activity in peripheral blood in HLH is of great value. Using flow cytometry following CD107a antibody labeling to measure NK activity is a simple, stability, reproducibility method and can be used for clinical diagnosis of HLH.
2040	Flow cytometric findings in hemophagocytic lymphohistiocytosis.	22523218	Hemophagocytic lymphohistiocytosis (HLH) is an often fatal hyperinflammatory syndrome. HLH may be inherited, but it more commonly arises secondary to Epstein-Barr virus (EBV) or other infections, hematologic malignancies, or rheumatologic diseases. We identified 17 patients diagnosed with HLH who had flow cytometric analysis of peripheral blood or bone marrow performed at the time of diagnosis. Two patients had primary HLH, and the others had HLH secondary to EBV infection, hematologic malignancies, rheumatologic conditions, or tuberculosis. The marrow typically showed a reactive lymphocytosis and a marked left shift in myelopoiesis regardless of the etiology. Qualitative abnormalities were also found in several cases, including T-cell abnormalities in the majority of the EBV-associated HLH cases. While not specific, flow cytometric findings in HLH are different from the findings in uninvolved marrow samples, and care should be taken not to overinterpret immunophenotypic findings in these cases as indicative of a primary marrow disorder or lymphoma.
2041	Salvage therapy of refractory hemophagocytic lymphohistiocytosis with alemtuzumab.	22522603	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome that remains difficult to treat. Even with current standard HLH therapy, only approximately half of patients will experience complete resolution of disease, and early mortality remains a significant problem. Salvage therapies have been described only in limited case reports, and there are no large studies of second-line therapies.We reviewed the charts of 22 pediatric and adult patients who received alemtuzumab for the treatment of refractory HLH at our center or in consultation with our group.Patients had received conventional therapies for a median of 8 weeks (range: 2-70) prior to alemtuzumab, and treatment immediately prior to alemtuzumab included dexamethasone (100%), etoposide (77%), cyclosporine (36%), intrathecal hydrocortisone ± methotrexate (23%), methylprednisolone (9%), and rituximab (14%). Patients received a median dose of 1 mg/kg alemtuzumab (range: 0.1-8.9 mg/kg) divided over a median of 4 days (range: 2-10). Fourteen patients experienced an overall partial response, defined as at least a 25% improvement in two or more quantifiable symptoms or laboratory markers of HLH 2 weeks following alemtuzumab (64%). Five additional patients had a 25% or greater improvement in a single quantifiable symptom or laboratory marker of HLH (23%). Seventy-seven percent of patients survived to undergo allogeneic hematopoietic cell transplantation. Patients experienced an acceptable spectrum of complications, including CMV and adenovirus viremia.Alemtuzumab appears to be an effective salvage agent for refractory HLH, leading to improvement and survival to HCT in many patients. Prospective trials to define optimal dosing levels, schedules, and responses are needed.
2042	Direct conversion of tenocytes into chondrocytes by Sox9.	22510437	Sox9 is a high-mobility group box-containing transcription factor that functions as a key regulator of chondrogenesis. We here report that Sox9 mediates the direct conversion of tenocytes to chondrocytes through an intermediate state in which both differentiation programs are active. Sox9 is abundantly expressed in cartilage but is undetectable in limb tendons that express Scleraxis (Scx) and Tenomodulin (Tnmd), tendon-specific early and late molecular markers, respectively. Upon forced expression of Sox9 in the chick forelimb, ectopic cartilage formation is preferentially observed in fibrous tissues including the tendons, ligaments, perichondrium/periosteum, dermis, and muscle connective tissues. Tnmd expression in tenocytes isolated from leg tendons was markedly upregulated by forced expression of basic helix-loop-helix (b-HLH) activators including Scx, Paraxis, Twist1 and Twist2. In contrast, the overexpression of Sox9 in monolayer tenocytes resulted in the downregulation of Tnmd and Scx expressions during passaging in culture, and the induction of cartilage molecular markers such as type II collagen (Col2a1) and Chondromodulin-I (ChM-I). This Sox9-driven switching from a tenocytic to a chondrocytic gene expression profile was associated with a dramatic change from a spindle to a polygonal cellular morphology. The extracellular accumulation of cartilage-characteristic proteoglycans was also observed. These data suggest that tenocytes have a strong potential for conversion into chondrocytes through the activities of Sox9 both in vitro and in vivo.
2043	Influence of collision energy and reagent rotation on the cross sections and product polarizations of the reaction F + HCl.	22502520	Quasiclassical trajectory calculations have been carried out for the F+HCl reaction in three dimensions on a recent DHSN PES of the ground 1(2)A' electronic state [M. P. Deskevich, M. Y. Hayes, K. Takahashi, R. T. Skodje, and D. J. Nesbitt, J. Chem. Phys. 124, 224303 (2006)]. The effects of the collision energy and the reagent initial rotational excitation on the cross sections and product polarization are studied for the v = 0 and j ≤ 10 states of HCl over a wide collision energy range. It has been found that either the collision energy or the HCl rotational excitation increase remarkably reaction cross sections. The QCT-calculated integral cross sections are in good agreement with previous QM results. A detailed study on product polarization for the title reaction is also performed. The calculated results show that the product rotational angular momentum j' is not only aligned, but also oriented along the direction perpendicular to the scattering plane. The orientation of the HF product rotational angular momentum vector j' depends very sensitively on the collision energy and also affected by the reagent rotation. The theoretical findings and especially the roles of the collision energy and initial rotational momentum on the product polarization are discussed and reasonably explained by the HLH mass combination, the property of the PES, as well as the reactive mechanism.
2044	[Monitoring of Epstein-Barr virus-infected cells by flow cytometer permits early diagnosis and evaluation of disease progression in EBV-associated hemophagocytic lymphohistiocytosis].	22499051	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is characterized by clonal expansion of EBV-infected CD8(+)T-cells. We have recently demonstrated that detection of a clonally expanded population of EBV-infected CD8(+)T-cells with CD5 down-regulation was a useful tool to distinguish EBV-HLH from EBV-related disorders such as severe infectious mononucleosis. A 5-year-old girl who presented with fever, pancytopenia and liver dysfunction was diagnosed by this method in addition to conventional diagnostic tests. Further, EBV-infected cells were identified as CD5(-)HLA-DR(+) TCR V β3(+) CD8(+)T cells, an increase or decrease of which over time reflected the disease severity in this patient. Treatment of patients with EBV-HLH varies from steroid alone to intensive chemotherapy or hematopoietic stem cell transplantation. Easy monitoring of EBV-infected cells by using flow cytometry over time may provide useful information to choose an appropriate treatment for each individual patient with EBV-HLH.
2045	Reduced-intensity conditioning in unrelated donor cord blood transplantation for familial hemophagocytic lymphohistiocytosis.	22488407	Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder of immune homeostasis characterized by fever, cytopenias, hepatosplenomegaly, and coagulopathy. We studied the outcomes of 13 FHL patients who underwent the first unrelated cord blood transplantation (UCBT) after non-myeloablative conditionings. The major regimen consisted of fludarabine (FLU; n = 12)+melphalan (MEL; n = 11)± low-dose total body irradiation (TBI 2-4 Gy; n = 6). The median age at presentation and period to UCBT were 6 and 5 months, respectively. Central nervous system (CNS) disease developed in one infant at diagnosis, and in two others until UCBT. HLH activity was controlled in all but one at the time of UCBT. Ten patients had early engraftment on median day 21 with no grade >2 treatment-related toxicity and two controllable grade >2 acute GVHD. Two patients with early rejection successfully underwent subsequent UCBT after myeloablative conditioning. Two others had late graft failure following mixed donor chimerism. Two deaths occurred from HLH; early liver failure and late CNS disease. Of 11 FLU+MEL-conditioned patients, the frequency of disease-free complete engraftment was higher for MEL (≥120 mg/m(2) )+TBI, or high-dose MEL (180 mg/m(2) ) than for others (83% vs. 25%, p = 0.036). The FLU+MEL-based non-myeloablative regimen was acceptable for FHL infants undergoing UCBT, although further studies will be needed for confirmation.
2046	Primary hemophagocytic lymphohistiocytosis in Iran: report from a single referral center.	22475297	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition characterized by fever, hepatosplenomegaly, and cytopenia, and widespread accumulation of lymphocytes and histiocytes, sometimes with hemophagocytosis, primarily involving the spleen, lymph nodes, bone marrow, and liver. HLH can either occur sporadically (secondary HLH) or as part of a familial syndrome (primary HLH), including familial HLH and the distinct immunodeficiency syndromes. Herein the authors report 6 Iranian patients with primary HLH and their outcome from a single tertiary-care center.
2047	Hemophagocytic lymphohistiocytosis in 2 pediatric patients secondary to hepatitis A virus infection.	22475296	Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by uncontrolled activation of cytotoxic T cells and antigen-presenting cells. Common clinical manifestations include high fever, maculopapular rash, neurological symptoms, coagulopathy, and abnormal liver function tests [1]. HLH can be either primary, that is, due to an underlying genetic defect, or secondary, associated with malignancies, autoimmune diseases, or infections. The true incidence of secondary HLH is difficult to define. Infection associated HLH are most commonly associated with viral infections mainly of the herpes group, with Epstein-Barr virus (EBV) that is proposed to be the most common cause [2]. Despite the high incidence of hepatitis A virus (HAV) infection in the pediatric population in general, there are few pediatric case reports in the literature about HAV-associated hemophagocytic syndrome [3]. We encountered 2 patients with HAV-associated hemophagocytic syndrome.
2048	Pitt-Hopkins syndrome-associated mutations in TCF4 lead to variable impairment of the transcription factor function ranging from hypomorphic to dominant-negative effects.	22460224	Transcription factor TCF4 (alias ITF2, SEF2 or E2-2) is a broadly expressed basic helix-loop-helix (bHLH) protein that functions as a homo- or heterodimer. Missense, nonsense, frame-shift and splice-site mutations as well as translocations and large deletions encompassing TCF4 gene cause Pitt-Hopkins syndrome (PTHS), a rare developmental disorder characterized by severe motor and mental retardation, typical facial features and breathing anomalies. Irrespective of the mutation, TCF4 haploinsufficiency has been proposed as an underlying mechanism for PTHS. We have recently demonstrated that human TCF4 gene is transcribed using numerous 5' exons. Here, we re-evaluated the impact of all the published PTHS-associated mutations, taking into account the diversity of TCF4 isoforms, and assessed how the reading frame elongating and missense mutations affect TCF4 functions. Our analysis revealed that not all deletions and truncating mutations in TCF4 result in complete loss-of-function and the impact of reading frame elongating and missense mutations ranges from subtle deficiencies to dominant-negative effects. We show that (i) missense mutations in TCF4 bHLH domain and the reading frame elongating mutation damage DNA-binding and transactivation ability in a manner dependent on dimer context (homodimer versus heterodimer with ASCL1 or NEUROD2); (ii) the elongating mutation and the missense mutation at the dimer interface of the HLH domain destabilize the protein; and (iii) missense mutations outside of the bHLH domain cause no major functional deficiencies. We conclude that different PTHS-associated mutations impair the functions of TCF4 by diverse mechanisms and to a varying extent, possibly contributing to the phenotypic variability of PTHS patients.
2049	Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.	22453338	Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.
2050	Hemophagocytic lymphohistiocytosis in a rheumatoid arthritis patient treated with infliximab.	22449679	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition with high mortality. We report a case of a 74-year-old woman with rheumatoid arthritis who developed HLH secondary to pyelonephritis due to Escherichia coli infection following infliximab treatment. Bone marrow aspiration showed proliferation of histiocytes with hemophagocytosis. The patient died despite treatment with intravenous antibiotics intravenous methylprednisolone and intravenous immunoglobulin. Cytokine levels were measured and are discussed.
2051	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Los Angeles County.	22431026	Data on Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH) in adults in the United States remain very limited. A cluster of four cases of EBV-HLH was observed in a 4-month period at a tertiary center in Los Angeles County (LA County) and the clinical and molecular characteristics identified in these cases are being described. EBV typing, immunophenotypic and molecular genetic studies were performed. Diagnostic criteria that may be used to identify EBV as a cause of HLH in adults are also being suggested. Finally, the crude incidence rate for HLH in LA County was determined and was compared to the worldwide crude incidence rate for HLH. The cases each occurred in young male adult residents of California and were associated with evidence of EBV reactivation and ferritin levels of >20,000 µg/L. A higher rate of cases of EBV-HLH in 2010 was found at UCLA Medical Center than for 2007-2009 (4.9/10,000 hospital discharges vs. 0.14/10,000 hospital discharges, respectively; P = 0.0017). The cases were associated with EBV type 1, and the insertion of the codon CTC (leucine) was found in numerous of the EBNA-2 gene sequences. The annual incidence of secondary, non-familial HLH was estimated to be 0.9 cases per million persons >15 years of age in LA County. Although EBV-HLH is a rare disease, the incidence in adults in Western countries may be underestimated.
2052	Treatment of hemophagocytic lymphohistiocytosis with alemtuzumab in systemic lupus erythematosus.	22426581	Hemophagocytic lymphohistiocytosis (HLH) is an immune disorder characterized by cytokine dysregulation and uncontrolled activation of T lymphocytes and macrophages. It is categorized as primary when associated with specific genetic mutations or secondary when associated with infections, malignancies, or autoimmune disorders. Clinical features of HLH include unexplained fever, hepatosplenomegaly, pancytopenia, and severe hyperferritinemia. Treatment of primary HLH has become standardized based on the HLH-2004 protocol using cyclosporine, etoposide, and dexamethasone with or without intrathecal methotrexate followed by hematopoietic stem cell transplantation. Treatment of secondary HLH is directed at control of the underlying condition. If unsuccessful, cytotoxic agents such as those in HLH-2004, steroids, intravenous γ-globulin, or targeted immune therapy have been used. Immunotherapy targeting CD52 expressed on immune effector cells of HLH is a rational therapeutic approach in patients too ill for traditional cytotoxic chemotherapy. We describe the successful use of alemtuzumab to treat HLH due to systemic lupus erythematosus.
2053	Mere exposure and flavour-flavour learning increase 2-3 year-old children's acceptance of a novel vegetable.	22425616	Vegetable consumption is low among many children. This study compared the efficacy of the exposure learning strategies mere exposure, flavour-flavour and flavour-nutrient learning in changing children's intake of a novel vegetable. An unmodified artichoke purée was served at pre-testing. Hereafter children were exposed 10 times to unmodified purée (mere exposure, n=32), a sweetened purée (flavour-flavour learning, n=33) or an energy dense purée with added fat (flavour-nutrient learning, n=39). Unmodified and sweet purée contained approximately 200 kJ/100g; the energy dense purée 580 kJ/100g. The unmodified purée was served again at post-testing, 3 and 6 months after last exposure to monitor long-term effects of learning. Intake of purée increased in the mere exposure and flavour-flavour condition, and was unchanged in the flavour-nutrient condition. Mere exposure changed children's intake by the 5th exposure, flavour-flavour learning by the 10th. Mere exposure led to the largest increase in intake of unmodified purée at post-test and over 6 months. Children following flavour-flavour learning consumed more of the sweet purée than of unmodified purée. About 30-40% of the children were resistant to acceptance changes. The results of this study imply that mere exposure and flavour-flavour learning are powerful strategies for changing children's acceptance of a novel vegetable, even though a substantial number of children are resistant to these types of exposure learning.
2054	CNS involvement at the onset of primary hemophagocytic lymphohistiocytosis.	2242289622422890	To differentiate onset of CNS involvement in primary hemophagocytic lymphohistiocytosis (HLH) from that of other CNS inflammatory diseases and to identify early symptoms linked to abnormal cognitive outcome.Forty-six children with primary HLH who had neurologic evaluation within 2 weeks and brain MRI within 6 months of diagnosis were included. Initial symptoms, CSF study, brain MRI, and neurologic outcome were assessed. Brain MRIs were compared with those of 44 children with acute disseminated encephalomyelitis (ADEM).At disease onset, 29 children (63%) had neurologic symptoms and 7 (15%) had microcephaly. Twenty-three (50%) children had abnormal CSF study, but only 15 (33%) had abnormal brain MRI. The latter showed that patients with HLH, unlike patients with ADEM, had symmetric periventricular lesions, without thalamic and brainstem involvement and with infrequent hyposignal intensity on T1. At the end of follow-up (3.6 ± 3.6 years), 17 of the 28 (61%) surviving patients had normal neurologic status, 5 (18%) had a severe neurologic outcome, and 6 (21%) had mild cognitive difficulties. Abnormal neurologic outcome was not influenced by age or type of genetic defect, but by the presence of neurologic symptoms, MRI lesions, or abnormal CSF study at onset. Early clinical and MRI symptoms may regress after treatment.Neurologic symptoms are frequent at the onset of primary HLH and are mostly associated with abnormal CSF findings, but with normal brain MRI. In cases of abnormal brain MRI, the observed lesions differ from those of ADEM.
2055	Lymphohistiocytosis in the brain: recognition of a potentially reversible aspect of primary HLH.	2242289022422896	NaN
2056	A genomewide survey of bHLH transcription factors in the coral Acropora digitifera identifies three novel orthologous families, pearl, amber, and peridot.	22419240	Decoding the genome of the coral, Acropora digitifera, enabled us to characterize a nearly full set of 70 basic helix-loop-helix (bHLH) transcription factors in this organism. This number is comparable to 68 bHLH genes in the sea anemone, Nematostella vectensis, and larger than those in most other invertebrate metazoans. The 70 bHLH genes were assigned to 29 orthologous families previously reported. In addition, we identified three novel HLH orthologous families, which we designated pearl, amber, and peridot, increasing the number of orthologous families to 32. Pearl and amber orthologues were found in genomes and expressed sequenced tags (ESTs) of Mollusca and Annelida in addition to Cnidaria. Peridot orthologues were found in genomes and ESTs of Cephalochordata and Hemichordata in addition to Cnidaria. These three genes were likely lost in the clades of Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens during animal evolution.
2057	[Hemophagocytic lymphohistiocytosis--a contemporary medical problem].	22400183	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immnoregulation caused by various inherited and acquired factors. Uncontrolled activation of T lymphocytes and macrophages, together with an impaired cytotoxic function of NK cells, results in the massive cytokine release and hyperinflammatory phenotype. HLH is characterized by fever, splenomegaly and peripheral blood cytopenia. Cytohistological examination shows accumulation of lymphocytes and macrophages, sometimes with hemophagocytic activity, in different tissues (e.g., bone marrow). Laboratory findings include hyperferritinemia (often 'sky-high' > 10.000 microg/l), hypertriglyceridemia, hypofibrinogenemia, and high levels of the alpha chain of the soluble interleukin-2 receptor (sIL-2R also called sCD25). In 2004 The Histiocyte Society updated current diagnostic guidelines for HLH. Therapy of HLH is based on suppression of the hyperinflammatory status by destruction of activated CD8+ T lymphocytes and macrophages, and treatment of any existing HLH triggers. However, treatment of HLH is associated with high morbidity and mortality. Therefore consideration should be given to referring HLH patients to centers with experience in the treatment and care of those with HLH. Due to aggressive course, awareness of HLH signs and symptoms is crucial among physicians for the early diagnosis and immediate introduction of adequate HLH treatment.
2058	Hemophagocytic lymphohistiocytosis can mimic the superior vena cava syndrome.	22395213	We report a case of a 3-year-old boy with hemophagocytic lymphohistiocytosis (HLH), with enlarged cervical lymph nodes causing internal jugular vein compression, who initially presented a condition similar to the superior vena cava syndrome. Laboratory data along with neck node and bone marrow biopsies confirmed the HLH. Genetic analysis revealed the patient to be compound heterozygous for 2 variations of the perforin gene, c.1620 A>G and c.562C>G. This case featuring a rare initial manifestation of HLH that has not been previously reported, points to the necessity of considering this disease when symptoms similar to superior vena cava syndrome are encountered.
2059	The Max b-HLH-LZ can transduce into cells and inhibit c-Myc transcriptional activities.	22384171	The inhibition of the functions of c-Myc (endogenous and oncogenic) was recently shown to provide a spectacular therapeutic index in cancer mouse models, with complete tumor regression and minimal side-effects in normal tissues. This was achieved by the systemic and conditional expression of omomyc, the cDNA of a designed mutant of the b-HLH-LZ of c-Myc named Omomyc. The overall mode of action of Omomyc consists in the sequestration of Max and the concomitant competition of the Omomyc/Max complex with the endogenous c-Myc/Max heterodimer. This leads to the inhibition of the transactivation of Myc target genes involved in proliferation and metabolism. While this body of work has provided extraordinary insights to guide the future development of new cancer therapies that target c-Myc, Omomyc itself is not a therapeutic agent. In this context, we sought to exploit the use of a b-HLH-LZ to inhibit c-Myc in a cancer cell line in a more direct fashion. We demonstrate that the b-HLH-LZ domain of Max (Max*) behaves as a bona fide protein transduction domain (PTD) that can efficiently transduce across cellular membrane via through endocytosis and translocate to the nucleus. In addition, we show that the treatment of HeLa cells with Max* leads to a reduction of metabolism and proliferation rate. Accordingly, we observe a decrease of the population of HeLa cells in S phase, an accumulation in G1/G0 and the induction of apoptosis. In agreement with these phenotypic changes, we show by q-RT-PCR that the treatment of HeLa cells with Max* leads to the activation of the transcription c-Myc repressed genes as well as the repression of the expression of c-Myc activated genes. In addition to the novel discovery that the Max b-HLH-LZ is a PTD, our findings open up new avenues and strategies for the direct inhibition of c-Myc with b-HLH-LZ analogs.
2060	Splenectomy as a therapeutic approach in refractory hemophagocytic lymphohistiocytosis.	2237743521873022	Zhang et al. have reported a case of an adult man with hemophagocytic lymphohistiocytosis (HLH) with an unknown etiology who has achieved a remission of HLH following comprehensive treatment based on splenectomy. In the present communication, the author critically discusses diagnostic and therapeutic issues related to the reported case, and questions utility of splenectomy in the treatment of active HLH.
2061	Killer cell immunoglobulin-like receptor gene polymorphisms predispose susceptibility to Epstein-Barr virus associated hemophagocytic lymphohistiocytosis in Chinese children.	22376216	Epstein-Barr virus associated hemophagocytic lymphohistiocytosis (EBV-HLH) has a high mortality rate among children. The pathogenesis of, and underlying predisposing factors for, EBV-HLH are as yet unclear; however, natural killer cells may play a key role in progression of the disease. This study attempted to determine whether killer cell immunoglobulin-like receptor (KIR) gene polymorphisms are responsible for susceptibility to EBV-HLH. Of the 125 children with EBV infection studied, 59 had EBV-HLH and 66 patients had EBV associated infectious mononucleosis (IM) without HLH. The control group was 146 normal children without immune deficiency. KIR polymorphisms were determined by polymerase chain reaction with sequence-specific primers. KIR polymorphism data were analyzed using the X(2) test or Fisher's exact test. The overall observed carrier frequency (OF) of KIR2DS5 was significantly higher in EBV-HLH patients than in IM patients and normal controls (49.2% versus 31.8%, P = 0.048; 49.2% versus 31.5%, P = 0.018, respectively), and the odds ratios (95% confidence interval) were 2.071 (1.001-4.286) and 2.101(1.132-3.900) respectively. The OF of KIR3DS1 was significantly higher in the EBV-HLH patients than in the IM patients (47.4% versus 24.6%, P = 0.012), but not different from normal controls. In summary, KIR polymorphisms may be involved in the development of EBV-HLH, with KIR2DS5 promoting susceptibility to this disease. The obtained KIR data will enrich the understanding of genetic relationships among diseases associated with EBV infection in children.
2062	Sensitivity and specificity of bone marrow hemophagocytosis in hemophagocytic lymphohistiocytosis.	22371906	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal disease characterized by abnormal activation of T-lymphocytes and macrophages. The diagnosis of HLH can be established if there is a family history of HLH, or evidence of genetic defects, or if 5 of 8 clinicopathologic criteria are fulfilled. This case-control study aimed to examine the extent of hemophagocytosis on the bone marrow examination of patients fulfilling diagnostic criteria for HLH. Hemophagocytosis in 6 bone marrow aspirates from 3 HLH patients was compared with 20 random control bone marrows. Macrophages with hemophagocytosis were counted using a Miller ocular disc in fields corresponding to 9,000 nucleated cells. Mean hemophagocytosis count in the HLH cases was estimated at 0.082% (range 0-0.31%), whereas in the controls it was 0.009% (range 0-0.04%). The sensitivity of hemophagocytosis was 83% with a specificity of only 60%. This demonstrates that rare hemophagocytosis can be seen in bone marrow aspirates from patients without a clinical diagnosis of HLH. It also shows that hemophagocytosis has too low a specificity to be a screening test for HLH. While the hemophagocytosis counts are significantly higher in HLH cases than in controls, overlap of the counts precludes using hemophagocytosis as a reliable indicator of HLH. A rise in the hemophagocytosis count threshold of 0.05-0.13% would increase the specificity to 100%. We suggest that the diagnostic scheme for HLH needs revision, and can be improved by addressing minimum hemophagocytosis count threshold.
2063	Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin.	22366321	The present work describes reversed-phase high performance liquid chromatographic methodologies (RP-HPLC) for the qualitative and quantitative analysis of the human glycoprotein hormones thyrotropin (hTSH), follitropin (hFSH), choriogonadotropin (hCG) and lutropin (hLH) in the presence of a large excess (up to 250:1) of human serum albumin (HSA). Chromatographic profiles with a good separation between the hormone and HSA were obtained by using a C4 column and specific gradient elution conditions for each hormone. Parameters such as resolution factor, tailing factor and relative retention time, were determined, and are useful for the evaluation of the quality of the separation obtained between the active pharmaceutical ingredient and the excipient present in the final formulation. The potential of each method for quantification of both HSA and the hormone was also demonstrated. Besides furnishing chromatographic quantifications that can substitute for in vivo bioassays and animal use, the chromatograms also provide a direct panorama of the quality and heterogeneity of the protein of interest.
2064	Antagonistic actions of HLH/bHLH proteins are involved in grain length and weight in rice.	22363621	Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/palea. Overexpression of an atypical bHLH, named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1.
2065	Adult hemophagocytic lymphohistiocytosis with severe pulmonary hypertension and a novel perforin gene mutation.	22359105	Adult hemophagocytic lymphohistiocytosis (HLH) is a rare and deadly hyperinflammatory syndrome presenting both diagnostic and therapeutic challenges. HLH may be primary, due to an underlying genetic abnormality, and/or secondary to infection, malignancy, or rheumatologic conditions. We describe a case of HLH-associated severe pulmonary hypertension paralleling Epstein-Barr virus (EBV) reactivation in a 52-year-old male in whom a novel perforin missense mutation was found (PRF1 1517C>T). Although intolerant of standard therapy (HLH-2004 protocol), a 6-week course of anti-CD52 (alemtuzumab) was associated with freedom-from-transfusion from weeks 4 to 13. However, 15 weeks after the onset of salvage therapy, he succumbed to polymicrobial sepsis despite treatment with prophylactic anti-infectives, with necropsy revealing disseminated blastomycosis and relapsed HLH. This case illustrates uncertainties in the relationships between pulmonary hypertension, a newly described PRF1 mutation, and possible pre-existing latent infectious risk factors (such as EBV or Blastomyces) in the pathogenesis and therapeutic perils of adult HLH.
2066	bHLH-O proteins are crucial for Drosophila neuroblast self-renewal and mediate Notch-induced overproliferation.	22357926	Drosophila larval neurogenesis is an excellent system for studying the balance between self-renewal and differentiation of a somatic stem cell (neuroblast). Neuroblasts (NBs) give rise to differentiated neurons and glia via intermediate precursors called GMCs or INPs. We show that E(spl)mγ, E(spl)mβ, E(spl)m8 and Deadpan (Dpn), members of the basic helix-loop-helix-Orange protein family, are expressed in NBs but not in differentiated cells. Double mutation for the E(spl) complex and dpn severely affects the ability of NBs to self-renew, causing premature termination of proliferation. Single mutations produce only minor defects, which points to functional redundancy between E(spl) proteins and Dpn. Expression of E(spl)mγ and m8, but not of dpn, depends on Notch signalling from the GMC/INP daughter to the NB. When Notch is abnormally activated in NB progeny cells, overproliferation defects are seen. We show that this depends on the abnormal induction of E(spl) genes. In fact E(spl) overexpression can partly mimic Notch-induced overproliferation. Therefore, E(spl) and Dpn act together to maintain the NB in a self-renewing state, a process in which they are assisted by Notch, which sustains expression of the E(spl) subset.
2067	Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle.	22349695	Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fast- and slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26(low) fast-dividing cells, whereas PKH26(high) slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26(high)Id1(high) population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26(high)Id1(high) population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.
2068	Portal-large terminase interactions of the bacteriophage T4 DNA packaging machine implicate a molecular lever mechanism for coupling ATPase to DNA translocation.	22345478	DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.
2069	[Clinical features and outcome analysis of 83 childhood Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with HLH-2004 protocol].	22339824	To investigate the clinical features of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH), to analysis the outcome of HLH-2004 protocol, and to explore the prognostic factors in EBV-HLH patients.The clinical features at onset and outcome of HLH-2004 protocol from 83 pediatric patients with EBV-HLH enrolled from January 2006 to December 2009 in our hospital were analyzed retrospectively. Univariate and multivariate COX regression analysis were used to identify statistically significant prognostic factors.(1) Among the 83 patients, 45 were males and 38 were females. The age of onset ranged from 6 months to 14 years 4 months. 44 patients were treated with HLH-2004, and 3-year overall survival (OS) was (55.8 ± 7.9)%. (2) The most common clinical features of EBV-HLH included high fever, cytopenia, hepatosplenomegaly, and coagulopathy; The respiratory symptoms, angina phlogistic, skin rashes, neurologic abnormality were rare. 97.3% of patients showed an elevation of serum ferritin, liver dysfunction and lipid metabolism disorders was found in most of EBV-HLH patients. 89.0% of patient had hemophagocytosis in bone marrow at diagnosis of EBV-HLH. (3) COX regression analysis revealed that anemia degree, serum albumin < 30 g/L, CD4:CD8 abnormity, NK cell < 3%, treatment protocol were related with the prognosis significantly (P < 0.05).EBV-HLH in pediatric patients has severe clinical feature and poor prognosis. HLH-2004 protocol is an effective treatment for patients with EBV-HLH. Symptomatic treatment can't rescue the patients of EBV-HLH.
2070	The haemophagocytic syndrome.	22338310	The haemophagocytic syndrome or haemophagocytic lymphohistiocytosis (HLH) is a syndrome encompassing a heterogeneous group of disorders characterized by a persistent activation of benign macrophages, leading to uncontrolled secretion of cytokines and phagocytosis of blood cells. The syndrome can be primary due to mutations in different genes crucially involved in lymphocyte cytotoxicity and secondary in association with infectious, autoimmune or malignant disorders. In most cases HLH displays an aggressive disease course with a high fatality rate without treatment. Early recognition of the syndrome and prompt initiation of appropriate treatment, in most cases consisting of immunochemotherapy, are mandatory to ensure long-term survival.
2071	Interactions between HLH and bHLH factors modulate light-regulated plant development.	22331621	Phytochromes (Phy) and phytochrome-interacting factor (PIF) transcription factors constitute a major signaling module that controls plant development in response to red and far-red light. A low red:far-red ratio is interpreted as shading by neighbor plants and induces cell elongation-a phenomenon called shade-avoidance syndrome (SAS). PAR1 and its closest homolog PAR2 are negative regulators of SAS; they belong to the HLH transcription factor family that lacks a typical basic domain required for DNA binding, and they are believed to regulate gene expressions through DNA binding transcription factors that are yet to be identified. Here, we show that light signal stabilizes PAR1 protein and PAR1 interacts with PIF4 and inhibits PIF4-mediated gene activation. DNA pull-down and chromatin immunoprecipitation (ChIP) assays showed that PAR1 inhibits PIF4 DNA binding in vitro and in vivo. Transgenic plants overexpressing PAR1 (PAR1OX) are insensitive to gibberellin (GA) or high temperature in hypocotyl elongation, similarly to the pifq mutant. In addition to PIF4, PAR1 also interacts with PRE1, a HLH transcription factor activated by brassinosteroid (BR) and GA. Overexpression of PRE1 largely suppressed the dwarf phenotype of PAR1OX. These results indicate that PAR1-PRE1 and PAR1-PIF4 heterodimers form a complex HLH/bHLH network regulating cell elongation and plant development in response to light and hormones.
2072	Characterization of Epstein-Barr virus (EBV)-infected cells in EBV-associated hemophagocytic lymphohistiocytosis in two patients with X-linked lymphoproliferative syndrome type 1 and type 2.	22325832	X-linked lymphoproliferative syndrome (XLP) is a rare inherited immunodeficiency by an extreme vulnerability to Epstein-Barr virus (EBV) infection, frequently resulting in hemophagocytic lymphohistiocytosis (HLH). XLP are now divided into type 1 (XLP-1) and type 2 (XLP-2), which are caused by mutations of SH2D1A/SLAM-associated protein (SAP) and X-linked inhibitor of apoptosis protein (XIAP) genes, respectively. The diagnosis of XLP in individuals with EBV-associated HLH (EBV-HLH) is generally difficult because they show basically similar symptoms to sporadic EBV-HLH. Although EBV-infected cells in sporadic EBV-HLH are known to be mainly in CD8+ T cells, the cell-type of EBV-infected cells in EBV-HLH seen in XLP patients remains undetermined.EBV-infected cells in two patients (XLP-1 and XLP-2) presenting EBV-HLH were evaluated by in EBER-1 in situ hybridization or quantitative PCR methods.Both XLP patients showed that the dominant population of EBV-infected cells was CD19+ B cells, whereas EBV-infected CD8+ T cells were very few.In XLP-related EBV-HLH, EBV-infected cells appear to be predominantly B cells. B cell directed therapy such as rituximab may be a valuable option in the treatment of EBV-HLH in XLP patients.
2073	Serum ferritin is a cost-effective laboratory marker for hemophagocytic lymphohistiocytosis in the developing world.	22322940	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease in children and presents many diagnostic difficulties. Without prompt intervention, the disease typically runs a rapidly fatal course. Diagnostic criteria were proposed by the Histiocyte Society in 1991 and have since been modified. Included in these criteria is a ferritin level >500 mcg/L. Although not diagnostic, a high ferritin level is highly suggestive of HLH. Serum ferritin assays are more accessible and cost-effective compared with other biochemical markers, particularly in resource-limited settings. Fifteen patients with HLH were treated at Red Cross War Memorial Children's Hospital between 1991 and 2010. Hyperferritinemia was a consistently reliable finding (93%) compared with either serum fibrinogen or triglycerides, which were elevated in only half of the patients. It is our contention that analysis of a complete blood count and serum ferritin (in addition to clinical criteria and tissue examination of marrow and/or cerebrospinal fluid) is probably the single most cost-effective and clinically helpful means to make the diagnosis of HLH when laboratory access is limited.
2074	Hemophagocytic lymphohistiocytosis associated with necrotizing enterocolitis in premature newborns.	22315234	Hemophagocytic lymphohistiocytosis (HLH) is a systemic disease resulting from the excessive release of inflammatory cytokines by macrophages under prolonged antigenic stimulation. If untreated, it leads to multiorgan failure and death. Necrotizing enterocolitis (NEC) has not previously been associated with HLH. Here we report four preterm infants who were diagnosed with HLH associated with NEC. Two patients received chemotherapy and one survived. The other two infants succumbed to multiorgan failure. These results suggest that NEC may be a common clinical manifestation of HLH in premature neonates.
2075	A dual mechanism controls nuclear localization in the atypical basic-helix-loop-helix protein PAR1 of Arabidopsis thaliana.	22311779	PAR1 is an atypical basic-helix-loop-helix (bHLH) protein that negatively regulates the shade avoidance syndrome in Arabidopsis thaliana acting as a transcriptional cofactor. Consistently with this function, PAR1 has to be in the nucleus to display biological activity. Previous structure-function analyses revealed that the N-terminal region of PAR1 drives the protein to the nucleus. However, truncated forms of PAR1 lacking this region still display biological activity, implying that PAR1 has additional mechanisms to localize into the nucleus. In this work, we compared the primary structure of PAR1 and various related and unrelated plant bHLH proteins, which led us to suggest that PAR1 contains a non-canonical nuclear localization signal (NLS) in the N-terminal region. By overexpressing truncated and mutated derivatives of PAR1, we have also investigated the importance of other regions of PAR1, such as the acidic and the extended HLH dimerization domains, for its nuclear localization. We found that, in the absence of the N-terminal region, a functional HLH domain is required for nuclear localization. Our results suggest the existence of a dual mechanism for PAR1 nuclear localization: (1) one mediated by the N-terminal non-consensus NLS and (2) a second one that involves interaction with other proteins via the dimerization domain.
2076	Treatment of hemophagocytic lymphohistiocytosis with cyclophosphamide, vincristine, and prednisone.	22307778	The goal of this study was to observe the therapeutic efficacy of cyclophosphamide, vincristine and prednisone combined chemotherapy (COP) in treating adult haemophagocytic lymphohistiocytosis (HLH). Fifteen cases diagnosed with HLH were enrolled in our study. Each of them was treated with the COP regimen. Two of the fifteen patients had autoimmune disease-associated HLH (13.3%), 2/15 had lymphoma-associated HLH (13.3%), 7/15 had infection-associated HLH (IAHLH) (46.7%), and the remaining 4/15 developed the disease in the absence of apparent underlying disease (26.7%). A complete response (CR) was achieved in 7/15 patients (46.7%), while a partial response (PR) was achieved in 5/15 patients (33.3%). With a mean follow-up of 72.5 weeks, the one-year overall survival was 66.7%. HLH of varying aetiology exhibited differing sensitivity and response to the COP regimen. In combination with aetiological and supportive treatment, the COP regimen as a mild and cost-effective chemotherapy is especially favourable for IAHLH, autoimmune disease-associated HLH, and disease severity precluding intensive treatment.
2077	Early-onset hemophagocytic lymphohistiocytosis after the start of chemotherapy for advanced neuroblastoma.	22304016	The authors report the clinical course of a 3-year-old boy with stage 4 neuroblastoma (NB) complicated by hemophagocytic lymphohistiocytosis (HLH) immediately after the start of chemotherapy. The NB responded very well to the chemotherapy, but the patient developed high fever on the 2nd day, and was diagnosed as having HLH of the 7th day of chemotherapy. No infections were demonstrated, and massive tumor cell destruction resulting from the rapid effect of chemotherapy was thought to be a cause of systemic cytokine response, resulting in HLH. Methylprednisolone pulse therapy was effective for the HLH, which did not recur thereafter. HLH should be recognized as a serious adverse event during chemotherapy for advanced NB that has a large malignant cell load.
2078	Forced homodimerization of the c-Fos leucine zipper in designed bHLHZ-like hybrid proteins MaxbHLH-Fos and ArntbHLH-Fos.	22301802	Although the c-Fos leucine zipper (LZ) does not form a homodimer in its native basic region/leucine zipper (bZIP) structure, we found that it is capable of homodimerization and promoting protein folding in engineered basic region/helix-loop-helix/leucine zipper (bHLHZ) hybrid proteins MaxbHLH-Fos and ArntbHLH-Fos, in which the bHLH subdomains of Max and Arnt are fused to the c-Fos LZ. By using the in vivo yeast one-hybrid system and in vitro circular dichroism and quantitative fluorescence anisotropy, we demonstrated that attachment of the c-Fos LZ to the otherwise unstructured MaxbHLH resulted in a hybrid bHLHZ-like protein now competent for homodimerization and DNA binding at the cognate E-box site, CACGTG. In ArntbHLH-Fos, the c-Fos LZ promoted proper folding of the HLH structure, although unlike MaxbHLH, ArntbHLH alone is capable of homodimerization and DNA binding. In addition, by comparing the E-box binding and secondary structures of MaxbHLH-Fos and two derivatives containing targeted mutations in the c-Fos LZ, we found that cooperative communication exists between the bHLH and LZ: proper folding of the four-helix bundle in the HLH region could be induced by the c-Fos LZ, and the HLH dimerization region could force homodimerization of the c-Fos LZ. These results demonstrate that although intrinsically unfavorable, the c-Fos LZ can homodimerize, demonstrating that the same c-Fos LZ element can yield orthogonal differences in structure and/or DNA-binding function within different transcription factor families, including the bZIP and bHLHZ.
2079	A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes.	22294731	Familial hemophagocytic lymphohistiocytosis (FHL) is a life-threatening disorder of immune regulation caused by defects in lymphocyte cytotoxicity. Rapid differentiation of primary, genetic forms from secondary forms of hemophagocytic lymphohistiocytosis (HLH) is crucial for treatment decisions. We prospectively evaluated the performance of degranulation assays based on surface up-regulation of CD107a on natural killer (NK) cells and cytotoxic T lymphocytes in a cohort of 494 patients referred for evaluation for suspected HLH. Seventy-five of 77 patients (97%) with FHL3-5 and 11 of 13 patients (85%) with Griscelli syndrome type 2 or Chediak-Higashi syndrome had abnormal resting NK-cell degranulation. In contrast, NK-cell degranulation was normal in 14 of 16 patients (88%) with X-linked lymphoproliferative disease and in 8 of 14 patients (57%) with FHL2, who were identified by diminished intracellular SLAM-associated protein (SAP), X-linked inhibitor of apoptosis protein (XIAP), and perforin expression, respectively. Among 66 patients with a clinical diagnosis of secondary HLH, 13 of 59 (22%) had abnormal resting NK-cell degranulation, whereas 0 of 43 had abnormal degranulation using IL-2-activated NK cells. Active disease or immunosuppressive therapy did not impair the assay performance. Overall, resting NK-cell degranulation below 5% provided a 96% sensitivity for a genetic degranulation disorder and a specificity of 88%. Therefore, degranulation assays allow a rapid and reliable classification of patients, benefiting treatment decisions.
2080	Human α/β hydrolase domain containing 10 (ABHD10) is responsible enzyme for deglucuronidation of mycophenolic acid acyl-glucuronide in liver.	22294686	Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is primarily metabolized by glucuronidation to a phenolic glucuronide (MPAG) and an acyl glucuronide (AcMPAG). It is known that AcMPAG, which may be an immunotoxic metabolite, is deglucuronidated in human liver. However, it has been reported that recombinant β-glucuronidase does not catalyze this reaction. AcMPAG deglucuronidation activity was detected in both human liver cytosol (HLC) and microsomes (HLM). In this study, the enzyme responsible for AcMPAG deglucuronidation was identified by purification from HLC with column chromatographic purification steps. The purified enzyme was identified as α/β hydrolase domain containing 10 (ABHD10) by amino acid sequence analysis. Recombinant ABHD10 expressed in Sf9 cells efficiently deglucuronidated AcMPAG with a K(m) value of 100.7 ± 10.2 μM, which was similar to those in HLM, HLC, and human liver homogenates (HLH). Immunoblot analysis revealed ABHD10 protein expression in both HLC and HLM. The AcMPAG deglucuronidation by recombinant ABHD10, HLC, and HLH were potently inhibited by AgNO(3), CdCl(2), CuCl(2), PMSF, bis-p-nitrophenylphosphate, and DTNB. The CL(int) value of AcMPAG formation from MPA, which was catalyzed by human UGT2B7, in HLH was increased by 1.8-fold in the presence of PMSF. Thus, human ABHD10 would affect the formation of AcMPAG, the immunotoxic metabolite.
2081	Regulation of protein function by interfering protein species.	25436525	Abstract Most proteins do not function alone but act in protein complexes. For several transcriptional regulators, it is known that they have to homo- or heterodimerize prior to DNA binding. These protein interactions occur through defined protein-protein-interaction (PPI) domains. More than two decades ago, inhibitor of DNA binding (ID), a small protein containing a single helix-loop-helix (HLH) motif was identified. ID is able to interact with the larger DNA-binding basic helix-loop-helix (bHLH) transcription factors, but due to the lack of the basic domain required for DNA binding, ID traps bHLH proteins in non-functional complexes. Work in plants has, in the recent years, identified more small proteins acting in analogy to ID. A hallmark of these small negative acting proteins is the presence of a protein-interaction domain and the absence of other functional domains required for transcriptional activation or DNA binding. Because these proteins are often very small and function in analogy to microRNAs (meaning in a dominant-negative manner), we propose to refer to these protein species as 'microProteins' (miPs). miPs can be encoded in the genome as individual transcription units but can also be produced by alternative splicing. Other negatively acting proteins, consisting of more than one domain, have also been identified, and we propose to call these proteins 'interfering proteins' (iPs). The aim of this review is to state more precisely how to discriminate miPs from iPs. Therefore, we will highlight recent findings on both protein species and describe their mode of action. Furthermore, miPs have the ability to regulate proteins of diverse functions, emphasizing their value as biotechnological tools.
2082	Distinct Caenorhabditis elegans HLH-8/twist-containing dimers function in the mesoderm.	22275075	The Caenorhabditis elegans basic helix-loop-helix (bHLH) factor HLH-8, the single Twist ortholog in the nematode genome, plays important roles in mesoderm development, including M lineage patterning and differentiation of vulval and enteric muscles. HLH-8 cooperates with HLH-2, the bHLH E/Daughterless ortholog, to regulate downstream target genes, but it is not known whether HLH-2 is an obligate partner for all HLH-8 functions.Using hlh-2 loss-of-function alleles and RNAi, we discovered that HLH-2 is required in the vulval muscles but not in M patterning or enteric muscle development. Additionally, we found that expressing tethered HLH-8/HLH-8 dimers in hlh-8 null animals rescued M patterning and enteric but not vulval muscle development.These results support a model whereby HLH-8/HLH-8 homodimers function in M lineage patterning and enteric muscles and HLH-8/HLH-2 heterodimers function in the M-derived vulval muscles. Interestingly, the different dimers function in the same M lineage cells and the switch in dimer function coincides with vulval muscle differentiation. The use of distinct Twist dimers is evolutionarily conserved, and C. elegans provides a paradigm for future dissection of differential promoter regulation by these dimers at a single cell resolution.
2083	HHV-8-related hemophagocytic lymphohistiocytosis in a boy with XLP phenotype.	22258354	We report a 2.5-year-old boy with an X-linked lymphoproliferative disease (XLP) phenotype who presented with human herpes virus-8 (HHV-8)-related hemophagocytic lymphohistiocytosis (HLH). XLP is a rare primary immunodeficiency characterized by extreme susceptibility to herpes viruses, mainly Epstein-Barr virus (EBV). Approximately 60% of patients with XLP present with fulminant mononucleosis associated with HLH, whereas remaining patients present with hypogammaglobulinemia or lymphoproliferative disease. Most commonly, one of the XLP phenotypes appears after exposure to EBV, but at least 12% of affected individuals developed symptoms without an evidence of EBV infection. Rarely, patients with XLP may present with central nervous system vasculitis or aplastic anemia. HHV-8 is lymphotrophic and it is associated with lymphoproliferative disorders and Kaposi sarcoma in immunodeficient hosts. Kaposi sarcoma rarely occurs in children with well-defined primary immunodeficiency. Also, HHV-8-related HLH was previously reported in 2 siblings with a perforin gene deficiency. Recently, it became evident that besides EBV, other viruses may trigger the symptoms in XLP. We report for the first time HHV-8-related HLH in EBV-negative pediatric patient with an XLP phenotype.
2084	Familial and acquired hemophagocytic lymphohistiocytosis.	22248322	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome in which an uncontrolled and ineffective immune response, triggered in most cases by infectious agents, leads to severe hyperinflammation. Familial forms of HLH (FHL), which are increasingly found also in adolescents and adults, are due to genetic defects leading to impaired function of natural killer cells and cytotoxic T cells. These mutations occur either in the perforin gene or in genes important for the exocytosis of cytotoxic granules. Cytotoxic granules contain perforin and granzymes, which induce apoptosis upon entering (infected) target cells. Additionally, perforin is important for the downregulation of the immune response. Acquired forms of HLH are encountered in association with (usually) viral infections, autoinflammatory/autoimmune diseases, malignant diseases, and acquired immune deficiency states (e.g., after organ transplantation). Treatment of HLH includes immune-suppressive and immune-modulatory agents, cytostatic drugs, and biological response modifiers. For patients with FHL, stem cell transplantation is indicated and can be curative.
2085	Hemophagocytic Lymphohistiocytosis (HLH) Associated with Plasmodium vivax Infection: Case Report and Review of the Literature.	22247919	Hemophagocytic lymphohistiocytosis (HLH) is an unusual syndrome characterized by fever, hepatosplenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, and pathologic findings of hemophagocytosis in the bone marrow and other tissues. HLH may be familial or associated with different types of infections, autoimmune disorders, or malignancies. Infection-associated HLH has been reported in various viral, bacterial, fungal, and parasitic infections, and case reports of parasitic infections implicated in HLH include rare cases from Plasmodium vivax infection, which occasionally affects both military personnel and civilians in Korea. We describe an unusual case of HLH resulting from Plasmodium vivax infection and review the literature. This case suggests that clinical suspicion of HLH is important when P. vivax infection is accompanied by cytopenias. Administration of antimalarial drugs may prevent irreversible end organ damage resulting from P. vivax-associated HLH.
2086	Perinatal tuberculosis associated hemophagocytic lymphohistiocytosis.	22246612	Hemophagocytic lymphohistiocytosis [HLH] is a reactive disorder characterized by generalised non-malignant histiocytic proliferation with prominent hemophagocytosis. It may be either primary [familial (FLH)] or secondary [infection or malignancy associated]. Organisms incriminated for infection associated hemophagocytic syndrome (IAHS) include viruses, bacteria, spirochetes, fungi and parasites. Reports of IAHS associated with tuberculosis in neonates are rare. The authors report a case of perinatal tuberculosis presenting as hemophagocytic lymphohistiocytosis.
2087	Analysis of EBNA-1 and LMP-1 variants in diseases associated with EBV infection in Chinese children.	22236445	In China, primary EBV infection occurs during childhood with seroprevalence reaching about 100% by 10 years of age. There are few studies on EBV variants in diseases associated with EBV infection in Chinese children. In this study, we investigated the diversity of the EBV genes (EBNA-1 and LMP-1) and the relationship between EBV variants and the clinical phenotypes in diseases associated with EBV infections in Chinese pediatric cases.The frequencies of EBV type I in the IM, HLH and HL samples were 98.4%, 100% and 95.8%, respectively. Three known EBNA-1 variants were identified, including V-val (all were V-val-v1 sub-variant), P-thr' and V-Leu (MT). The frequency of V-val-v1 was 98.6% in the IM samples, 100% in the HLH samples and 97.1% in the HL samples. There were no significant differences of the distribution of EBNA-1 variants between IM, HLH and HL samples (P > 0.05). Three known LMP-1 variants, including China 1, China 2 and Med, were identified and China 1 was predominant in all groups (IM 88.6%, HLH 100% and HL 100%). The frequency of del-LMP-1 was 88.6% in the IM samples, 100% in the HLH samples and 96.0% in the HL samples. There were no significant differences in the frequency of del-LMP-1 between the IM, HLH and HL samples (P > 0.05). The frequency of XhoI loss was 90.6% in the IM samples, 100% in the HLH samples and 100% in the HL samples, with no significant difference in frequency (P > 0.05). In the EBV type I strain, V-val-v1 variant (EBNA-1) was linked with China1 variant (LMP-1) in 88.9% of the IM samples, 100% of the HLH samples and 80.0% of the HL samples in this study.Type I EBV was the most prevalent subtype EBV in Chinese pediatric cases and V-val-v1 (EBNA-1) and China1 (LMP-1) variants were the most dominant variants. There was a strong linkage between V-val-v1 (EBNA-1) variant and China1 (LMP-1) variant in type I EBV. The sequence variation in EBV genes may represent a geographic polymorphism since no preferential associations were found between specific EBV variants and specific diseases in this study.
2088	Inherited defects causing hemophagocytic lymphohistiocytic syndrome.	22236431	Hemophagocytic lymphohistiocytosis (HLH) manifests as the uncontrolled activation of T lymphocytes and macrophages infiltrating multiple organs. Molecular studies of individuals with HLH have demonstrated in most of these conditions a critical role of granule-dependent cytotoxic activity in the regulation of lymphocyte homeostasis, and have allowed the characterization of key effectors regulating cytotoxic granule release. The cytolytic process may now be considered a multistep process, including cell activation; the polarization of cytotoxic granules toward the conjugated target cell; the tethering, priming, and fusion of the cytotoxic granules with the plasma membrane; and the release of their contents (perforin and granzymes) into the intercellular cleft, leading to target cell death. Cytolytic cells have a second effector function involving the production of cytokines, principally γ-interferon, which is secreted independently of the exocytosis cytotoxic granule pathway. An analysis of the mechanisms underlying HLH has identified γ-interferon as a key cytokine inducing uncontrolled macrophage activation, and thus represents a potential therapeutic target.
2089	The effects of methylmercury on Notch signaling during embryonic neural development in Drosophila melanogaster.	22230562	Methylmercury (MeHg) is a ubiquitous toxicant that targets the developing fetal nervous system. MeHg interacts with the Notch signaling pathway, a highly-conserved intercellular signaling mechanism required for normal development. Notch signaling is conveyed by activation of the genes in the enhancer of split (E(spl)) locus in Drosophila. We have previously shown that acute high doses of MeHg upregulate several E(spl) genes in Drosophila neural-derived C6 cells. Furthermore, MeHg induction of E(spl) can occur independent of the Notch receptor itself. We now show that MeHg, unlike inorganic mercury (HgCl2), preferentially upregulates E(spl)mδ and E(spl)mγ in Drosophila C6 cells. This is distinct from Delta ligand-induced Notch signaling in which no induction of E(spl)mδ is seen. MeHg is also seen to specifically upregulate E(spl)mδ in Drosophila embryos where HgCl2 showed no such effect. Additionally, treatment of embryos with MeHg caused a consistent failure in axonal outgrowth of the intersegmental nerve (ISN). This ISN phenotype was partially replicated by genetic activation of the Notch pathway, but was not replicated by increasing expression of E(spl)mδ. These data suggest a role for Notch signaling and the E(spl)mδ target gene in MeHg toxicity, however, the site of action for E(spl)mδ in this system remains to be elucidated.
2090	Clinical and genetic characteristics of XIAP deficiency in Japan.	22228567	Deficiency of X-linked inhibitor of apoptosis (XIAP) caused by XIAP/BIRC4 gene mutations is an inherited immune defect recognized as X-linked lymphoproliferative syndrome type 2. This disease is mainly observed in patients with hemophagocytic lymphohistiocytosis (HLH) often associated with Epstein-Barr virus infection. We described nine Japanese patients from six unrelated families with XIAP deficiency and studied XIAP protein expression, XIAP gene analysis, invariant natural killer T (iNKT) cell counts, and the cytotoxic activity of CD8(+) alloantigen-specific cytotoxic T lymphocytes. Of the nine patients, eight patients presented with symptoms in infancy or early childhood. Five patients presented with recurrent HLH, one of whom had severe HLH and died after cord blood transplantation. One patient presented with colitis, as did another patient's maternal uncle, who died of colitis at 4 years of age prior to diagnosis with XIAP deficiency. Interestingly, a 17-year-old patient was asymptomatic, while his younger brother suffered from recurrent HLH and EBV infection. Seven out of eight patients showed decreased XIAP protein expression. iNKT cells from patients with XIAP deficiency were significantly decreased as compared with age-matched healthy controls. These results in our Japanese cohort are compatible with previous studies, confirming the clinical characteristics of XIAP deficiency.
2091	Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children.	22226576	The study goal was to determine the diagnostic accuracy of a specific cytokine pattern including interferon-gamma (IFN-γ), interleukin (IL)-10, and IL-6 for hemophagocytic lymphohistiocytosis (HLH) in febrile children.In this prospective study, 756 patients with fever admitted to a hematology-oncology unit were enrolled. The causes of fever were documented and the serum cytokines, including IFN-γ, tumor necrosis factor-alpha (TNF-α), IL-10, IL-6, IL-4, and IL-2, were determined using cytometric bead array techniques.Of 1474 episodes of fever that were analyzed, 71 episodes of HLH manifested a specific cytokine pattern of highly increased levels of IFN-γ (median level: 1088.5 pg/mL) and IL-10 (623.5 pg/mL) but a moderately increased level of IL-6 (51.1 pg/mL). IL-6 was predominantly increased to varied extents in patients in the sepsis group (244.6 pg/mL) and the nonsepsis infection group (34.7 pg/mL). The diagnostic accuracy of IFN-γ and IL-10 for HLH was 99.5% and 92.8%, respectively. By applying the cutoff point of 100 pg/mL, IFN-γ had a sensitivity of 94.4% and a specificity of 97.2% for HLH. When using the criteria of IFN-γ >75 pg/mL and IL-10 >60 pg/mL, the specificity reached 98.9% and the sensitivity was 93.0%.The specific cytokine pattern of markedly elevated levels of IFN-γ and IL-10 with only modestly elevated IL-6 levels has high diagnostic accuracy for HLH and may be a useful approach to differentiate HLH from infection.
2092	Hemophagocytic syndrome in classic dengue Fever.	22224008	A 24-year-old previously healthy girl presented with persistent fever, headache, and jaundice. Rapid-test anti-dengue virus IgM antibody was positive but anti-dengue IgG was nonreactive, which is suggestive of primary dengue infection. There was clinical deterioration during empiric antibiotic and symptomatic therapy. Bone marrow examination demonstrated the presence of hemophagocytosis. Diagnosis of dengue fever with virus-associated hemophagocytic syndrome was made according to the diagnostic criteria of the HLH 2004 protocol of the Histiocyte Society. The patient recovered with corticosteroid therapy. A review of literature revealed only a handful of case reports that showed the evidence that this syndrome is caused by dengue virus. Our patient is an interesting case of hemophagocytic syndrome associated with classic dengue fever and contributes an additional case to the existing literature on this topic. This case highlights the need for increased awareness even in infections not typically associated with hemophagocytic syndrome.
2093	Development of an osteosarcoma following dental extraction after allogeneic stem cell transplantation.	24006835	Radio-induced sarcoma is known to occur several years following bone irradiation especially when this treatment is combined to high dose chemotherapy regimens prior to allogeneic haematopoietic stem cell transplantation (HSCT) in very young children. However, little is known about the stimulus of aggressive bony surgery in the development of these tumours.We report the case of a young girl in whom dental extraction was rapidly followed by the occurrence of a localized tumour 11 years after allogeneic haematopoietic stem cell transplantation using total body irradiation (TBI) for a haemophagocytic lymphophistiocytosis (HLH).This tumour involved tooth socket and all the right side of the mandible and was diagnosed as an osteogenic osteosarcoma of the zygomatic bone.This tumour had the characteristics of a radio-induced sarcoma. Thanks to the very short time between the dental extraction and the occurrence of the osteosarcoma at the same location, we discuss the role of the dental extraction as a trigger of osteosarcoma development.
2094	A case of hemophagocytic lymphohistiocytosis in a patient with chronic lymphocytic leukemia after treatment with fludarabine, cyclophosphamide, and rituximab chemotherapy, with autopsy findings.	23533846	Hemophagocytic lymphohistiocytosis (HLH) is rarely described in association with chronic lymphocytic leukemia (CLL), mostly triggered by disease progression or concurrent infection. A 68-year-old male received 4 cycles of fludarabine, cyclophosphamide, and rituximab (FCR) for CLL and achieved a complete response. Twenty-four days after the last chemotherapy, he presented with febrile neutropaenia and was diagnosed with HLH. The diagnosis was based upon persistent fever, pancytopenia, hyperferritinemia, splenomegaly, and hemophagocytosis on bone marrow aspirate. He began treatment with dexamethasone, cyclosporine, and etoposide. Fever resolved and hyperferritinemia improved but pancytopenia persisted. He died 13 days later from septic shock with positive blood cultures. A limited postmortem examination was performed and biopsies were taken from bone marrow, liver, and spleen. Biopsies demonstrated abundant hemophagocytosis by the activated macrophage as stained by CD68. There was no evidence of residual CLL as demonstrated by the lack of lymphocytes which was confirmed by the negative staining of CD79a. Chemotherapy appears to be responsible for the development of HLH in this patient. This is the second reported case of HLH developing after a rituximab-containing chemotherapy.
2095	Infection-associated haemophagocytic lymphohistiocytosis: a case series using steroids only protocol for management.	22193223	Haemophagocytic lymphohistiocytosis (HLH) is a heterogeneous group of clinical syndromes characterised by activation and subsequent uncontrolled non-malignant proliferation of T lymphocytes and macrophages, leading to a cytokine storm that accounts for most of its clinical features such as acute febrile illness, hepatosplenomegaly, multi-organ dysfunction and fulminant pancytopenia-resembling severe sepsis. Here, we present a series of 23 cases of infection-associated HLH diagnosed in our hospital within a time period of last three and half years. Though the presentation and progression of disease was variable, the patients shared some common features like prolonged fever unresponsive to broad spectrum antibiotics, organomegaly and cytopenias. In most of the cases, however, the triggering infectious agent could not be identified. They were treated using a steroid only protocol along with supportive measures and showed an excellent response.
2096	Hemophagocytic lymphohistiocytosis: updates and evolving concepts.	22189397	Hemophagocytic lymphohistiocytosis (HLH) is an immune dysregulatory syndrome that is associated with underlying defects of perforin-dependent cytotoxic function. This review seeks to update readers on new scientific insights and evolving clinical concepts related to this rare but fatal disorder.Clinically, HLH is defined by severe inflammation and potentially fatal damage to a variety of organ systems including the bone marrow, liver, or brain. Recent preclinical studies have increasingly defined HLH as a syndrome of abnormal and excessive T-cell activation, which leads to toxic activation of macrophages and other innate immune cells. Although macrophages have long been suspected to be important for disease development, recent studies have for the first time demonstrated their central role in the development of inflammation-associated cytopenias. In addition to defining new therapeutic targets, these scientific insights suggest significant overlap between HLH and severe inflammation in a variety of clinical contexts. Recent clinical observations are also changing how HLH is conceptualized. Increased recognition of HLH in older children and adults, sometimes in association with classic disease-associated mutations, is challenging the traditional view of HLH as either a distinctly familial or a sporadic disorder.Recent scientific and clinical insights are expanding understanding and recognition of HLH, driving an evolution in how it is defined, and suggesting future directions for improving therapy of this disorder.
2097	Treatment choice of immunotherapy or further chemotherapy for Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	22183955	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) leads to an aggressive and often fatal course without appropriate treatment. Etoposide therapy is crucial for the better prognosis, although it remains unknown what patients need cytotoxic agents. Since we have complied with step-up strategy in a tertiary center, treatment outcomes were studied to search predictors for disease course.The study enrolled 22 EBV-HLH patients treated between 1999 and 2010 in Kyushu University. Immunotherapy, chemotherapy and stem cell transplantation (SCT) proceeded in stages unless patients attained a consecutive >21 days-afebrile remission. Clinical and laboratory data and outcomes were retrospectively analyzed.Median age of 9 males and 13 females was 5 years (range: 9 months-41 years). Sixteen patients (73%) presented at age <15 years. Two patients remitted spontaneously, 12 attained remissions after immunotherapy, 5 after chemotherapy, and 1 after successful SCT. The remaining two patients died after chemotherapy and SCT, respectively. Median EBV load was 1 × 10(5) copies/ml of peripheral blood (range: 200-5 × 10(7)). T-cells were exclusively targeted (94%; 15/16 examined) often with EBV/T-cell receptor clonality. EBV status indicated 19 primary infections and 3 reactivations. Either death occurred in EBV-reactivated patients who underwent chemotherapy ± SCT. Age at primary infection in pediatric patients increased in the last 5 years. Patients having prolonged fever (P = 0.017) or high soluble CD25 levels (P = 0.017) at diagnosis were at higher risk for requiring chemotherapy assessed by multivariate analyses.No cytotoxic agents were needed for >60% of EBV-HLH patients. Early immunotherapy may modulate T-cell activation and reduce the chance of unnecessary chemotherapy.
2098	[Diagnostic significance of Th1/Th2 cytokine pattern in childhood hemophagocytic lymphohistiocytosis].	22176904	To illustrate the diagnostic value of Th1/Th2 cytokine pattern in childhood hemophagocytic lymphohistiocytosis (HLH) and its diagnostic accuracy.The BD(TM) CBA Human Th1/Th2 Cytokine Kit II was used to measure the serum Th1 and Th2 cytokines, including Interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin (IL)-10, IL-6, IL-4 and IL-2 in 50 patients with de novo HLH admitted to our hospital from Oct. 2005 to Aug. 2009. The above cytokine levels were also determined in 250 healthy volunteers and 235 patients with sepsis as controls.The primary features of these patients were prolonged high-grade fever (50/50), hepatomegaly (44/50), splenomegaly (38/50), hemocytopenia (47/50), hyperferritinemia (49/50), coagulopathy (44/50), hemophagocytosis in bone marrow (42/50), liver dysfunction (42/50) and hypertriglyceridemia (42/50). The IFN-γ, TNF, IL-10, IL-6, IL-4 and IL-2 levels for healthy children were (4.6 ± 1.8) ng/L, (4.0 ± 1.2) ng/L, (6.5 ± 1.3) ng/L, (6.0 ± 1.5) ng/L, (2.9 ± 0.8) ng/L and (2.6 ± 0.7) ng/L, while the median levels of them in acute phase of HLH children were 1138.5 (49.2 - 5000.0) ng/L, 3.4 (1.0 - 25.1) ng/L, 740.5 (26.5 - 5000.0) ng/L, 66.1 (3.9 - 4472.6) ng/L, 3.9 (1.0-32.8) ng/L and 4.0 (1.0 - 51.1) ng/L, respectively. The cytokine levels decreased to 9.1 (1.9 - 180.1) ng/L, 2.9 (1.0 - 11.0) ng/L, 11.4 (2.9 - 184.2) ng/L, 6.5 (1.0 - 44.8) ng/L, 2.7 (1.0 - 6.5) ng/L and 4.1 (1.0 - 12.0) ng/L respectively after remission. The IFN-γ, IL-10 and IL-6 levels in acute phase were significantly higher than those after remission and those of the healthy control (P all < 0.001). IL-4, IL-2 and TNF slightly elevated or at normal range in acute phase of HLH. The patients with sepsis showed a different cytokine pattern, with an extremely high level of IL-6 (median: 251.3 ng/L, range: 8.4- > 5000.0 ng/L) and moderately elevated level of IL-10 (median: 46.5 ng/L, range: 3.1 - 5000.0 ng/L), whereas IFN-γ was only slightly elevated (median: 9.2 ng/L, range: 1.3 - 498.8 ng/L). When the criteria for HLH set as the following: IFN-γ > 100 ng/L, IL-10 > 60 ng/L and the concentration of IFN-γ higher than that of IL-6, the specificity reached as high as 98.7% and the sensitivity was 88.0% for the diagnosis of HLH among patients with HLH and sepsis. Meanwhile, the positive predictive value (PPV) and negative predictive value (NPV) could reach 93.6% and 97.5%, respectively.The significant increase of IFN-γ and IL-10 with slightly increased level of IL-6 is a sensitive and specific cytokine pattern for childhood HLH, which is helpful for its diagnosis and differential diagnosis.
2099	[Research advance on Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis].	22169321	Epstein-Barr virus (EBV) is the major triggering factor producing virus-associated hemophagocytic syndrome (VAHS). The clinical manifestations were various and it's diagnosis should meet HLH-2004 criteria as well as EBV-positive, and familial hemophagocytic lymphohistiocytosis and lymphoma-associated hemophagocytic syndrome must be ruled out, too. In terms of treatment, combination of dexamethasone, VP-16 and cyclosporin A is the first choice, if they do not work, hematopoietic stem cell transplantation can be done. In this review, the recent advance on pathogenesis, clinical features, diagnostic problems, treatment strategies and prognosis of EBV-HLH have been described.
2100	Successful engraftment and survival following allogeneic hematopoietic stem cell transplant in a child with familial hemophagocytic lymphohistiocytosis.	22161639	Hemophagocytic syndrome is a rare disorder mainly affecting children. Symptoms include prolonged fever, hepatosplenomegaly and cytopenias. Allogeneic stem cell transplant appears to provide the best overall cure rate in this disease. The authors report a young boy, the second child of consanguineous parents, diagnosed with familial hemophagocytic lymphohistiocytosis (HLH) who underwent allogeneic stem cell transplant form HLA matched father.
2101	Approach to hemophagocytic syndromes.	22160031	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyperinflammatory condition. It may occur as a primary (genetic) condition due to mutations in genes important in the cytolytic secretory pathway that cause perforin and granzymes to induce apoptosis in target cells. Primary HLH is divided into familial HLH (FHLH1-5), in which HLH is the only manifestation of disease, and other genetic causes in which HLH is one of several clinical manifestations. The identical clinical findings may arise secondary to infectious, rheumatologic, malignant, or metabolic conditions. Whether primary or secondary, HLH therapy needs to be instituted promptly to prevent irreversible tissue damage. It is helpful to think of HLH as the severe end of the spectrum of hyperinflammatory disorders when the immune system starts to damage host tissues (immunopathology). Therefore, no single clinical feature alone is diagnostic for HLH, and it is important that the entire clinical presentation be considered in making the diagnosis. This article contains a discussion of the genetic background, clinical presentation, diagnostic dilemmas, and features that are helpful in making the diagnosis of HLH, along with a discussion of common problems in its management.
2102	Clinical features of adult patients with secondary hemophagocytic lymphohistiocytosis from causes other than lymphoma: an analysis of treatment outcome and prognostic factors.	22147006	Although hemophagocytic syndrome (HS) featuring secondary hemophagocytic lymphohistiocytosis (HLH) has a grave prognosis, little is known about the natural course of the disease. Patients who showed the clinical features of HLH as well as tissue-proven hemophagocytosis when seen at Asan Medical Center between 1999 and 2010 were included in this analysis. Patients with proven lymphoma were excluded. The median age of our 23 study patients was 49 years. Epstein-Barr virus was suspected to have caused HS in 16 (70%) patients and hepatitis A virus in one patient. Twenty-two patients were treated, 13 according to the HLH protocol and nine using immunosuppressive agents such as corticosteroid and/or cyclosporine. Five patients undertook allogeneic hematopoietic cell transplantation (HCT) during their treatment-dependent relapse (n = 4) or responsive status (n = 1). After the median follow-up of 180 days, 17 (74%) died and six (26%) were alive. The median time from initial presentation until death was 41 days among those patients who died. The serum fibrinogen level ≥166 mg/dL determined at the initial visit was significantly associated with the survival time according to univariate analysis. The low histiocyte proportion in bone marrow and early initiation of treatment tended to correlate with a favorable outcome. On multivariate analysis, serum fibrinogen ≥166 mg/dL (hazard ratio, 0.175, P = 0.018) was an independent clinical factor for determining the patient survival time. Despite appropriate patient management, the outcome of HS featuring HLH was grave. The serum fibrinogen level at the initial presentation was significant, and selected patients obtained some benefit from allogeneic HCT.
2103	Prevalence of coinfection in children with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	22146525	The pathology of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) has not been elucidated. The progression of the disease could be influenced by coinfection with other pathogens. This study investigates the prevalence of cytomegalovirus (CMV), hepatitis B and C, bacteria, mycoplasma, fungi, and tuberculosis among EBV-HLH children.Clinical and laboratory records of EBV-HLH patients at Beijing Children's Hospital between June 2007 and June 2010 were retrospectively reviewed.Forty-seven children diagnosed with EBV-HLH were included. The average age at diagnosis was 4 years 1 months ± 3 years 9 months (mean ± SD). CMV-Ab-IgM was positive in 6.7% (3 of 45), bacteria culture was positive in 14.3% (6 of 42), fungi culture was positive in 10.7% (3 of 28) patients. Hepatitis B virus surface antigen, hepatitis C virus antibody, purified protein derivative, and myoplasma antibody-IgM were negative in these patients. Two patients were coinfected by 2 pathogens: fungi and bacteria (n = 1), CMV and bacteria (n = 1). Four patients died in hospital, among them 2 patients had bacteria culture positive results. Only a fraction of the EBV-HLH patients had genetic defects in PRF1, UNC13D, and XIAP.The overall coinfection ratio with EBV-HLH was 21.3% in our series (10 of 47), 50% of in hospital deaths (2 of 4) were attributable to sepsis. More attention should pay on coinfections in EBV-HLH patients, especially bacterial coinfection.
2104	Intravenous immunoglobulin treatment for macrophage activation syndrome complicating chronic granulomatous disease.	22119934	Chronic granulomatous disease is a rare phagocyte disorder characterized by an increased susceptibility to infections and inflammatory complications. We describe two patients with chronic granulomatous disease (CGD) complicated by macrophage activation syndrome (MAS) (secondary hemophagocytic lymphohistiocytosis) treated with intravenous immunoglobulin (IVIG).A report of two cases of CGD complicated by MAS who were successfully treated with IVIG was made, and a comparison was made with ten other cases reported in the literature.MAS is a severe potentially fatal complication of CGD. Most cases are associated with Burkholderia cepacia and leishmaniasis infection. The treatment of these patients varies between centers, and one example is the use of the HLH-2004 protocol. IVIG could be an effective first line option for this complication in CGD patients.The exaggerated inflammatory response characteristic of CGD patients could play a role in the development of this complication. IVIG appears to be a safe and effective first line treatment in these patients.
2105	Recombinant human LH supplementation versus supplementation with urinary hCG-based LH activity during controlled ovarian stimulation in the long GnRH-agonist protocol: a matched case-control study.	22115012	An observational, matched, case-control study was carried out to compare the efficacy of recombinant human luteinizing hormone (r-hLH) supplementation with that of urinary human menopausal gonadotrophin (u-hMG)-based LH activity during controlled ovarian stimulation (COS) for assisted reproductive technology (ART) using a long gonadotrophin-releasing hormone (GnRH)-agonist protocol. A total of 4719 women, 1573 per group, matched by age, body mass index, indication and number of previous ART cycles, were treated with either recombinant human follicle-stimulating hormone (r-hFSH) and r-hLH in a fixed 2:1 ratio or u-hMG, either alone or in combination with r-hFSH, after down-regulation in a long GnRH-agonist protocol. Compared with the two u-hMG groups (u-hMG alone or in combination with r-hFSH, respectively), r-hFSH consumption was significantly lower (p < 0.001; p < 0.001), and pregnancy rates per cycle (p = 0.006; p = 0.022) and per embryo transfer (p = 0.025; p = 0.008), and implantation rate per embryo transferred (p < 0.001; p < 0.001) were significantly higher in the group treated with the fixed combination of r-hFSH and r-hLH. In COS protocols with r-hFSH, supplementation with r-hLH appears to be more effective than supplementation with u-hMG using the long GnRH-agonist protocol for ART.
2106	Pulmonary lymphomatoid granulomatosis in Griscelli syndrome type 2.	22111599	Griscelli syndrome type 2 (GS2) is a rare autosomal-recessive disorder associated with a RAB27A gene mutation, and clinically manifesting as hypopigmentation, disseminated chronic encephalitis, and severe immunological disorders characterized by an accelerated hematological phase, also referred to as hemophagocytic syndrome (HS), or hemophagocytic lymphohistiocytosis (HLH). The authors report the diagnosis of GS2 in an 11-year-old girl with hypopigmentation, immunodeficiency, hepatosplenomegaly, severe neurological impairments, and fatal multiorgan failure. In this patient a diagnosis of pulmonary lymphomatoid granulomatosis (LG), an Epstein-Barr virus (EBV)-related lymphoproliferative disorder, was established from radiological and histological findings. Although EBV-related malignancies are common in immunocompromised patients, this is the first report of a diagnosis of pulmonary LG in a patient with GS2.
2107	Neuroradiologic findings and follow-up with magnetic resonance imaging of the genetic forms of haemophagocytic lymphohistiocytosis with CNS involvement.	22106034	Haemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome caused by deficient down-regulation of the immune response. Presence of central nervous system (CNS) involvement at diagnosis is a poor prognostic sign, and should be carefully investigated. Herein, we describe the neuroradiological findings, clinical data, and treatment outcome in 12 patients with genetic HLH and CNS complications. Neuroimaging was important in identifying CNS involvement, monitoring treatment responses, and detecting treatment complications.
2108	[Visceral leishmaniasis associated hemophagocytic lymphohistiocytosis: report of four childhood cases].	22088188	The clinical features of four cases of visceral leishmaniasis (VL)-associated hemophagocytic lymphohistiocytosis (VL-HLH) were retrospectively analyzed for the purpose of helping the diagnosis of secondary HLH.Clinical data of three childhood cases of VL-HLH documented in our hospital and one case diagnosed in the Capital Institute of Pediatrics was reviewed retrospectively, with particular emphasis on peculiar clinical manifestations and on clues to the diagnosis of this relatively rare disease entity.Three children were from endemic areas of VL, and the other one had lived in endemic area for one year, which was revealed by detailed history-taking. Clinically, VL-HLH is characterized by persistent fever, hepatosplenomegaly and pancytopenia, which is similar to those of HLH, and is one of the important reasons of delayed diagnosis or misdiagnosis. Based on the HLH-2004 protocol, all the four cases met the diagnostic criteria of HLH. In addition, bone marrow aspirate and immunologic detection of VL-specific antibody via rk39 dipstick test during the early disease course of VL-HLH yielded negative results. Two cases who received HLH-targeted therapy responded reasonably well, with rapid temperature normalization and spleen retraction. Nevertheless, Hb remained lower than normal, which we believed to be related to persistent red cell destruction by the invading parasite Leishmania donovani.VL, a parasitic disease caused by Leishmania donovani, which is currently endemic just in 6 provinces in China, shares similar clinical picture of HLH and is an easily ignored underlying cause of secondary HLH. We suggest that VL should be in the list of differential diagnosis for any patients with HLH who lives in or has a definite travel history to endemic areas. Repeated bone marrow studies are highly warranted to make a definite diagnosis of VL, because bone marrow aspirate or rk39 dipstick test during early disease course might yield negative results. Although VL-HLH responds quite well to HLH-tailored chemotherapy, specific therapy against VL must be given to prevent disease recurrence, and HLH-targeted chemotherapy might be discontinued to prevent chemotherapy-related toxicities.
2109	A network of broadly expressed HLH genes regulates tissue-specific cell fates.	22078884	Spatial and temporal expression of specific basic-helix-loop-helix (bHLH) transcription factors defines many types of cellular differentiation. We find that a distinct mechanism regulates the much broader expression of the heterodimer partners of these specific factors and impinges on differentiation. In Drosophila, a cross-interacting regulatory network links expression of the E protein Daughterless (Da), which heterodimerizes with bHLH proteins to activate them, with expression of the Id protein Extramacrochaetae (Emc), which antagonizes bHLH proteins. Coupled transcriptional feedback loops maintain the widespread Emc expression that restrains Da expression, opposing bHLH-dependent differentiation while enhancing growth and cell survival. Where extracellular signals repress emc, Da expression can increase. This defines regions of proneural ectoderm independently from the proneural bHLH genes. Similar regulation is found in multiple Drosophila tissues and in mammalian cells and therefore is likely to be a conserved general feature of developmental regulation by HLH proteins.
2110	Somatic mutations in NKX2–5, GATA4, and HAND1 are not a common cause of tetralogy of Fallot or hypoplastic left heart.	22043484	The majority of congenital heart disease (CHD) occurs as a sporadic finding, with a minority of cases associated with a known genetic abnormality. Combinations of genetic and environmental factors are implicated, with the recent and intriguing hypothesis that an apparently high rate of somatic mutations might explain some sporadic CHD. We used samples of right ventricular myocardium from patients undergoing surgical repair of tetralogy of Fallot (TOF) and hypoplastic left heart (HLH) to examine the incidence of somatic mutation in cardiac tissue. TOF is a common form of cyanotic CHD, occurring in 3.3 per 10,000 live births. HLH is a rare defect in which the left side of the heart is severely under-developed. Both are severe malformations whose genetic etiology is largely unknown. We carried out direct sequence analysis of the NKX2–5 and GATA4 genes from fresh frozen cardiac tissues and matched blood samples of nine TOF patients. Analysis of NKX2–5, GATA4, and HAND1 was performed from cardiac tissue of 24 HLH patients and three matched blood samples. No somatic or germline mutations were identified in the TOF or HLH patients. Although limited by sample size, our study suggests that somatic mutations in NKX2–5 and GATA4 are not a common cause of isolated TOF or HLH.
2111	Recombinant activated factor VII in hemophagocytic lymphohistiocytosis with disseminated intravascular coagulation.	22040579	Hemophagocytic lymphohistiocytosis (HLH) is a lifethreatening disorder due to hyperinflammation resulting in infiltration of different organs with extensive hemophagocytosis. Severe coagulopathy was one of the main reasons for death in HLH. Over secretion of plasminogen activator by activated macrophages leads to hyperfibrinolysis. We reported a 36-year-old woman who was diagnosed as HLH probably secondary to lymphoma. Massive bleeding from gut and retroperitoneal area were not able to be controlled by conventional hemostatic treatments. This patient received one dose recombinant activated factor VII (rFVIIa) 3.6 mg (70 μg/kg). Hemostatic effect was achieved in 0.5 hour and lasted 24 hours. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were quickly corrected to normal ranges. Fibrinogen level elevated from 0.5 g/L before using rFVIIa to 1.8 g/L 20 hours after. Although dexamethasone and etopside were administrated to treat HLH, this patient died from septic shock after persistent neutropenia. This suggests that rFVIIa may be effective in the management of intractable hemorrhage in patients with HLH.
2112	Hemophagocytic lymphohistiocytosis after hematopoietic stem cell transplantation in children: a nationwide survey in Japan.	22038983	Hemophagocytic lymphohistiocytosis (HLH) is associated with hypercytokinemia in children. Although HLH can be also observed after hematopoietic stem cell transplantation (HSCT), the incidence and clinical features of HLH after HSCT remain obscure.The clinical features of HLH after HSCT (post-HSCT HLH) were investigated in children with malignancies, immune deficiencies, or aplastic anemia. The HLH/Langerhans Cell Histiocytosis (LCH) Committee of the Japanese Society of Pediatric Hematology (JSPH) sent questionnaires to hospitals with JPSH members asking for details of cases in which HLH occurred after HSCT between 1998 and 2008.Among 42 children who were diagnosed with post-HSCT HLH between 1998 and 2008 in Japan, 37 fulfilled our inclusion criteria; of these, 26 were classified as early-onset (onset <30 days after HSCT) and 11 were classified as late-onset (onset >30 days after HSCT). In the early-onset group, the presence of respiratory symptoms, high levels of total bilirubin, and triglycerides at onset and the lack of control of GVHD with tacrolimus were significantly associated with non-resolution of HLH (P < 0.05). The survival rate was significantly higher in patients with resolution of HLH than in those without resolution (59% vs. 14%, P < 0.05).These findings suggest that early-onset post-HSCT HLH is a specific entity of HLH, and appropriate diagnosis and prompt management need to be established.
2113	Glomerular hemophagocytic macrophages in a patient with proteinuria and clinical and laboratory features of hemophagocytic lymphohistiocytosis (HLH).	22038068	Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous disorder characterized by excessive activation and proliferation of nonmalignant histiocytes, which are commonly found in bone marrow, lymph nodes, spleen and liver in affected patients. Here, we report the presence of glomerular macrophages, including one showing erythrophagocytosis, on renal biopsy in a 25-year-old patient with clinical presentation and laboratory changes consistent with HLH. The clinical course was marked by persistent fever for 2 months, pleural and pericardial effusion, splenomegaly, lymphadenopathy, pancytopenia, cardiac arrhythmias, multiple organ dysfunction, and proteinuria, with demise after a 2-month hospitalization. Positive assay for Epstein-Barr virus (EBV), marked hyperferritinemia, hypofibrinogenemia, hypertriglyceridemia, elevated anti-nuclear antibody, proteinuria, and decreased circulating NK cells by flow cytometry were compatible with the diagnosis of HLH. We suggest that the glomerular hemophagocytic macrophages, which have not heretofore been described in the kidney of a patient with HLH, may have contributed to renal dysfunction manifest as proteinuria.
2114	Novel mouse xenograft models reveal a critical role of CD4+ T cells in the proliferation of EBV-infected T and NK cells.	22028658	Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients' PBMC to immunodeficient mice of the NOD/Shi-scid/IL-2Rγ(null) strain. In these models, EBV-infected T, NK, or B cells proliferated systemically and reproduced histological characteristics of the two diseases. Analysis of the TCR repertoire expression revealed that identical predominant EBV-infected T-cell clones proliferated in patients and corresponding mice transplanted with their PBMC. Expression of the EBV nuclear antigen 1 (EBNA1), the latent membrane protein 1 (LMP1), and LMP2, but not EBNA2, in the engrafted cells is consistent with the latency II program of EBV gene expression known in CAEBV. High levels of human cytokines, including IL-8, IFN-γ, and RANTES, were detected in the peripheral blood of the model mice, mirroring hypercytokinemia characteristic to both CAEBV and EBV-HLH. Transplantation of individual immunophenotypic subsets isolated from patients' PBMC as well as that of various combinations of these subsets revealed a critical role of CD4+ T cells in the engraftment of EBV-infected T and NK cells. In accordance with this finding, in vivo depletion of CD4+ T cells by the administration of the OKT4 antibody following transplantation of PBMC prevented the engraftment of EBV-infected T and NK cells. This is the first report of animal models of CAEBV and EBV-HLH that are expected to be useful tools in the development of novel therapeutic strategies for the treatment of the diseases.
2115	Risk factors for early death in children with haemophagocytic lymphohistiocytosis.	22017632	Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening disturbance of immunoregulation. HLH comprises primary and acquired forms with different disease severity. A large proportion of deaths occur early into treatment. We investigated association with early death for laboratory and clinical parameters before the start of and 2 weeks into therapy.A total of 232 children from Scandinavia, Germany or Italy, fulfilling diagnostic criteria and/or with familial disease and/or HLH-causing mutations, receiving HLH treatment 1994-2008 were included. The relation between clinical findings and early pre-transplant death was examined using the Cox proportional hazards model, with a 4-month right-truncation of the outcome. Patients were censored at last follow-up or transplant. Statistically significant predictors were adjusted for sex, age and each other.The following features were significantly associated with adverse outcome: hyperbilirubinaemia (>50 μmol/L; adjusted hazard ratio (aHR) 3.2; 95% confidence interval 1.3-8.1, p = 0.011), hyperferritinaemia (>2000 μg/L; aHR 3.2; 1.2-8.6, p = 0.019), cerebrospinal fluid pleocytosis (>100 × 10(6) /L; aHR 5.1; 1.4-18.5, p = 0.012) at diagnosis, and thrombocytopenia (<40 × 10(9) /L; aHR 3.4; 1.1-10.7, p = 0.033), and hyperferritinaemia (>2000 μg/L; aHR 10.6; 1.2-96.4, p = 0.037) 2 weeks into therapy. Non-improvement of fever, anaemia and/or thrombocytopenia also had adverse impact.There seem to be easily available clinical predictors of early mortality in HLH patients, which may help guide treatment decisions.
2116	Identification and characterization of a putative basic helix-loop-helix transcription factor involved in the early stage of conidiophore development in Aspergillus oryzae.	22008745	The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes. HLH proteins form homo- and/or heterodimers with other HLH proteins and bind to E-box motifs. The regulation and functions of these proteins can be complex due to their bifunctional roles as activators and repressors of gene transcription. In this study, we isolated and characterized a novel predicted bHLH protein-encoding gene, AO090023000902, designated ecdR (early conidiophore development regulator), in Aspergillus oryzae. The ecdR gene disruptant produced very few conidia. Conversely, the overexpression of ecdR resulted in the formation of a large number of conidia at an early stage, suggesting that the EcdR protein is required for early asexual development. Additionally, when serially diluted conidia were spread-cultivated onto malt agar medium, we found that conidial number of the control strain depended on the cultivated conidium density, while the ecdR-overexpressing strain showed no significant change in conidiation. These phenotypes of ecdR-disruptant and ecdR-overexpressing strains are partially similar to those of the sclR-overexpressing strain and sclR-disruptant, respectively. Yeast two-hybrid assays and sclR, ecdR-double deletion experiment indicated that EcdR plays a major role in conidiation, and SclR represses this function by competitively interacting with EcdR in A. oryzae.
2117	Relative humidity hysteresis in solid-state chemical reactivity: a pharmaceutical case study.	21993854	The chemical reaction rate for solid-state product formation in a pharmaceutical case study was monitored by equilibration with either a 75%, 21.5%, 75% relative humidity (RH) cycle ("high-low-high", HLH) or a 21.5%, 75%, 21.5% RH cycle ("low-high-low", LHL). For the HLH cycle, it was found that the degradant formation rate was reversible; that is, the rate at the final 75% RH step was equivalent to the rate at the initial 75% RH step. For samples equilibrated with the LHL cycle, a significantly higher rate of product formation was seen when the low RH condition was re-established than for the initial sample that had never been exposed to high RH. The observed hysteresis in degradant formation rate as a function of RH is not explained by hysteresis in the bulk moisture sorption isotherm, which is minimal in the case studied. It is suggested that high RH exposure impacts the solid-solid interface by either changing the amount of solid-solution present, by altering the mechanical properties of the material such that there is a greater mobility even when moisture is removed, or by altering the interfacial material to have a greater amount of moisture present even when dried.
2118	The expanding spectrum of hemophagocytic lymphohistiocytosis.	21971331	Hemophagocytic lymphohistiocytosis (HLH) is more widely recognized by clinicians. No longer viewed as a disorder of young children, adult patients are now being identified and treated. In this review, I summarize clinical features of patients with recently identified genetic causes, discuss a new paradigm for understanding the clinical evolution of HLH, and update current results with hematopoietic cell transplantation.The list of genetic defects underlying HLH continues to grow. Among the autosomal recessive defects underlying HLH, we add STX11 (Syntaxin 11) - a snare protein, and MUNC18-2 (also known as STXBP2 - Syntaxin-binding protein). These two proteins now join MUNC 13-4 as components of the degranulation machinery in cytotoxic lymphocytes, responsible for the delivery of Perforin and Granzyme B to selectively kill target cells. The mechanism of action in the newest X-linked disorder associated with HLH, XIAP deficiency (also termed XLP 2), is currently unknown. Treatment of HLH has also improved in recent years, at least in experienced centers where a significant number of patients are seen. Clinicians who are familiar with the dynamic evolution of the disease are learning how to modify treatment when initial or continuation therapy fails to achieve a stable clinical status, preferably clinical remission. Use of reduced intensity conditioning protocols pretransplant has resulted in superior short-term and long-term survival rates of greater than 85%.Substantial progress continues to be made in exploring the complex cause and pathophysiology of HLH. Hand in hand, a greater recognition of the condition has led to improved treatments.
2119	Secondary hemophagocytosis in 3 patients with organic acidemia involving propionate metabolism.	21970506	Hemophagocytic lymphohistiocytosis (HLH) may develop secondary to infections, malignancies, immune deficiency syndromes, and rheumatologic and metabolic disorders. Associations between HLH and inborn errors of metabolism, including lysinuric protein intolerance, multiple sulfatase deficiency, galactosemia, Gaucher disease, Pearson syndrome, and galactosialidosis, have previously been reported in the literature. In this report the authors present 3 children with disorders of propionate metabolism--1 with methylmalonic acidemia and 2 with propionic acidemia--who developed secondary HLH during their metabolic attacks. All patients fulfilled the 5 HLH criteria of the Histiocyte Society. Familial HLH was ruled out by molecular analysis. Plasma exchange was performed for 2 of them. Unfortunately 1 died of multiorgan failure despite intensive therapy. This is the first report of such an association.
2120	PUF-8 suppresses the somatic transcription factor PAL-1 expression in C. elegans germline stem cells.	21968099	RNA-binding proteins of the PUF family are well conserved post-transcriptional regulators that control a variety of developmental processes. The C. elegans protein PUF-8 is essential for several aspects of germ cell development including the maintenance of germline stem cells (GSCs). To explore the molecular mechanisms underlying its function, we have identified 160 germline-expressed mRNAs as potential targets of PUF-8. We generated GFP::H2B-3' UTR fusions for 17 mRNAs to assay their post-transcriptional regulation in germ cells. Twelve transgenes were not expressed in the mitotic germ cells, and depletion of PUF-8 led to misexpression of six of them in these cells. In contrast, the expression of 3' UTR fusion of hip-1, which encodes the HSP-70 interacting protein, was dependent on PUF-8. These results indicate that PUF-8 may regulate the expression of its targets both negatively as well as positively. We investigated the PUF-8-mediated post-transcriptional control of one mRNA, namely pal-1, which encodes a homeodomain transcription factor responsible for muscle development. Our results show that PUF-8 binds in vitro to specific sequences within pal-1 3' UTR that are critical for post-transcriptional suppression in GSCs. Removal of PUF-8 resulted in PAL-1 misexpression, and PAL-1-dependent misexpression of the myogenic promoter HLH-1 in germ cells. We propose that PUF-8 protects GSCs from the influence of somatic differentiation factors such as PAL-1, which are produced in the maternal germline but meant for embryogenesis.
2121	Familial hemophagocytic lymphohistiocytosis in a pediatric patient diagnosed by brain magnetic resonance imaging.	21959744	Familial hemophagocytic lymphohistiocytosis (fHLH) is an autosomal recessive disorder characterized by proliferation and infiltration of several organs by activated lymphocytes and macrophages. Without allogeneic stem cell transplantation, fHLH is fatal. We describe a previously healthy 11-month-old boy with a rapidly progressive encephalopathy. An older brother died at 8 months following a subacute encephalopathy diagnosed as meningoencephalitis. The family history led to the suspicion of a metabolic disease, but metabolic studies were unrevealing. MRI showed multiple inhomogeneous signal abnormalities in the cortex and white matter, most prominent in the cerebral hemispheres and around the dentate nucleus. Gadolinium-enhanced T1-weighted images showed a multitude of enhancing foci, suggestive of perivascular enhancement. Based on MRI pattern with multiple lesions, perivascular enhancement and family history, fHLH was suspected. DNA analysis showed that the patient was compound-heterozygous for the c.445 G>A (p.Gly149Ser) mutation in exon 1 and the c.757 G>A (p.Glu253Lys) mutation in exon 2 of the perforin 1 gene. The patient was treated according to the international HLH-2004 protocol (dexamethasone, etoposide, cyclosporine, intrathecal methotrexate and prednisolone) followed by allogeneic cord blood transplantation. He showed a significant neurological and radiological improvement. The reported case demonstrates that MRI pattern recognition can lead to early diagnosis of fHLH, with subsequent adequate treatment.
2122	Differences between adult and pediatric septic shock.	21952599	Sepsis is a significant public health problem that affects children and adults alike. Despite some similarities in the approach to pediatric and adult septic shock, there are key differences as it relates to pathophysiology, clinical presentation, and therapeutic approaches. In this review article, we discuss these differences under 4 headings: a) Developmental differences in the hemodynamic response, b) Activated Protein C, c) Thrombocytopenia associated multiple organ failure and d) Hemophagocytic Lymphohistiocytosis (HLH).
2123	The ETS family member TEL binds to nuclear receptors RAR and RXR and represses gene activation.	21949683	Retinoic acid receptor (RAR) signaling is important for regulating transcriptional activity of genes involved in growth, differentiation, metabolism and reproduction. Defects in RAR signaling have been implicated in cancer. TEL, a member of the ETS family of transcription factors, is a DNA-binding transcriptional repressor. Here, we identify TEL as a transcriptional repressor of RAR signaling by its direct binding to both RAR and its dimerisation partner, the retinoid x receptor (RXR) in a ligand-independent fashion. TEL is found in two isoforms, created by the use of an alternative startcodon at amino acid 43. Although both isoforms bind to RAR and RXR in vitro and in vivo, the shorter form of TEL represses RAR signaling much more efficiently. Binding studies revealed that TEL binds closely to the DNA binding domain of RAR and that both Helix Loop Helix (HLH) and DNA binding domains of TEL are mandatory for interaction. We have shown that repression by TEL does not involve recruitment of histone deacetylases and suggest that polycomb group proteins participate in the process.
2124	Molecular study of the perforin gene in familial hematological malignancies.	21936944	Perforin gene (PRF1) mutations have been identified in some patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH) and in patients with lymphoma. The aim of the present study was to determine whether patients with a familial aggregation of hematological malignancies harbor germline perforin gene mutations. For this purpose, 81 unrelated families from Tunisia and France with aggregated hematological malignancies were investigated. The variants detected in the PRF1 coding region amounted to 3.7% (3/81). Two of the three variants identified were previously described: the p.Ala91Val pathogenic mutation and the p.Asn252Ser polymorphism. A new p.Ala 211Val missense substitution was identified in two related Tunisian patients. In order to assess the pathogenicity of this new variation, bioinformatic tools were used to predict its effects on the perforin protein structure and at the mRNA level. The segregation of the mutant allele was studied in the family of interest and a control population was screened. The fact that this variant was not found to occur in 200 control chromosomes suggests that it may be pathogenic. However, overexpression of mutated PRF1 in rat basophilic leukemia cells did not affect the lytic function of perforin differently from the wild type protein.
2125	Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis.	21928113	Phytochrome Interacting Factor 1 (PIF1), a basic helix-loop-helix (bHLH) protein, functions as a negative regulator of various facets of photomorphogenesis. To indentify PIF1-interacting proteins, we performed yeast two-hybrid screening using PIF1 as a bait and identified a group of proteins including PIF1 itself, PIF3 and long hypocotyl in far-red 1 (HFR1), an atypical HLH protein. Directed yeast two-hybrid interaction assays showed that PIF1 can form heterodimers with all other PIFs as well as with HFR1. PIF1 and PIF3 interacted with each other in both in vitro and in vivo co-immunoprecipitation assays. PIF1-PIF3 heterodimer also bound to a G-box DNA sequence element in vitro. To understand the biological significance of these interactions, a pif1pif3 double mutant was obtained and characterized. Analyses of the single and double mutants showed that PIF3 plays a prominent role in repressing photomorphogenesis under continuous blue light conditions. pif1 and pif3 showed additive phenotypes more prominently under discontinuous blue light conditions. Similar to PIF1, PIF3 was also rapidly phosphorylated, poly-ubiquitylated and degraded in response to blue light. PIF3 also interacted with phytochromes in response to blue light. A PIF3 mutant defective in interaction with both phyA and phyB displayed reduced degradation under blue light, suggesting that phy-interaction was necessary for the blue light-induced degradation of PIF3. Taken together, these data suggest a combinatorial control of photomorphogenesis by bHLH proteins in response to light in Arabidopsis.
2126	A case of haemophagocytic syndrome in HIV-associated disseminated histoplasmosis.	21904709	Disseminated histoplasmosis is an opportunistic infection which is commonly associated with HIV. Haemophagocytic lymphohistiocytosis (HLH) has been described as a secondary phenomenon to infection, collagen-vascular disorders and malignancies. In patients with HIV, cases of reactive haemophagocytic syndrome associated with disseminated histoplasmosis have been reported with CD4 counts of less than 50 cells/µl (450-1660 cells/µl). We report a case of a 25 year old man with HIV who presented with a CD4 count of 153 cells/µl and would suggest that this diagnosis should be considered at higher CD4 counts than previously reported.
2127	Chemoimmunotherapy for hemophagocytic lymphohistiocytosis: long-term results of the HLH-94 treatment protocol.	21900192	Hemophagocytic lymphohistiocytosis (HLH) used to have a dismal prognosis. We report the final results of HLH-94, the largest prospective diagnostic/therapeutic HLH study so far. The treatment includes immunosuppressive and cytotoxic therapy aiming at clinical remission, followed by HSCT in patients with familial, persistent, or recurrent disease. Altogether, 249 patients fulfilled inclusion criteria and started HLH-94 therapy (July 1994-December 2003); 227 (91%) were followed-up for ≥ 5 years. At 6.2 years median follow-up, estimated 5-year probability of survival was 54% ± 6%. Seventy-two patients (29%) died before HSCT, 64 within 1 year, 97% of whom had active disease. In 124 patients who underwent HSCT, 5-year survival was 66 ± 8%; tendency to increased survival (P = .064) in patients with nonactive disease at HSCT. Patients with familial disease had a 5-year survival of 50% ± 13%; none survived without HSCT. Patients deceased during the first 2 months more often had jaundice, edema, and elevated creatinine. Forty-nine patients (20%) were alive without signs of HLH activity and off-therapy > 1-year without HSCT; they presented at older age (P < .001), were more often female (P = .011), and less often had CNS disease (P < .001) or hepatomegaly (P = .007). To conclude, HLH-94 chemoimmunotherapy has considerably improved outcome in HLH. Collaborative efforts are needed to further reduce early mortality, HSCT-related mortality, and neurologic late effects.
2128	Association of IRF5 polymorphisms with susceptibility to hemophagocytic lymphohistiocytosis in children.	21898142	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome and has a varied genetic background. The polymorphism of interferon regulatory factor 5 gene (IRF5) was reported to be associated with susceptibility to macrophage activation syndrome. IRF5 acts as a master transcription factor in the activation of pro-inflammatory cytokines. We assessed associations of IRF5 gene polymorphisms with susceptibility to secondary HLH.Three IRF5 single nucleotide polymorphisms (rs729302, rs2004640, and rs2280714) were genotyped using TaqMan assays in 82 secondary HLH patients and 188 control subjects.There was a significant association of the GT/TT genotype at rs2004640 with secondary HLH susceptibility (p < 0.01). The IRF5 haplotype (rs729302 A, rs2004640 T, and rs2280714 T) was associated with secondary HLH susceptibility (p < 0.01).These findings indicate that IRF5 is a genetic factor influencing the susceptibility to secondary HLH and that the IRF5-associated immune response contributes to the pathogenesis of HLH.
2129	Fatal hemophagocytic syndrome in a patient with a previously well-controlled asymptomatic HIV infection after EBV reactivation.	21888928	We present a rare case of hemophagocytic lymphohistiocytosis (HLH) in a 70 year-old male patient with previously well-controlled HIV infection. We could confirm HLH in different organs post mortem. Since the diagnosis of HLH was delayed, the patient died despite initiation of chemotherapy. As cause for HLH, an EBV reactivation could be confirmed. In HIV infection, HLH may be the first HIV manifestation, often triggered by an opportunistic infection or immune reconstitution syndrome, but it is uncommon in the state of a well-controlled or aviremic HIV infection.
2130	Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH.	2188104322123903	Familial hemophagocytic lymphohistiocytosis (HLH) is a rare primary immunodeficiency disorder characterized by defects in cell-mediated cytotoxicity that results in fever, hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but rarely diagnosed in adults. We conducted a retrospective review of genetic and immunologic test results in patients who developed HLH in adulthood. Included in our study were 1531 patients with a clinical diagnosis of HLH; 175 patients were 18 years or older. Missense and splice-site sequence variants in PRF1, MUNC13-4, and STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was found in 12 of these patients (48%). The preponderance of hypomorphic mutations in familial HLH-causing genes correlates with the later-onset clinical symptoms and the more indolent course in adult patients. We conclude that late-onset familial HLH occurs more commonly than was suspected previously.
2131	Subtle differences in CTL cytotoxicity determine susceptibility to hemophagocytic lymphohistiocytosis in mice and humans with Chediak-Higashi syndrome.	21878672	Perforin-mediated cytotoxicity is important for controlling viral infections, but also for limiting immune reactions. Failure of this cytotoxic pathway leads to hemophagocytic lymphohistiocytosis (HLH), a life-threatening disorder of uncontrolled T-cell and macrophage activation. We studied susceptibility to HLH in 2 mouse strains (souris and beige(J)) and a cohort of patients with partial defects in perforin secretion resulting from different mutations in the LYST gene. Although both strains lacked NK-cell cytotoxicity, only souris mice developed all clinical and histopathologic signs of HLH after infection with lymphocytic choriomeningitis virus. The 2 strains showed subtle differences in CTL cytotoxicity in vitro that had a large impact on virus control in vivo. Whereas beige(J) CTLs eliminated lymphocytic choriomeningitis virus infection, souris CTLs failed to control the virus, which was associated with the development of HLH. In LYST-mutant patients with Chediak-Higashi syndrome, CTL cytotoxicity was reduced in patients with early-onset HLH, whereas it was retained in patients who later or never developed HLH. Thus, the risk of HLH development is set by a threshold that is determined by subtle differences in CTL cytotoxicity. Differences in the cytotoxic capacity of CTLs may be predictive for the risk of Chediak-Higashi syndrome patients to develop HLH.
2132	Splenectomy for an adult patient with refractory secondary hemophagocytic lymphohistiocytosis.	2187302222377435	Treatment regimens of secondary hemophagocytic lymphohistiocytosis (sHLH) are complicated and individualized. CHOP regimen is well known for the treatment of adult sHLH, but it was not so effective for the 56-year-old male patient in our study. Splenomegaly, one of clinical manifestations of HLH, has urged us to investigate the role of splenectomy in HLH patients. Splenectomy is not only beneficial to confirm the underlying diseases, but also beneficial for the treatment of HLH. Here, we present a case diagnosed as sHLH who has recovered from HLH following comprehensive treatment based on splenectomy. The therapeutic value of splenectomy in sHLH needs further study.
2133	Cell architecture: surrounding muscle cells shape gland cell morphology in the Caenorhabditis elegans pharynx.	21868609	The acquisition and maintenance of shape is critical for the normal function of most cells. Here we investigate the morphology of the pharyngeal glands of Caenorhabditis elegans. These unicellular glands have long cellular processes that extend discrete lengths through the pharyngeal musculature and terminate at ducts connected to the pharyngeal lumen. From a genetic screen we identified several mutants that affect pharyngeal gland morphology. The most severe such mutant is an allele of sma-1, which encodes a β-spectrin required for embryonic elongation, including elongation of the pharynx. In sma-1 mutants, gland projections form normally but become increasingly abnormal over time, acquiring additional branches, outgrowths, and swelling, suggestive of hypertrophy. Rather than acting in pharyngeal glands, sma-1 functions in the surrounding musculature, suggesting that pharyngeal muscles play a critical role in maintenance of gland morphology by restricting their growth, and analysis of other mutants known to affect pharyngeal muscles supports this hypothesis. We suggest that gland morphology is maintained by a balance of forces from the muscles and the glands.
2134	Effects of repeated exposure on acceptance of initially disliked and liked Nordic snack bars in 9-11 year-old children.	21868139	Children's food choices are guided by their preferences. It is established, however, that repeated exposure to a novel food increases children's acceptance. This study investigated how acceptance of an initially liked and disliked snack bar develops in 9-11 year-old children.315 children were randomised into three groups: A control group (n = 111) and two groups exposed to an initially liked kamut bar (n = 94) and an initially disliked sea buckthorn bar (n = 110). Acceptance of both bars was tested before and after the exposure period, and on the 9th exposure.Intake of both bars increased significantly in the exposure groups. There was no difference in the control groups' intake or liking of the bars between pre and post-testing. Liking rose significantly for children exposed to the disliked sea buckthorn bar, while this was not observed in children exposed to the liked kamut bar. In a post-test children exposed to kamut bars had higher intake of that bar than non-exposed children. This was also observed for the sea buckthorn bar that was also given significantly higher liking scores by the exposure group.The majority of children exposed to an initially disliked bar increase acceptance after nine exposures to the same level as an initially liked bar. Children repeatedly exposed to a liked bar show stable acceptance.
2135	The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum.	21851852	Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α(D)-HlH, α(N)-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α(D)-HlH and β-HlH was obtained, including the 5' and 3' UTR, 180bp of the 5' end and around 900bp at the 3' end are missing for the third subunit. The subunits α(D)-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α(N)-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α(D)-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.
2136	Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.	21851100	Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.
2137	Expression of HES and HEY genes in infantile hemangiomas.	21834989	Infantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs.HemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR.HEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes.HES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells.
2138	How I treat hemophagocytic lymphohistiocytosis.	21828139	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of pathologic immune activation, occurring as either a familial disorder or a sporadic condition, in association with a variety of triggers. This immune dysregulatory disorder is prominently associated with cytopenias and a unique combination of clinical signs and symptoms of extreme inflammation. Prompt initiation of immunochemotherapy is essential for survival, but timely diagnosis may be challenging because of the rarity of HLH, its variable presentation, and the time required to perform diagnostic testing. Therapy is complicated by dynamic clinical course, high risk of treatment-related morbidity, and disease recurrence. Here, we review the clinical manifestations and patterns of HLH and describe our approach to the diagnosis and therapy for this elusive and potentially lethal condition.
2139	Severe CMV-related pneumonia complicated by the hemophagocytic lymphohistiocytic (HLH) syndrome in quiescent Crohn's colitis: harmful cure?	218267671680439419177429	NaN
2140	The action mechanism of the Myc inhibitor termed Omomyc may give clues on how to target Myc for cancer therapy.	21811581	Recent evidence points to Myc--a multifaceted bHLHZip transcription factor deregulated in the majority of human cancers--as a priority target for therapy. How to target Myc is less clear, given its involvement in a variety of key functions in healthy cells. Here we report on the action mechanism of the Myc interfering molecule termed Omomyc, which demonstrated astounding therapeutic efficacy in transgenic mouse cancer models in vivo. Omomyc action is different from the one that can be obtained by gene knockout or RNA interference, approaches designed to block all functions of a gene product. This molecule--instead--appears to cause an edge-specific perturbation that destroys some protein interactions of the Myc node and keeps others intact, with the result of reshaping the Myc transcriptome. Omomyc selectively targets Myc protein interactions: it binds c- and N-Myc, Max and Miz-1, but does not bind Mad or select HLH proteins. Specifically, it prevents Myc binding to promoter E-boxes and transactivation of target genes while retaining Miz-1 dependent binding to promoters and transrepression. This is accompanied by broad epigenetic changes such as decreased acetylation and increased methylation at H3 lysine 9. In the presence of Omomyc, the Myc interactome is channeled to repression and its activity appears to switch from a pro-oncogenic to a tumor suppressive one. Given the extraordinary therapeutic impact of Omomyc in animal models, these data suggest that successfully targeting Myc for cancer therapy might require a similar twofold action, in order to prevent Myc/Max binding to E-boxes and, at the same time, keep repressing genes that would be repressed by Myc.
2141	Structure-activity relationships of a peptidic antagonist of Id1 studied by biosensor method, circular dichroism spectroscopy, and bioassay.	21805537	Id1 proteins, inhibitors of differentiation or DNA binding, act as dominant negative antagonists of the bHLH family of transcription factors, which play an important role in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by forming high-affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation-associated genes. Our goal is to study the SARs of a peptidic antagonist of Id1, peptide 3C, which exhibits high affinity for Id1 and inhibitory effect on the proliferation of cancer cells. A series of N-terminal- and C-terminal-deleted analogs of peptide 3C were designed, synthesized, and characterized. Affinity of each peptide for Id1 or Id1-HLH domain was determined by SPR-based biosensor. The secondary structure of each peptide was studied by CD spectroscopy. Biological effect of each peptide in breast cancer cell (MCF-7) was analyzed by the MTT cell viability assay. Results demonstrated that peptide 3C and peptide 3C-CtD4 exhibited higher affinity for Id1-HLH and the equilibrium dissociation constants (K(D) ) were 3.16 and 2.77 µM, respectively. CD results indicated that the percentage of α-helix (%) in the secondary structure of deleted peptides were different, ranging from 7.93 to 10.45%. Although MTT results showed that treatment of MCF-7 with these peptides did not cause antiproliferative effects in cancer cells, SPR results demonstrated that the high-affinity peptides 3C and 3C-CtD4 are promising for further modifications to enhance their affinity for Id1-HLH and antiproliferative effects in cancer cells and for the development of peptidic antagonists as anticancer agents.
2142	New insights into the Orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment.	21789514	CK2 is a Ser/Thr protein kinase that regulates the activity of the Drosophila basic-helix-loop-helix (bHLH) repressor M8 encoded by the Enhancer of split Complex (E(spl)C) during neurogenesis. Specifically, phosphorylation appears to elicit a conformational change in an autoinhibited state of M8 to one that is permissive for repression. We describe biochemical and molecular modeling studies that provide new insights into repression by M8. Our studies implicate the phosphorylation domain in autoinhibition, and indicate that binding of the co-repressor Groucho (Gro) is context-dependent. Molecular modeling indicates that the Orange domain, proposed to be a specificity-determinant, may instead play a structural role, and that a conformational rearrangement of this domain may be necessary for repression. This model also provides a structural mechanism for the behavior of mutant alleles of the m8 gene. The insights gained from these studies should be applicable to the conserved metazoan bHLH repressors of the Hairy and Enhancer of Split (HES) family that are related to Drosophila M8.
2143	A case of acute encephalopathy with hemophagocytic lymphohistiocytosis and clonal T-cell expansion.	21784590	We report on a 9-year-old boy who presented with acute encephalopathy and hemophagocytic lymphohistiocytosis (HLH). The patient was referred to our hospital because of fever, seizures, and decreased consciousness. He showed moderately elevated levels of proinflammatory cytokines in the cerebrospinal fluid and plasma, and clonal expansion of highly activated CD8⁺ T cells in the peripheral blood. These CD8⁺ T cells were found to be larger cells that stained positive for T-cell receptor Vβ13.6, and decreased shortly after steroid therapy. Our findings suggest that his acute encephalopathy was likely a clinical manifestation of HLH, and that immunophenotypic analysis may be helpful for early recognition of HLH in such rare encephalopathy.
2144	The transcription factor HLH-2/E/Daughterless regulates anchor cell invasion across basement membrane in C. elegans.	21784067	Cell invasion through basement membrane is a specialized cellular behavior critical for many developmental processes and leukocyte trafficking. Invasive cellular behavior is also inappropriately co-opted during cancer progression. Acquisition of an invasive phenotype is accompanied by changes in gene expression that are thought to coordinate the steps of invasion. The transcription factors responsible for these changes in gene expression, however, are largely unknown. C. elegans anchor cell (AC) invasion is a genetically tractable in vivo model of invasion through basement membrane. AC invasion requires the conserved transcription factor FOS-1A, but other transcription factors are thought to act in parallel to FOS-1A to control invasion. Here we identify the transcription factor HLH-2, the C. elegans ortholog of Drosophila Daughterless and vertebrate E proteins, as a regulator of AC invasion. Reduction of HLH-2 function by RNAi or with a hypomorphic allele causes defects in AC invasion. Genetic analysis indicates that HLH-2 has functions outside of the FOS-1A pathway. Using expression analysis, we identify three genes that are transcriptionally regulated by HLH-2: the protocadherin cdh-3, and two genes encoding secreted extracellular matrix proteins, mig-6/papilin and him-4/hemicentin. Further, we show that reduction of HLH-2 function causes defects in polarization of F-actin to the invasive cell membrane, a process required for the AC to generate protrusions that breach the basement membrane. This work identifies HLH-2 as a regulator of the invasive phenotype in the AC, adding to our understanding of the transcriptional networks that control cell invasion.
2145	Treatment of recurrent CNS disease post-bone marrow transplant in familial HLH.	21755594	CNS involvement in Hemophagocytic Lymphohistiocytosis (HLH) has been reported in 63-73% of children at diagnosis [Haddad et al. (1997); Blood 89: 794-800; Horne et al. (2008); Br J Haematol 140: 327-335]. Patients can present with neurologic symptoms, abnormal CSF cytology, abnormal neuro-imaging, or a combination of these findings. CNS involvement is usually associated with a poor prognosis and increased mortality. The 3 year overall survival is 44% in patients with CNS involvement compared to 67% in patients without CNS involvement at diagnosis [Horne et al. (2008); Br J Haematol 140: 327-335]. We describe a treatment strategy employing systemic dexamethasone to control CNS disease in a patient with familial HLH and persistent CNS disease post Bone Marrow Transplant.
2146	Hemophagocytic lymphohistiocytosis: a potentially underrecognized association with systemic inflammatory response syndrome, severe sepsis, and septic shock in adults.	2173749222396580	Hemophagocytic lymphohistiocytosis (HLH) was originally described as a genetic disorder of immune regulation, presenting in neonates with protracted fever, hepatosplenomegaly, and cytopenia. A secondary form of HLH, triggered by serious infections, was subsequently described in adults.We report three adult patients who presented with systemic inflammatory response syndrome and features consistent with severe sepsis and septic shock, who subsequently received a diagnosis of secondary HLH. We reviewed the relationship between infection-triggered HLH and septic shock from the perspective of the adult intensivist.The hyperinflammatory pathophysiologic characteristics of HLH and septic shock are closely intertwined. Clinical and laboratory features of HLH and septic shock overlap in some patients, making the syndromes difficult to distinguish. In our experience and review, progressive pancytopenia was the feature most likely to suggest secondary HLH in the adult patient with presumed (or definite) septic shock. Use of other HLH-2004 diagnostic criteria is hindered by the poor operating characteristics of these tests in critically ill adults. Bone marrow aspiration is the most useful diagnostic test, but may yield an initial false-negative result.The HLH-2004 treatment protocol is not of proven benefit in critically ill adults, but observational data suggest that aggressive immunosuppressive therapy should not be delayed. Further study of HLH in the critical care setting might provide important insights into the pathogenesis and clinical treatment of sepsis.
2147	Notch-dependent induction of left/right asymmetry in C. elegans interneurons and motoneurons.	21737278	Although nervous systems are largely bilaterally symmetric on a structural level, they display striking degrees of functional left/right (L/R) asymmetry. In Caenorhabditis elegans, two structurally symmetric pairs of sensory neurons, ASE and AWC, display two distinctly controlled types of functional L/R asymmetries (stereotyped versus stochastic asymmetry). Beyond these two cases, the extent of neuronal asymmetry in the C. elegans nervous system was unclear. Here, we report that the Beta3/Olig-type bHLH transcription factor hlh-16 is L/R asymmetrically expressed in several distinct, otherwise bilaterally symmetric interneuron and motoneuron pairs that are part of a known navigation circuit. We find that hlh-16 asymmetry is generated during gastrulation by an asymmetric LAG-2/Delta signal originating from the mesoderm that promotes hlh-16 expression in neurons on the left side through direct binding of the Notch effector LAG-1/Su(H)/CBF to a cis-regulatory element in the hlh-16 locus. Removal of hlh-16 reveals an unanticipated asymmetry in the ability of the axons of the AIY interneurons to extend into the nerve ring, with the left AIY axon requiring elevated hlh-16 expression for correct extension. Our study suggests that the extent of molecular L/R asymmetry in the C. elegans nervous system is broader than previously anticipated, establishes a novel signaling mechanism that crosses germ layers to diversify bilaterally symmetric neuronal lineages, and reveals L/R asymmetric control of axonal outgrowth of bilaterally symmetric neurons.
2148	Association of Crohn's disease, thiopurines, and primary epstein-barr virus infection with hemophagocytic lymphohistiocytosis.	21722918	To assess the incidence of hemophagocytic lymphohistiocytosis (HLH) in a well-defined population of children with inflammatory bowel disease (IBD) and evaluate the common clinical and laboratory characteristics of individuals with IBD who developed HLH.We conducted a retrospective study of all children who developed HLH over an 8-year period. The incidence of HLH in patients with IBD was calculated using US census data and a statewide project examining the epidemiology of pediatric IBD.Among children in Wisconsin, 20 cases of HLH occurred during the study period; 5 cases occurred in children with IBD. Common characteristics include: Crohn's disease (CD), thiopurine administration, fever lasting more than 5 days, lymphadenopathy, splenomegaly, anemia, lymphopenia, and elevated serum triglycerides and ferritin. Of the patients, 4 had primary Epstein-Barr virus infections. The incidence of HLH among all children in Wisconsin was 1.5 per 100 000 per year. The risk was more than 100-fold greater for children with CD (P < .00001).Pediatric patients with CD are at increased risk for developing HLH; primary Epstein-Barr virus infection and thiopurine administration may be risk factors.
2149	The tailless ortholog nhr-67 functions in the development of the C. elegans ventral uterus.	21718694	The development of the C. elegans uterus provides a model for understanding the regulatory pathways that control organogenesis. In C. elegans, the ventral uterus develops through coordinated signaling between the uterine anchor cell (AC) and a ventral uterine (VU) cell. The nhr-67 gene encodes the nematode ortholog of the tailless nuclear receptor gene. Fly and vertebrate tailless genes function in neuronal and ectodermal developmental pathways. We show that nhr-67 functions in multiple steps in the development of the C. elegans uterus. First, it functions in the differentiation of the AC. Second, it functions in reciprocal signaling between the AC and an equipotent VU cell. Third, it is required for a later signaling event between the AC and VU descendants. nhr-67 is required for the expression of both the lag-2/Delta signal in the AC and the lin-12/Notch receptor in all three VU cells and their descendants, suggesting that nhr-67 may be a key regulator of Notch-signaling components. We discuss the implications of these findings for proposed developmental regulatory pathways that include the helix-loop-helix regulator hlh-2/daughterless and transcription factor egl-43/Evi1 in the differentiation of ventral uterine cell types.
2150	Reduced-intensity conditioning haematopoietic cell transplantation for haemophagocytic lymphohistiocytosis: an important step forward.	21707584	Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunodeficiency characterized by severe systemic hyper-inflammatory responses to infectious or other triggers of the immune system. In many patients, the underlying cause of HLH is a genetic defect leading to defective CD8(+) T cell and natural killer cell granule-mediated cytotoxicity. The treatment of HLH consists principally of immune suppression followed by allogeneic haematopoietic cell transplantation (HCT) to cure the underlying defect and prevent relapse of HLH. Initial treatment regimens consist of steroids coupled with either etoposide or antithymocyte globulin, ± ciclosporin. Complete responses are observed in only 50-75% of patients and even after a complete response, relapse and death still occur. The only definitive, long-term cure for patients with genetic forms of HLH is allogeneic HCT. Unfortunately, allogeneic HCT for patients with HLH is often complicated by critical illness, extensive organ involvement, active infections, or refractory HLH. For these reasons, patients are unusually prone to developing transplant-related toxicities and complications. In recent years, great strides have been made with regard to the care and transplantation of patients with HLH. Here we review the current state of the treatment of patients with HLH with allogeneic HCT, highlighting the important steps forward that have been made with reduced-intensity conditioning.
2151	A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans.	21698137	One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.
2152	Modulation of dopamine-dependent behaviors by the Caenorhabditis elegans Olig homolog HLH-17.	21688290	In vertebrates and invertebrates, dopamine signaling modulates a wide variety of physical and behavioral functions and exerts these effects through heterotrimeric G proteins. The soil nematode Caenorhabditis elegans has been used to model dopamine signaling and reacts reproducibly to alterations in dopamine levels through eight well-characterized dopaminergic neurons located in the head. In C. elegans, the basic helix-loop-helix transcription factor HLH-17 is strongly and constitutively expressed in the glia cells that ensheath four of the dopaminergic neurons, yet it is not required for specification or development of either the glia or the neurons. In this study, we sought to determine whether HLH-17 functions in dopamine signaling. We found that, unlike wild-type animals, hlh-17 animals are resistant to the effects of exogenous dopamine on egg laying and mobility. hlh-17 animals are also defective in the basal slowing and gustatory plasticity behaviors that require functional dopamine signaling. We also found that the expression of the dopamine receptor genes dop-1, dop-2, and dop-3 and the RGS protein gene egl-10 is significantly reduced in hlh-17 animals. Together these results point to a role for HLH-17 in dopamine signaling in C. elegans.
2153	Rapamycin does not control hemophagocytic lymphohistiocytosis in LCMV-infected perforin-deficient mice.	21681935	Hemophagocytic lymphohistiocytosis (HLH) is an immunodysregulatory disorder for which more effective treatments are needed. The macrolide rapamycin has immunosuppressive properties, making it an attractive candidate for controlling the aberrant T cell activation that occurs in HLH. To investigate its therapeutic potential, we used rapamycin to treat Lymphocytic Choriomeningitis Virus (LCMV)-infected perforin-deficient (Prf1(-/-)) mice according to a well-established model of HLH. At the regimens tested, rapamycin did not improve weight loss, splenomegaly, hemophagocytosis, cytopenias, or proinflammatory cytokine production in LCMV-infected Prf1(-/-) animals. Thus, single agent rapamycin appears ineffective in treating the clinical and laboratory manifestations of LCMV-induced HLH.
2154	Screening the PRF1, UNC13D, STX11, SH2D1A, XIAP, and ITK gene mutations in Chinese children with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	21674762	This study aimed to investigate the prevalence of mutations in the PRF1, UNC13D, STX11, SH2D1A, XIAP, and ITK in Chinese pediatric patients with EBV-HLH.Sixty-seven pediatric patients diagnosed with EBV-HLH in Beijing Children's Hospital were recruited. Nucleotide sequences of all exons and their flanking intronic sequences of PRF1, UNC13D, STX11, SH2D1A, XIAP, and ITK were amplified by PCR followed by direct sequencing.Eight patients were identified with heterozygous, compound heterozygous, or homozygous mutations in PFR1, UNC13D, and XIAP. Three missense mutations (c.83G>A, c.503G>A, c.632C>T) were found in PRF1 of two males and two females. Compound heterozygous c.93C>G and c.1066C>T were found in PRF1 of a 2.5-year-old female. Four different mutations were found in UNC13D of two patients: compound nonsense heterozygous mutations c.766C>T and c.1215C>G were found in one male and two splicing mutations c.1596+1G>C and c.2709+1G>A were found in another male. A heterozygous mutation c.1099+2T>C in XIAP was found in a 4-year-old male. No detrimental mutations were identified in STX11, SH2D1A, or ITK. NK cell activity did not differ between the eight FHL patients and the remaining patients. There was no statistical difference in clinical features and laboratory data for these two subgroups with biallelic and heterozygous mutations.Seven novel mutations in PRF1, UNC13D, and XIAP were identified in EBV-HLH patients. Only a fraction of the Chinese children with EBV-HLH have genetic defects in PRF1, UNC13D, and XIAP. There were no gene mutations of PRF1/UNC13D/STX11/SH2D1A/XIAP/ITK in the majority of Chinese child patients with EBV-HLH.
2155	Unrelated bone marrow transplantation induced long-term remission in a patient with life-threatening Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	21628862	We present a case of life-threatening Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) with severe hepatitis that was successfully treated by allogeneic stem cell transplantation from an unrelated donor. A 26-year-old woman was admitted to hospital with a high fever and liver dysfunction. Laboratory tests, including bone marrow aspiration, revealed severe HLH that occurred after EBV infection. High-dose methylprednisolone and etoposide therapy did not control the disease. We could control the HLH, but the EBV viremia continued following the CHOPE (cyclophosphamide, doxorubicin, vincristine, prednisone, and etoposide) chemotherapy regimen. Therefore, the patient underwent allogeneic bone marrow transplantation from an HLA-matched, unrelated donor. The patient has remained in good condition without disease recurrence for 2 years after bone marrow transplantation. Although there is no consensus regarding allogeneic stem cell transplantation for EBV-HLH, it is the treatment of choice for aggressive EBV-HLH when the patient is refractory to intensive chemotherapy.
2156	Hemophagocytic Lymphohistiocytosis in Association With Clostridium difficile Infection and Cutaneous T-Cell Lymphoma.	29147237	Hemophagocytic lymphohistiocytosis (HLH) or Macrophage Activation Syndrome (MAS) is a potentially life threatening disorder that presents with fever, suppressed blood cell counts, hepatosplenomegaly and multi-organ failure. HLH has been reported in association with genetic mutations, infections, autoimmune disorders, and various malignancies. However to the best of our knowledge, HLH in association with Clostridium difficile infection has never been reported. We present a case of HLH in a patient with Epstein-Barr virus (EBV) positive natural killer T (NKT) cell cutaneous lymphoma and active Clostridium difficile infection. A 35-year-old male with recently diagnosed EBV positive NKT cell lymphoma was admitted for Clostridium difficile associated diarrhea. During the course of hospitalization he gradually developed pancytopenia and multi-organ failure leading to death. Post-mortem examination confirmed the diagnosis of hemophagocytic lymphohistiocytosis.
2157	Parvovirus B19-associated hemophagocytic lymphohistiocytosis in a child with precursor B-cell acute lymphoblastic leukemia under maintenance chemotherapy.	21617562	Development of hemophagocytic lymphohistiocytosis (HLH) is quite rare among acute lymphoblastic leukemia (ALL) patients. We present a 3-year-old boy with precursor B-cell ALL, who was complicated by HLH because of parvovirus B19 infection during maintenance chemotherapy. Remarkable erythroid hypoplasia, giant normoblasts, and hemophagocytosed macrophages in bone marrow were important clues for the diagnosis. The patient was successfully treated with high-dose steroids and intravenous immunoglobulins. To our knowledge, this is the first report describing parvovirus B19-associated HLH in ALL. Our case highlights that parvovirus B19 can cause HLH, a potentially fatal disorder, and prolonged unexpected cytopenia in childhood ALL.
2158	Primary haemophagocytic syndrome in a young girl.	21608353	Haemophagocytic syndrome is a life-threatening systemic illness characterized by an uncontrolled inflammatory response. Patients present with fever, hepatosplenomegaly, jaundice and liver dysfunction, neurological manifestations and often pancytopenla. Bone marrow, lymph node, hepatic or splenic biopsy shows macrophages with Ingested blood cells or their precursors. Laboratory markers include elevated triglycerides and ferritin, low fibrinogen with normal or low erythrocyte sedimentation rate (ESR). Familial haemophagocytic lymphohistiocytosis (HLH) is an autosomal recessive disorder. Secondary haemophagocytic syndrome results from infections, malignancy and collagen vascular disorders. We describe a young girl with primary haemophagocytic syndrome.
2159	Perforin is a critical physiologic regulator of T-cell activation.	21606480	Individuals with impaired perforin-dependent cytotoxic function (Ctx(-)) develop a fatal inflammatory disorder called hemophagocytic lymphohistiocytosis (HLH). It has been hypothesized that immune hyperactivation during HLH is caused by heightened infection, defective apoptosis/responsiveness of Ctx(-) lymphocytes, or enhanced antigen presentation. Whereas clinical and experimental data suggest that increased T-cell activation drives HLH, potential abnormalities of T-cell activation have not been well characterized in Ctx(-) hosts. To define such abnormalities and to test these hypotheses, we assessed in vivo T-cell activation kinetics and viral loads after lymphocytic choriomeningitis virus (LCMV) infection of Ctx(-) mice. We found that increased T-cell activation occurred early during infection of Ctx(-) mice, while they had viral burdens that were identical to those of WT animals, demonstrating that T-cell hyperactivation was independent of viral load. Furthermore, cell transfer and signaling studies indicated that increased antigenic stimulation, not a cell-intrinsic defect of responsiveness, underlay heightened T-cell activation in vivo. Finally, direct measurement of viral antigen presentation demonstrated an increase in Ctx(-) mice that was proportional to abnormal T-cell activation. We conclude that perforin-dependent cytotoxicity has an immunoregulatory role that is distinguishable from its pathogen clearance function and limits T-cell activation in the physiologic context by suppressing antigen presentation.
2160	[The study of gene mutations in unknown refractory viral infection and primary hemophagocytic lymphohistiocytosis].	21600143	To study the type and corresponding clinical characteristics of primary hemophagocytic lymphohistiocytosis (HLH) associated immune gene mutations in the refractory virus infection or HLH of unknown causes.From December 2009 to July 2010, the patients with refractory virus infection or HLH of unknown causes were screened for the primary HLH associated immune genes mutations by DNA sequence analysis, including PRF1, UNC13D, STX11, STXBP2, SH2D1A and XIAP. The clinical characteristics and outcomes were followed up.Totally 25 patients with refractory virus infection or HLH of unknown causes were investigated for the 6 genes and 13 cases were found carrying gene mutations, composing of 6 of PRF1 mutation, 3 of UNC13D, and each one of STX11, XIAP, SH2D1A and STXBP2, respectively. Among the 13 cases with gene mutations, 5 suffered from Epstein-Barr virus associated HLH (EBV-HLH), 1 human herpes virus 7 associated HLH (HHV7-HLH), 1 HLH without causes, 4 chronic activated EB virus infection (CAEBV) with 1 progressing to Hodgkin's lymphoma carrying abnormal chromosome of t(15;17) (q22;q25) and hyperdiploid, 2 EBV associated lymphoma. Among the other 12 patients without gene mutation, 4 suffered from EBV-HLH with 1 progressing to peripheral T lymphoma, 8 suffered from CAEBV.Primary HLH associated immune gene mutations are critical causes of refractory virus infection of unknown causes, most patients manifest as HLH, some cases appear in CAEBV and EBV associated lymphoma. DNA sequence analysis is helpful to early diagnosis and correct decision-making for treatment.
2161	Mid-follicular LH supplementation in women aged 35-39 years undergoing ICSI cycles: a randomized controlled study.	2157584920031032	This single-centre, randomized, parallel group, comparative study aimed to identify potential benefits of mid-follicular recombinant human LH (r-HLH) supplementation in women aged 35-39 years undergoing ovarian stimulation for intracytoplasmic sperm injection (ICSI). The main endpoint was the number of metaphase II oocytes retrieved. After pituitary suppression with a gonadotrophin-releasing hormone agonist, ovarian stimulation was initiated with recombinant human FSH (r-HFSH; 300-450 IU/day). On stimulation day 6, patients were randomized to receive r-HFSH alone or r-HFSH + r-HLH (r-HLH 150 IU/day) for the remainder of the stimulation period. Final follicular maturation was triggered with 250 μg of recombinant human chorionic gonadotrophin. After assessing oocyte nuclear maturity, oocyte were fertilized by ICSI and afterwards embryo quality was analyzed. Of the 131 women enrolled, 68 were allocated to r-HFSH alone and 63 to r-HFSH + r-HLH. No significant differences were observed in markers of either oocyte or embryo quality or quantity. However, higher rates of implantation and live birth per started cycle were observed with r-HLH supplementation than with r-HFSH alone. Although additional large studies are required to further investigate these findings, r-HLH supplementation for women aged 35-39 years undergoing ICSI is recommended as it may have a beneficial action on implantation.
2162	Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice.	21576823	Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are 2 similar diseases characterized by a cytokine storm, overwhelming inflammation, multiorgan dysfunction, and death. Animal models of HLH suggest that disease is driven by IFN-γ produced by CD8⁺ lymphocytes stimulated by persistent antigen exposure. In these models and patients with "primary" HLH, the antigen persists due to genetic defects, resulting in ineffective cytotoxic responses by CD8⁺ T cells and poor pathogen clearance. However, infectious triggers are often not identified in patients with MAS, and some patients with HLH or MAS lack defects in cytotoxic T cell killing. Herein, we show that repeated stimulation of TLR9 produced an HLH/MAS-like syndrome on a normal genetic background, without exogenous antigen. Like previous HLH models, TLR9-induced MAS was IFN-γ dependent; however, unlike other models, disease did not require lymphocytes. We further showed that IL-10 played a protective role in this model and that blocking IL-10 signaling led to the development of hemophagocytosis. IL-10 may therefore be an important target for the development of effective therapeutics for MAS. Our data provide insight into MAS-like syndromes in patients with inflammatory diseases in which there is chronic innate immune activation but no genetic defects in cytotoxic cell function.
2163	Haemophagocytic lymphohistiocytosis: a case series from Mumbai.	21575318	A retrospective review of ten patients (8 girls, 2 boys) admitted over a 9-month period with haemophagocytic lymphohistiocytosis (HLH) is presented. Presenting features included fever and hepatosplenomegaly (10), bleeding manifestations (7), lymphadenopathy (4), skin rash (4), shock (4), jaundice (3), CNS disorder (3), renal failure (2) and arthritis (2). Three infants had familial HLH (FHL) while the other seven patients had acquired (secondary) HLH. Two patients with FHL had very low perforin levels (0 and 0.05%). There was secondary HLH owing to systemic onset juvenile idiopathic arthritis in two patients, and one each had anaplastic large cell lymphoma, measles with pneumonia, disseminated tuberculosis, dengue hemorrhagic fever and lymphoproliferative disorder. Cytopenia affecting two or three lineages in peripheral blood was present in all while haemophagocytosis in bone marrow was documented in nine patients .Other important laboratory parameters were raised ferritin (9), raised LDH (9), hypertriglyceridaemia (7) and hypofibrinogenaemia (5). The patients were treated according to the HLH2004 protocol. Diagnosis of HLH should be considered early in patients presenting with unremitting fever, hepatosplenomegaly and cytopenias as without appropriate treatment HLH is usually fatal.
2164	Detection of T-cell receptor gene rearrangement in children with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis using the BIOMED-2 multiplex polymerase chain reaction combined with GeneScan analysis.	21570959	Hemophagocytic lymphohistiocytosis (HLH) is a serious immune disorder. Epstein-Barr virus (EBV) is the most common trigger of secondary HLH. T-cell receptor (TCR) gene rearrangement is detectable in half of patients with EBV-associated HLH using Southern blotting and conventional polymerase chain reaction (PCR) analyses. However, its clinical significance is unclear. The impact of TCR gene clonality on the outcome of patients with EBV-HLH should be evaluated using more sensitive methods. In this study, we used BIOMED-2 multiplex PCR and GeneScan analysis to evaluate TCR gene rearrangement in childhood EBV-HLH.Six children with EBV-HLH were enrolled in this study. EBV load in blood was quantified by real-time PCR. TCR gene rearrangement was analyzed by BIOMED-2 multiplex PCR.All 6 patients showed monoclonal peaks in TCRβ and/or TCRγ at diagnosis. Serial monitoring of one patient disclosed a change in the rearrangement pattern of the TCR genes in response to immunochemotherapy.These findings suggest that BIOMED-2 multiplex PCR, a highly sensitive method for detecting T-cell clonality, is useful for predicting the therapeutic response in childhood EBV-HLH.
2165	Hemophagocytic Lymphohistiocytosis Induced by Severe Pandemic Influenza A (H1N1) 2009 Virus Infection: A Case Report.	21559246	After early outbreaks in North America in April 2009, the pandemic influenza A (H1N1) virus spread rapidly around the world, and even some patients developed certain severe complications. We reported one case of hemophagocytic lymphohistiocytosis (HLH) induced by severe pandemic influenza A (H1N1) virus infection. A 17-year-old girl had acute onset of fever, dry cough, rhinorrhea, and sore throat Her family members and close friends also had the similar symptoms. Anti-infection treatment with penicillin was given after 8 days of the onset of symptoms in the local hospital, and her chest radiograph showed consolidation of the left lung. Then, she was sent to the People's Hospital of Guizhou Province in China and endotracheal intubation were underwent on the ninth day for acute hypoxic respiratory failure. She was diagnosed with HLH induced by severe pandemic influenza A (H1N1) 2009 virus. Oseltamivir, steroids, immunoglobulin, and plasmapheresis were given immediately after admission. After being treated in the People's Hospital of Guizhou Province for 16 days, she was discharged. This experience shows that HLH may be a life-threatening complication for severe pandemic influenza A (H1N1) 2009 virus infection and responds well to therapy.
2166	Disruption of MyD88 signaling suppresses hemophagocytic lymphohistiocytosis in mice.	21551232	Hemophagocytic lymphohistiocytosis (HLH) is a rare inflammatory disorder with a poor prognosis for affected individuals. To find a means of suppressing the clinical phenotype, we investigated the cellular and molecular mechanisms leading to HLH in Unc13d(jinx/jinx) mice, in which cytolytic function of NK and CD8(+) T cells is impaired. Unc13d(jinx/jinx) mutants infected with lymphochoriomeningitis virus (LCMV) present typical clinical features of HLH, including splenomegaly, elevated serum IFNγ, and anemia. Proteins mediating cell-cell contact, cytokine signaling or Toll-like receptor (TLR) signaling were analyzed. We show that neither the integrin CD18, which is involved in adhesion between antigen-presenting cells and effector T cells, nor tumor necrosis factor (TNF) made nonredundant contributions to the disease phenotype. Disruption of IFNγ signaling reduced immune cell activation in Unc13d(jinx/jinx) mice, but also resulted in uncontrolled viral proliferation and exaggerated release of inflammatory cytokines. Abrogating the function of myeloid differentiation primary response gene 88 (MyD88) in Unc13d(jinx/jinx) mice suppressed immune cell activation and controlled cytokine production in an IL-1 receptor 1 (IL-1R1)-independent way. Our findings implicate MyD88 as the key initiator of myeloid and lymphoid proliferation in HLH, and suggest that blockade of this signaling molecule may reduce immunopathology in patients.
2167	Current opinion on use of luteinizing hormone supplementation in assisted reproduction therapy: an Asian perspective.	21550853	LH and FSH have complementary functions in ensuring optimal oocyte maturation and ovulation. In women undergoing assisted reproduction technology protocols with gonadotrophin-releasing hormone analogues, LH and FSH concentrations are reduced. While FSH use in assisted reproduction technology is well established, there is no published consensus on the need for exogenous LH in Asian patients. Having reviewed the concept of the LH therapeutic window and differences between recombinant human LH (r-HLH) and human menopausal gonadotrophin, a consensus was reached on which patient subgroups may benefit from LH supplementation. Adjuvant r-HLH gives clinicians precise control over the dose of LH bioactivity administered to target the therapeutic window. The use of r-HLH is recommended in women with poor response in a previous cycle or suboptimal follicular progression in a current cycle by day 6-8 of stimulation. r-HLH should also be considered in women at risk of suboptimal response, specifically age > 35 years. Other risk markers that suggest the need for LH supplementation, which include baseline/day-6 serum LH and anti-Müllerian hormone concentrations, antral follicle count and LH polymorphisms require further research and verification. For measurement of LH response adequacy, the monitoring of follicular progression, oestradiol concentrations and endometrial thickness is recommended.
2168	An atypical HLH protein OsLF in rice regulates flowering time and interacts with OsPIL13 and OsPIL15.	21549224	In plants, flowering as a crucial developmental event is highly regulated by both genetic programs and environmental signals. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flowering induction. In this study, we isolated the OsLF gene by its association with the T-DNA insertion in the rice late flowering mutant named A654. The OsLF gene encodes an atypical HLH protein composed of 419 amino acids (aa). Overexpression of the OsLF gene in wild type rice recapitulated the late flowering phenotype of A654, indicating that the OsLF gene negatively regulates flowering. Flowering genes downstream of OsPRR1 such as OsGI and Hd1 were down regulated in the A654 mutant. Yeast two hybrid and colocalization assays revealed that OsLF interacts strongly with OsPIL13 and OsPIL15 that are potentially involved in light signaling. In addition, OsPIL13 and OsPIL15 colocalize with OsPRR1, an ortholog of the Arabidopsis APRR1 gene that controls photoperiodic flowering response through clock function. Together, these results suggest that overexpression of OsLF might repress expression of OsGI and Hd1 by competing with OsPRR1 in interacting with OsPIL13 and OsPIL15 and thus induce late flowering.
2169	Successful treatment of recurrent malignancy-associated hemophagocytic lymphohistiocytosis with a modified HLH-94 immunochemotherapy and allogeneic stem cell transplantation.	21533602	Acquired hemophagocytic lymphohistiocytosis (HLH) triggered by a known or still to be recognized malignancy is a life-threatening hyperinflammatory syndrome due to massive cytokine release from activated lymphocytes and macrophages. Malignancy-associated HLH (M-HLH) often impedes adequate treatment of malignancy and has the worst outcome compared with any other form of HLH. The incidence of M-HLH is unknown, and there are no published treatment recommendations addressed to this HLH form. Here, we report the case of a young woman with recurrent ALK1-positive anaplastic large T-cell lymphoma and M-HLH successfully treated with a modified HLH-94 protocol, allogeneic stem cell transplantation (alloSCT) and donor lymphocyte infusion (DLI). More than 3 years after DLI, the patient is alive, in complete remission from her malignancy and HLH-free, although suffering from extensive chronic graft-versus-host disease. AlloSCT and, if needed, DLI performed to consolidate remission of malignancy and HLH may have a curative impact on both entities. We propose that when discussing possible treatment options for patients with M-HLH, alloSCT should be considered in eligible individuals.
2170	Causes, clinical symptoms, and outcomes of infectious diseases associated with hemophagocytic lymphohistiocytosis in Taiwanese adults.	21524613	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon but a potentially life-threatening condition. Few systematic reviews have been published on the clinical manifestations, causes, and indicators for prognosis of HLH caused by infections.We retrospectively reviewed the medical records of patients diagnosed with HLH documented by bone marrow study at a teaching hospital between 2000 and 2007. HLH was defined according to the HLH-2004 diagnostic guidelines, which include fever; splenomegaly; cytopenia; hypertriglyceridemia; hypofibrinogenemia; and hemophagocytosis evident on pathological examination of bone marrow, spleen, or lymph node tissue; low or absent natural killer cell activity; hyperferritinemia; and high serum levels of soluble CD25. The demographic characteristics, clinical presentations, laboratory results, and final outcomes were recorded. The cause of HLH was diagnosed by microbiological, pathological, serological, and molecular biological methods.Among the studied patients, 66 had HLH because of noninfectious causes and 30 because of infections. Compared with patients with HLH related to noninfectious causes, those with HLH related to infections had lower mortality (70% vs. 47%, p=0.03). The most common causative pathogens causing HLH were virus (41%), mycobacteria (23%), bacteria (23%), and fungi (13%), in that order of frequency. Clinical presentations of HLH were variable and included fever (90%), tachypnea (83%), tachycardia (80%), hepatosplenomegaly (40%), lymphadenopathy (27%), and altered consciousness (23%). Laboratory findings revealed thrombocytopenia in 93%, hyperferritinemia in 90%, elevated serum lactate dehydrogenase levels in 80%, anemia in 67%, and leukopenia in 60% of the patients. Fourteen patients (47%) died. In multivariate analysis, age more than 50 years (p=0.05; odds ratio [OR], 3.46; 95% confidence interval [CI], 1.00-15.73), fever not subsiding within 3 days of diagnosing HLH (p=0.003; OR, 2.38; 95% CI, 1.21-11.25), and occurrence of disseminated intravascular coagulation as a complication (p=0.009; OR, 3.22; 95% CI, 1.68-10.01) were found to be statistically significant indicators of mortality in patients with HLH.The infectious diseases associated with HLH were diverse and resulted in a high mortality rate. Cases in which the patients were aged more than 50 years, developed DIC, and had persistent fever even after 3 days of being diagnosed with HLH showed poor prognosis.
2171	Induction of Id-1 by FGF-2 involves activity of EGR-1 and sensitizes neuroblastoma cells to cell death.	21506108	Inhibitor of differentiation-1 (Id-1) is a member of helix-loop-helix (HLH) family of proteins that regulate gene transcription through their inhibitory binding to basic-HLH transcription factors. Similarly to other members of this family, Id-1 is involved in the repression of cell differentiation and activation of cell growth. The dual function of Id-1, inhibition of differentiation, and stimulation of cell proliferation, might be interdependent, as cell differentiation is generally coupled with the exit from the cell cycle. Fibroblast growth factor-2 (FGF-2) has been reported to play multiple roles in different biological processes during development of the central nervous system (CNS). In addition, FGF-2 has been described to induce "neuronal-like" differentiation and trigger apoptosis in neuroblastoma SK-N-MC cells. Although regulation of Id-1 protein by several mitogenic factors is well-established, little is known about the role of FGF-2 in the regulation of Id-1. Using human neuroblastoma cell line, SK-N-MC, we found that treatment of these cells with FGF-2 resulted in early induction of both Id-1 mRNA and protein. The induction occurs within 1 h from FGF-2 treatment and is mediated by ERK1/2 pathway, which in turn stimulates expression of the early growth response-1 (Egr-1) transcription factor. We also demonstrate direct interaction of Egr-1 with Id-1 promoter in vitro and in cell culture. Finally, inhibition of Id-1 expression results in G(2) /M accumulation of FGF-2-treated cells and delayed cell death.
2172	A novel single point mutation of the LYST gene in two siblings with different phenotypic features of Chediak Higashi syndrome.	21488161	Chediak Higashi syndrome (CHS) is an autosomal-recessive disorder characterized by oculocutaneous albinism, recurrent infections and a progressive primary neurological disease. Here, we describe two siblings with CHS due to a novel homozygous R1836X mutation in the LYST gene associated with loss of NK cell degranulation and cytotoxicity. While one sibling was born with fair skin and hair and died of hemophagocytic lymphohistiocytosis (HLH) at 5 months of age, the other sibling had dark black hair and skin and developed HLH at the age of 4 years.
2173	The Drosophila STUbL protein Degringolade limits HES functions during embryogenesis.	21486924	Degringolade (Dgrn) encodes a Drosophila SUMO-targeted ubiquitin ligase (STUbL) protein similar to that of mammalian RNF4. Dgrn facilitates the ubiquitylation of the HES protein Hairy, which disrupts the repressive activity of Hairy by inhibiting the recruitment of its cofactor Groucho. We show that Hey and all HES family members, except Her, interact with Dgrn and are substrates for its E3 ubiquitin ligase activity. Dgrn displays dynamic subcellular localization, accumulates in the nucleus at times when HES family members are active and limits Hey and HES family activity during sex determination, segmentation and neurogenesis. We show that Dgrn interacts with the Notch signaling pathway by it antagonizing the activity of E(spl)-C proteins. dgrn null mutants are female sterile, producing embryos that arrest development after two or three nuclear divisions. These mutant embryos exhibit fragmented or decondensed nuclei and accumulate higher levels of SUMO-conjugated proteins, suggesting a role for Dgrn in genome stability.
2174	A novel animal model of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in humanized mice.	21467545	EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a rare yet devastating disorder caused by EBV infection in humans. However, the mechanism of this disease has yet to be elucidated because of a lack of appropriate animal models. Here, we used a human CD34(+) cell-transplanted humanized mouse model and reproduced pathologic conditions resembling EBV-HLH in humans. By 10 weeks postinfection, two-thirds of the infected mice died after exhibiting high and persistent viremia, leukocytosis, IFN-γ cytokinenemia, normocytic anemia, and thrombocytopenia. EBV-infected mice also showed systemic organ infiltration by activated CD8(+) T cells and prominent hemophagocytosis in BM, spleen, and liver. Notably, the level of EBV load in plasma correlated directly with both the activation frequency of CD8(+) T cells and the level of IFN-γ in plasma. Moreover, high levels of EBV-encoded small RNA1 were detected in plasma of infected mice, reflecting what has been observed in patients. These findings suggest that our EBV infection model mirrors virologic, hematologic, and immunopathologic aspects of EBV-HLH. Furthermore, in contrast to CD8(+) T cells, we found a significant decrease of natural killer cells, myeloid dendritic cells, and plasmacytoid dendritic cells in the spleens of infected mice, suggesting that the collapse of balanced immunity associates with the progression of EBV-HLH pathogenesis.
2175	Evaluation of Th1/Th2 cytokines as a rapid diagnostic tool for severe infection in paediatric haematology/oncology patients by the use of cytometric bead array technology.	21463393	Haematology/oncology children are usually at risk for various infections after intensive chemotherapy. We evaluated the quantification of Th1/Th2 cytokines with a flow cytometric bead array (CBA) in 795 hospitalized haematology/oncology children (309 febrile and 486 afebrile patients) to seek for a diagnostic method for determination of the type and the severity of infection. Three hundred and nine febrile patients developed a total of 505 febrile episodes. Microbiological examination demonstrated a positive blood culture (microbiologically documented infection (MDI)) in 145/505 febrile episodes. The controls included 550 healthy children, 43 haemophagocytic lymphohistiocytosis (HLH) patients, 35 cytomegalovirus infection patients and 19 Epstein-Barr virus infection patients. Interleukin (IL)-4, IL-6, IL-10, tumour necrosis factor (TNF)-α and interferon (IFN)-γ levels in febrile episodes were significantly higher than those in healthy children, and the cytokine profile was different from that of the HLH controls or the viral infection controls. IL-6 levels were much higher in MDI patients (usually >1000.0 pg/mL, 60/145) than in HLH patients (2/43); however, IFN-γ levels were only slightly increased in MDI patients, rarely being more than 100.0 pg/mL (8/145 vs. 39/43 in HLH patients). The median levels of IL-4, IL-6, IL-10, TNF-α and IFN-γ in febrile patients before antibiotic therapy were 3.9, 660.1, 122.7, 6.9 and 11.4 pg/mL, respectively, and returned to 3.3, 22.8, 9.6, 4.1 and 6.4 pg/mL, respectively, after infection was controlled. IL-6 and IL-10 levels were positively associated with septic shock and mortality rates. In conclusion, our results have demonstrated the usefulness of IL-6/IL-10/TNF-α/IFN-γ determination with CBA technology for the early rapid diagnosis, severity evaluation and assessment of therapy effect in febrile haematology/oncology children.
2176	Advances in hemophagocytic lymphohistiocytosis: pathogenesis, early diagnosis/differential diagnosis, and treatment.	21442147	Hemophagocytic lymphohistiocytosis (HLH) is a histiocytic disorder characterized by a highly stimulated, but ineffective, immune response to antigens, which results in life-threatening cytokine storm and inflammatory reaction. Considerable progress has been made during the past 2 decades. Detection of molecular genetic abnormalities in genes involved in immune response pathways, such as PRF1, STX11, UNC13D, STXBP2, RAB27A, LYST, AP3B1, SH2D1A, and BIRC4, is confirmatory for the diagnosis. Clinical diagnosis is largely made according to HLH-2004 criteria. However, a new finding of the Th1/Th2 cytokine pattern (significant increase of IFN-γ and IL-10 with slightly increased or normal level of IL-6) is a useful biomarker for the early diagnosis, differential diagnosis, and the monitoring of the disease. Intensive immunosuppressive therapy is generally accepted as treatment for the relief of clinical symptoms/signs, while allogeneic hematopoietic stem cell transplantation is currently the only potentially curative therapy option for severe familial forms of HLH.
2177	Diverse roles of inhibitor of differentiation 2 in adaptive immunity.	21437223	The helix-loop-helix (HLH) transcription factor inhibitor of DNA binding 2 (Id2) has been implicated as a regulator of hematopoiesis and embryonic development. While its role in early lymphopoiesis has been well characterized, new roles in adaptive immune responses have recently been uncovered opening exciting new directions for investigation. In the innate immune system, Id2 is required for the development of mature natural killer (NK) cells, lymphoid tissue-inducer (LTi) cells, and the recently identified interleukin (IL)-22 secreting nonconventional innate lymphocytes found in the gut. In addition, Id2 has been implicated in the development of specific dendritic cell (DC) subsets, decisions determining the formation of αβ and γδ T-cell development, NK T-cell behaviour, and in the maintenance of effector and memory CD8(+) T cells in peripheral tissues. Here, we review the current understanding of the role of Id2 in lymphopoiesis and in the development of the adaptive immune response required for maintaining immune homeostasis and immune protection.
2178	DNA methylation of germ-cell-specific basic helix-loop-helix (HLH) transcription factors, Sohlh2 and Figlα during gametogenesis.	21427160	Regulation of germ-cell-specific transcription is essential for the differentiation and other physiological processes of germ cells. Germ-cell-specific transcription factors, Sohlh2 and Figla, play key roles in gametogenesis. To elucidate whether an epigenetic mechanism is involved in the controlled expression of Sohlh2 and Figlα, we examined the dynamics of DNA methylation at two gene loci. Results showed changes in methylation patterns in the promoter regions of both genes during the period of germ cell differentiation, while the methylation patterns in first exons and first introns (near the transcription initiation sites) remained constant. The methylation reprogramming at the cytosine-phosphate-guanine (CpG) locus in the Figlα promoters (P1: -812 to -568 bp and P2: -692 to -438 bp) and in the Sohlh2 promoter (sohlh2-P: -202 to 173 bp) was found to correlate with the expression of Figlα and Sohlh2 transcripts, respectively. Data therefore suggested that a dynamic DNA CpG methylation in the Sohlh2 and Figlα promoters, but not in the intron and exon sequences, is linked to the regulation of gene expressions, even though CpG islands are also present in their introns or exons.
2179	HMMerThread: detecting remote, functional conserved domains in entire genomes by combining relaxed sequence-database searches with fold recognition.	21423752	Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak sequence similarity. Our predictions open up new avenues for biological and medical studies. Genome-wide HMMerThread domains are available at http://vm1-hmmerthread.age.mpg.de.
2180	The Hammersmith hospital hematopathology case of the month: hemophagocytic lymphohistiocytosis secondary to Epstein-Barr virus associated T-cell lymphoma.	21417828	We discuss the clinical presentation, investigations carried out, and the management issues in an elderly patient with hemophagocytic lymphohistiocytosis (HLH). The HLH was associated with an underlying Epstein-Barr virus positive peripheral T-cell lymphoma, not otherwise specified, and the lymphomatous cells harbored translocation t(5;9)(q11.2:p24). The 9p24 region contains the JAK2 gene. The disease had a fulminant course.
2181	A new allele of Lyl1 confirms its important role in hematopoietic stem cell function.	21387538	Lyl1 codes for a bHLH protein that is an important regulator of hematopoietic stem cell function. An existing mutant allele of Lyl1 features a lacZ gene inserted in-frame into the fourth exon, leaving behind the N-terminus and the DNA-binding basic region, resulting in a translated chimeric protein. Here, we have generated a null allele, which allowed us to examine residual function of the N-terminus in the absence of a bHLH region. The new Lyl1-/- mouse model exhibited a reduced ability to generate lymphoid lineages and a somewhat more severe hematopoietic repopulation defect when transplanting purified hematopoietic stem cells. Our data show that in the absence of the HLH but presence of the N-terminus, residual function of the Lyl1 is detectable but relatively minor. The new model may be of use for studies of Lyl1 in which a null allele is required, or for which presence of the LacZ may complicate the combined use of additional mouse models bearing the lacZ marker.
2182	Clinical characteristics of hemophagocytic lymphohistiocytosis related to Kawasaki disease.	2138187121381868	It is difficult to predict the prognosis or clinical course of secondary hemophagocytic lymphohistiocytosis (HLH) due to the various underlying causes. The authors analyzed the clinical and laboratory findings and outcomes in patients with HLH who had initially been diagnosed with Kawasaki disease (KD), and evaluated the clinical significance of each factor. Among the 21 patients with HLH, 5 had initially been diagnosed with KD and 16 had other etiologies. A comparative analysis was performed for fever duration, presence of cytopenia, serum ferritin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride, fibrinogen, hyponatremia, reactivation, and survival rate in those HLH patients associated with KD (group I) and other causes (group II). In patients in group I, a higher level of reactivation (20%), a lower survival rate (P = .001), higher AST (P = .031) and ferritin (P = .005), and frequent hyponatremia (P = .000) were found compared to patients in group II. Interestingly, patients in group I was older than the average of age of most KD patients. A high index of suspicion on the progression from KD to HLH would be mandatory when the KD patients show elevated AST and ferritin and the presence of hyponatremia, and especially so if the patient is of older age.
2183	Successful treatment with combined chemotherapy of two adult cases of hemophagocytic lymphohistiocytosis in recipients of umbilical cord blood cell transplantation.	21380927	Hemophagocytic lymphohistiocytosis (HLH), which occurs during the early period following allogeneic hematopoietic stem cell transplantation (HSCT), is often difficult to diagnose. It is characterized by severe clinical manifestations and high mortality. Despite current therapeutic approaches, outcomes remain poor. Here, we summarize the cases of two patients who experienced engraftment failure and subsequently developed hematopoietic stem cell transplantation-hemophagocytic lymphohistiocytosis (HSCT-HLH). Both patients had high-risk hematological malignancies for which they underwent umbilical cord blood transplantation (UCBT) at our institution. Based on the presence of massive hemophagocytosis in their bone marrow specimens and the results of chimerism analysis, diagnosis of HSCT-HLH was made in both cases. A single infusion of low-dose etoposide was administered immediately to each patient. Four days after the injection, therapeutic efficacy was evaluated. As both cases showed an insufficient response to etoposide, we added vincristine and a medium dose of prednisolone. Clinical symptoms rapidly improved and stable engraftments were observed within a week. The first and second patients were alive 1008 and 232 days after UCBT, respectively. The combined use of low-dose etoposide and vincristine plus prednisolone appears to be a promising treatment option for HSCT-HLH, without serious adverse side effects.
2184	Hemophagocytic lymphohistiocytosis with MUNC13-4 gene mutation or reduced natural killer cell function prior to onset of childhood leukemia.	21370424	Hemophagocytic lymphohistiocytosis (HLH) is a rare histiocytic reactive process due to mutations in the perforin, MUNC13-4 or syntaxin 11 genes, or secondary to malignancy, infection or autoimmune disorder. HLH as a preceding diagnosis to leukemia is rare. We report two cases with progression to acute leukemia, one heterozygous for MUNC13-4 and the other with reduced natural killer (NK) cell function and perforin expression. These defects may predispose to a secondary HLH-like presentation of pre-clinical leukemia or confer increased susceptibility to malignancy. HLH patients with genetic mutations or NK cell function abnormalities need monitoring for future malignancy even if the HLH resolves.
2185	Chlamydia pneumoniae infection-related hemophagocytic lymphohistiocytosis and acute encephalitis and poliomyelitis-like flaccid paralysis.	21370423	A 3-year-old male presented with Chlamydia pneumoniae infection-related hemophagocytic lymphohistiocytosis (HLH). The patient developed an episode of HLH with severe skin eruption following C. pneumoniae pneumonia. Symptoms responded to steroid/cyclosporine A therapy, but the patient slowly lost consciousness and developed systemic flaccid paralysis. He was diagnosed with encephalitis/myelitis by brain and spinal MRI. Neurological symptoms and signs gradually resolved. We thought that the immune response to C. pneumoniae infection triggered the development of HLH, associated with unusual neurological complications. This report describes a novel case of C. pneumoniae-associated HLH and with poliomyelitis like flaccid paralysis.
2186	Acute promyelocytic leukemia following chemotherapy for EBV-associated hemophagocytic lymphohistiocytosis.	21370422	We report a case of chemotherapy-related acute promyelocytic leukemia (APL) following therapy with VP-16/etoposide for EBV-associated hemophagocytic lymphohistiocytosis (HLH). A 17-month-old male presented with fever and lymphadenopathy. Bone marrow and liver biopsies showed hemophagocytosis. He responded well to chemotherapy including dexamethasone, VP-16/etoposide, and cyclosporine. One and a half year later, he developed fever and pancytopenia. Clinical work-up revealed APL with t(15;17)(q22;q12);PML-RARα translocation. He underwent chemotherapy for APL and is in remission 8 years after diagnosis. Alternative non-leukemogenic agents to effectively treat HLH would be desirable.
2187	[Anaplastic large cell lymphoma associated with hemophagocytic lymphohistiocytosis: a case report and review of the literature].	21344777	Malignancy-associated hemophagocytic syndrome has been reported mostly in adults, in children it is very rare. It can develop before or during treatment, in association with an infection or without a known triggering factor. Here we present a case report of a 14 years old boy diagnosed with HLH before recognition of anaplastic large cell lymphoma (ALCL). Because of progression of symptoms characteristic for HLH, therapy according to the HLH 2004 protocol was introduced. Finally, histopathological examination of a subsequently excised enlarged lymph node demonstrated lesions typical for ALCL. First-line therapy, according to protocol for this type of lymphoma, was introduced. Because of resistance to standard therapy, change of protocol was necessary. Finally the patients achieved complete remission. He was qualified for allogeneic stem cell transplantation. The patient died in peritransplantation period because of infection complications. Occurrence of HLH can cause diagnostic difficulties, because it often masks the underlying disease, especially when it is associated with infection. Therefore, each case of HLH requires accurate evaluation for malignancy. Poor outcome in patients with malignancy-associated HLH is emphasized in the literature. There is necessity of establishment of more effective therapy.
2188	[Hemophagocytic syndrome in children with different underlying conditions].	21344776	Hemophagocytic syndrome (HS) is a life-threatening condition of hyperinflammation. Main symptoms are: prolonged fever, cytopenia, hepatosplenomegaly, hemophagocytosis, hyperferritinemia, hypertriglyceridemia and hypofibrinogenemia. Primary genetic form and secondary HS associated with infections, malignancies or autoimmune disorders can be distinguished. Untreated HS in most cases leads to death. We analyzed retrospectively 7 cases of HS in children (3 girls, 4 boys; aged 10 days -14 years) treated in 3 different pediatric centers from 2004 to 2009. In 3 cases HS was associated with infections (EBV, CMV, Bacillus Calmette Guerin - BCG), in 1 child with non-Hodgkin anaplastic large cell lymphoma (ALCL), in 1 patients probably with side effect of antiepileptic drug. In 2 cases cause of HS remained unknown. Fever, hepatomegaly, pan- or bicytopenia and hyperferritinemia were present in all children. In addition, splenomegaly was noted in 6 cases, hemophagocytosis in 6 children, impaired function or decreased number of NK cells in 4 cases, hypofibrino-genemia in 5 and hypotriglyceridemia in 4 patients. Among other symptoms and signs we observed: lymphadenopathy, hepatic failure, oedema, rash, neurological symptoms, increased level of LDH and inflammatory markers. In one child acute pancreatitis occurred. Among others, antibiotics, antiviral and immunosuppressive drugs were used in therapy. HLH-2004 protocol was applied in 4 cases. Patient with ALCL was treated with chemotherapy and allogeneic stem cell transplantation. Four patients are alive, 2 died because of HS, child with ALCL died because of generalized infection in peritrans-plantation period. In case of prolonged fever, splenomegaly and cytopenia diagnosis of HS should be considered. Following tests are recommended: complete blood count, ferritin, triglycerides, fibrinogen, bone marrow aspiration and NK cell assessment. Patients should be also screened for infections and malignancies. Early diagnosis of HS and underlying condition is crucial to start lifesaving therapy.
2189	[Hemophagocytic lymphohistiocytosis: diagnostic problems in pediatrics].	21344773	Hemophagocytic lymphohistiocytosis (HLH), also known as hemophagocytic syndrome (HS), is a life-threatening hyperinflammatory condition caused by uncontrolled proliferation of activated lymphocytes and histiocytes producing excessively proinflammatory cytokines. HLH can occur in all age groups. The most common syndromes are: prolonged fever, hepatosplenomegaly, unspecific neurological symptoms, pancytopenia, hemophagocytosis. Characteristic biochemical markers include high triglycerides, ferritin and decreased fibrinogen. There are two forms of HLH, primary (genetic) and acquired (secondary to infections, malignant and autoimmune diseases). Occurrence of both forms of HLH, acquired and genetic, is induced by infections, usually viral or by other triggering agents. Despite established diagnostic criteria many cases probably remain unrecognized. The main diagnostic difficulty is low specificity of symptoms, and in secondary form contemporary occurrence of symptoms of underlying disease. The aim of HLH treatment is to suppress hyperinflammation, what can be achieved by use of immunosuppressive/immunomodulating agents or cytostatics. Patients with genetic form of HLH require hematopoietic stem cell transplantation. Awareness of the symptoms and diagnostic criteria of HLH is important for early diagnosis and immediate application of life-saving therapy.
2190	Malignancy-associated hemophagocytic lymphohistiocytosis in adults: a retrospective population-based analysis from a single center.	21299462	A retrospective, population-based study was conducted to evaluate the incidence, clinical features, and outcome of malignancy-associated hemophagocytic lymphohistiocytosis (M-HLH) in adults. Between January 1996 and December 2009, eight out of 887 (0.9%) patients diagnosed with hematological malignancies developed aggressive M-HLH in an area inhabited by approximately 160,000 people. Thus the estimated annual incidence of M-HLH in adulthood was 0.36/100,000 individuals/year. The clinical course of M-HLH was aggressive in all patients. Six patients were treated with a modified HLH-94 protocol; three achieved remission (durable in one case) while the others did not respond and died within an average of 2.4 months (range 1.5-3.5) after M-HLH diagnosis. Infection complicating the course of M-HLH occurred in four (50%) patients, all of whom developed fulminant M-HLH and died. Although the small study population limits the results, the long observation period strengthens its value.
2191	Ehrlichia-induced hemophagocytic lymphohistiocytosis in two children.	21298756	Two children presented with a history of fever and rash. Lab values revealed pancytopenia, elevated ferritin, coagulopathy, and elevated triglycerides. Both children quickly developed respiratory distress and hypotension requiring admission to the ICU. Bone marrow biopsies revealed hemophagocytosis. Studies for Ehrlichia returned positive. The patients were started on doxycycline and treated for hemophagocytic lymphohistiocytosis (HLH). Each made a full recovery. In both patients, testing for MUNC and perforin genes were found to have no mutation. These two cases demonstrate the importance of considering Ehrlichiosis as a possible trigger of HLH.
2192	Hemophagocytic lymphohistiocytosis associated with precursor B acute lymphoblastic leukemia.	21298755	We report a case of a child with precursor-B acute lymphoblastic leukemia (ALL) who experienced refractory thrombocytopenia and massive splenomegaly during standard induction chemotherapy. He was diagnosed with hemophatocytic lymphohistiocytosis (HLH) during induction. Clinical and laboratory evaluation showed no evidence of infectious cause to HLH. Pancytopenia and HLH persisted after consolidation therapy even with remission from leukemia. After failure to control HLH with ALL-directed therapy and HLH-directed therapy, the patient underwent unrelated donor hematopoietic stem cell transplantation 8 months after diagnosis. He is 34 months post-transplant and in remission from leukemia and HLH.
2193	Unusual functional manifestations of a novel STX11 frameshift mutation in two infants with familial hemophagocytic lymphohistiocytosis type 4 (FHL4).	21298754	Familial hemophagocytic lymphohistiocytosis (FHL) is typically an autosomal recessive, early-onset, life-threatening immune disorder. Loss-of-function mutations in STX11 have been found to impair NK cell degranulation and cytotoxicity. Here, we describe two unrelated infants of Punjabi descent presenting with FHL and carrying a novel, homozygous STX11 frameshift mutation [c.867dupG]. Western blot analysis indicated absence of syntaxin-11. Unexpectedly, degranulation by NK cells from one of the patients was not impaired, although patient NK cells showed mildly and significantly decreased cytotoxicity, respectively. Importantly, these observations imply that STX11 should be sequenced in HLH patients even when impaired NK cell degranulation is not found.
2194	Primary Epstein-Barr virus infection associated with Kikuchi's disease and hemophagocytic lymphohistiocytosis: a case report and review of the literature.	21291855	An association between hemophagocytic lymphohistiocytosis (HLH) and Kikuchi's disease is rarely seen in children. Here, we present the case of a male adolescent (age 16 years and 3 months) who suffered from spiking fever for more than 1 week, and multiple nodules over the neck and bilateral axilla for 2 months. A skin rash also developed over all four limbs, abdomen and face. Laboratory data and skin biopsy gave results compatible with a diagnosis of Kikuchi's disease. Hemophagocytosis in the bone marrow, hyper-triglyceridemia, elevated ferritin, and splenomegaly were also found, fulfilling the criteria for HLH. A recent primary Epstein-Barr virus infection was also diagnosed by serology. The patient ran a relatively benign course. Intravenous immunoglobulins, steroids or etoposide-containing regimens were not used, and his recovery was uneventful. A review of the literature showed that up to February 2009, 11 additional cases of Kikuchi's disease presenting simultaneously with, or mimicking, HLH had been reported. Complete resolution within several weeks, with no recurrence, was seen in all but one patient (a pregnant woman).
2195	Hemophagocytic lymphohistiocytosis associated with 2009 pandemic influenza A (H1N1) virus infection.	21285906	Hemophagocytic lymphohistiocytosis (HLH) has not been described earlier in the context of 2009 pandemic influenza A (H1N1) virus infection, although certain populations are thought to be at risk for complicated pandemic influenza A disease. Here, we report the second case of HLH after infection with the influenza A H1N1 virus treated with peroral oseltamivir successfully.
2196	Role of helix-loop-helix proteins during differentiation of erythroid cells.	21282467	Helix-loop-helix (HLH) proteins play a profound role in the process of development and cellular differentiation. Among the HLH proteins expressed in differentiating erythroid cells are the ubiquitous proteins Myc, USF1, USF2, and TFII-I, as well as the hematopoiesis-specific transcription factor Tal1/SCL. All of these HLH proteins exhibit distinct functions during the differentiation of erythroid cells. For example, Myc stimulates the proliferation of erythroid progenitor cells, while the USF proteins and Tal1 regulate genes that specify the differentiated phenotype. This minireview summarizes the known activities of Myc, USF, TFII-I, and Tal11/SCL and discusses how they may function sequentially, cooperatively, or antagonistically in regulating expression programs during the differentiation of erythroid cells.
2197	Potocki-Lupski syndrome: an inherited dup(17)(p11.2p11.2) with hypoplastic left heart.	2127165621271654	Low copy repeat (LCR) sequences in 17p11.2 predispose this region to genomic deletions and duplications. Duplication of 17p11.2, also known as Potocki-Lupski syndrome (PTLS), is a well-described microduplication syndrome featuring cognitive and language deficits, developmental delay, autistic behavior, structural cardiovascular anomalies, hypotonia, failure to thrive, apnea, and dysmorphism. We present a mother and her two children who share both dysmorphic features and the dup(17)(p11.2p11.2); the first child was born with hypoplastic left heart (HLH). The dup(17)(p11.2p11.2) was identified by GTG-banding analysis of peripheral blood specimens from all three individuals and confirmed by fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). Here we provide a thorough description of the phenotypes of the affected individuals, as well as describe physical features not reported previously for PTLS.
2198	Serum neopterin levels as a diagnostic marker of hemophagocytic lymphohistiocytosis syndrome.	21270283	The objective of this study was to retrospectively evaluate the utility of serum neopterin as a diagnostic marker of hemophagocytic lymphohistiocytosis (HLH). The medical records of patients diagnosed with HLH (familial and secondary) between January 2000 and May 2009 were reviewed retrospectively, and clinical and laboratory information related to HLH criteria, in addition to neopterin levels, was recorded. A group of 50 patients with active juvenile dermatomyositis (JDM) (who routinely have neopterin levels assessed) served as controls for the assessment of the accuracy, sensitivity, and specificity of neopterin as a diagnostic test for HLH. The Pearson correlation was used to measure the association between serum neopterin levels and established HLH-related laboratory data. Serum neopterin levels were measured using a competitive enzyme immunoassay. During the time frame of the study, 3 patients with familial HLH and 18 patients with secondary HLH were identified as having had serum neopterin measured (all HLH patients were grouped together). The mean neopterin levels were 84.9 nmol/liter (standard deviation [SD], 83.4 nmol/liter) for patients with HLH and 21.5 nmol/liter (SD, 10.13 nmol/liter) for patients with JDM. A cutoff value of 38.9 nmol/liter was 70% sensitive and 95% specific for HLH. For HLH patients, neopterin levels correlated significantly with ferritin levels (r = 0.76, P = 0.0007). In comparison to the level in a control group of JDM patients, elevated serum neopterin was a sensitive and specific marker for HLH. Serum neopterin has value as a diagnostic marker of HLH, and prospective studies are under way to further evaluate its role as a marker for early diagnosis and management of patients.
2199	What intensivists need to know about hemophagocytic syndrome: an underrecognized cause of death in adult intensive care units.	2125762725966495	Hemophagocytic syndrome, also known as hemophagocytic lymphohistiocytosis (HLH), is a rare and frequently fatal disorder caused by an uncontrollable and ineffective systemic immune response. Patients initially present with fever, cytopenia, and hepatosplenomegaly, and subsequently develop multiorgan failure (MOF). Hemophagocytosis can be found on biopsy specimen but is not required. Acquired forms of HLH can occur in apparently healthy adults, while children present more often with an inherited form of the disease. Since HLH often presents with sepsis-like symptoms and organ dysfunction, patients are usually treated for presumed sepsis, which inevitably leads to delayed diagnosis and treatment. Intensivists need to have a low threshold for suspecting this disorder when previously healthy individuals present with a fulminant sepsis-like syndrome, which are unresponsive to conventional treatment. We present 3 patients with HLH who were admitted to our adult medical intensive care unit (MICU) over a 2-year period with fatal outcomes and emphasize the diagnostic importance of markedly elevated serum ferritin levels and the need for tissue biopsy in making an accurate diagnosis in a timely manner.
2200	Hemophagocytic lymphohistiocytosis with neurological presentation: MRI findings and a nearly miss diagnosis.	21234777	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease with rarer neurological presentation. When this occurs, diagnosis may be delayed. This report aims to call attention to clinical, laboratory, and radiological features that should prompt the correct diagnosis. A 13-year-old girl presented with progressive increase in intracranial pressure and ataxia. MRI showed a diffuse tumor-like swelling of the cerebellum with tonsillar herniation and patchy white matter post-contrast enhancement. Regression of swelling with steroids ruled out glioma and medulloblastoma, and brain lymphoma was considered. Diagnosis of HLH was reached 2 months after onset when uncontrolled fever and severe elevation of liver enzymes occurred. Two bone marrow biopsies were needed to demonstrate hemophagocytosis. Familial HLH was confirmed by perforin gene mutations. Bone marrow transplantation was performed. The early diagnosis of HLH may be life saving. Awareness of the disease is necessary to investigate its characteristic findings, thus avoiding a delay in diagnosis.
2201	Hemophagocytic lymphohistiocytosis in children with chronic granulomatous disease.	21225928	Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever, cytopenias, splenomegaly, and hemophagocytosis by macrophages activated by high cytokine levels. Chronic granulomatous disease (CGD) is characterized by recurrent infections, hyperinflammation, and excessive cytokine release. This may predispose patients with CGD to developing HLH during an infection. We conducted a retrospective review of patients with CGD, treated at our institution between 1999 and 2008. Three out of 17 patients developed HLH. Patients with CGD may be at increased risk for developing HLH. Remission of HLH was achieved after treatment with antimicrobials, steroids, and intravenous immunoglobulin This approach to treatment appears to be effective.
2202	Large eddy simulation of pollutant gas dispersion with buoyancy ejected from building into an urban street canyon.	21216525	The dispersion of buoyancy driven smoke soot and carbon monoxide (CO) gas, which was ejected out from side building into an urban street canyon with aspect ratio of 1 was investigated by large eddy simulation (LES) under a perpendicular wind flow. Strong buoyancy effect, which has not been revealed before, on such pollution dispersion in the street canyon was studied. The buoyancy release rate was 5 MW. The wind speed concerned ranged from 1 to 7.5m/s. The characteristics of flow pattern, distribution of smoke soot and temperature, CO concentration were revealed by the LES simulation. Dimensionless Froude number (Fr) was firstly introduced here to characterize the pollutant dispersion with buoyancy effect counteracting the wind. It was found that the flow pattern can be well categorized into three regimes. A regular characteristic large vortex was shown for the CO concentration contour when the wind velocity was higher than the critical re-entrainment value. A new formula was theoretically developed to show quantitatively that the critical re-entrainment wind velocities, u(c), for buoyancy source at different floors, were proportional to -1/3 power of the characteristic height. LES simulation results agreed well with theoretical analysis. The critical Froude number was found to be constant of 0.7.
2203	A widespread distribution of genomic CeMyoD binding sites revealed and cross validated by ChIP-Chip and ChIP-Seq techniques.	21209968	Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites.
2204	The shade avoidance syndrome in Arabidopsis: a fundamental role for atypical basic helix-loop-helix proteins as transcriptional cofactors.	21205034	The shade avoidance syndrome (SAS) refers to a set of plant responses aimed at anticipating eventual shading by potential competitors. The SAS is initiated after perception of nearby vegetation as a reduction in the red to far-red ratio (R:FR) of the incoming light. Low R:FR light is perceived by the phytochromes, triggering dramatic changes in gene expression that, in seedlings, eventually result in an increased hypocotyl elongation to overgrow competitors. This response is inhibited by genes such as PHYTOCHROME RAPIDLY REGULATED 1 (PAR1), PAR2 and LONG HYPOCOTYL IN FR 1 (HFR1), which are transcriptionally induced by low R:FR. Although PAR1/PAR2 and HFR1 proteins belong to different groups of basic helix-loop-helix (bHLH) transcriptional regulators, they all lack a typical basic domain required for binding to E-box and G-box motifs in the promoter of target genes. By overexpressing derivatives of PAR1 and HFR1 we show that these proteins are actually transcriptional cofactors that do not need to bind DNA to directly regulate transcription. We conclude that protein-protein interactions involving the HLH domain of PAR1 and HFR1 are a fundamental aspect of the mechanism by which these proteins regulate gene expression, most likely through interaction with true transcription factors that do bind to the target genes and eventually unleash the observed SAS responses.
2205	Overwhelming primary Epstein-Barr virus infection requiring corticosteroid treatment.	22675096	A 20-year-old woman presented with a 2-week history of fever and malaise. Physical examination was unremarkable. Viral infection was suspected and Epstein-Barr virus serology confirmed acute infectious mononucleosis. During admission, she gradually developed pancytopenia and liver enzyme abnormalities. The patient clinically deteriorated with persisting fever, orthostatic hypotension and hepatosplenomegaly. Bone marrow examination showed haemophagocytic lymphohistiocytosis (HLH). Treatment with high-dose corticosteroids was started and patient recovered quickly. Ferritin decreased immediately, fever resolved within 3 days, viral clearance was reached within 3 weeks. Steroid therapy was gradually tapered off in three months. The Histiocyte Society recommends immunochemotherapy with steroids, etoposide and cyclosporine. Potential side effects of etoposide are severe bone marrow depression and leukaemia. Our patient survived on corticosteroids alone. Early recognition of HLH and prompt treatment are of utmost importance for survival. Treatment with steroids alone can be life-saving.
2206	Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans.	21177963	Regulation of gene expression by sequence-specific transcription factors is central to developmental programs and depends on the binding of transcription factors with target sites in the genome. To date, most such analyses in Caenorhabditis elegans have focused on the interactions between a single transcription factor with one or a few select target genes. As part of the modENCODE Consortium, we have used chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) to determine the genome-wide binding sites of 22 transcription factors (ALR-1, BLMP-1, CEH-14, CEH-30, EGL-27, EGL-5, ELT-3, EOR-1, GEI-11, HLH-1, LIN-11, LIN-13, LIN-15B, LIN-39, MAB-5, MDL-1, MEP-1, PES-1, PHA-4, PQM-1, SKN-1, and UNC-130) at diverse developmental stages. For each factor we determined candidate gene targets, both coding and non-coding. The typical binding sites of almost all factors are within a few hundred nucleotides of the transcript start site. Most factors target a mixture of coding and non-coding target genes, although one factor preferentially binds to non-coding RNA genes. We built a regulatory network among the 22 factors to determine their functional relationships to each other and found that some factors appear to act preferentially as regulators and others as target genes. Examination of the binding targets of three related HOX factors--LIN-39, MAB-5, and EGL-5--indicates that these factors regulate genes involved in cellular migration, neuronal function, and vulval differentiation, consistent with their known roles in these developmental processes. Ultimately, the comprehensive mapping of transcription factor binding sites will identify features of transcriptional networks that regulate C. elegans developmental processes.
2207	[Clinical analysis of 10 cases of secondary hemophagocytic lymphohistiocytosis treated with HLH-2004 chemotherapy].	21176364	This study was aimed to investigate the therapeutic efficacy of HLH-2004 chemotherapy in patients with secondary hemophagocytic lymphohistiocytosis (sHLH). 10 cases of sHLH treated with HLH-2004 regimen at our department were analyzed retrospectively. The results showed that 7 patients had clinical response to HLH-2004 regimen, while other 3 patients had no clinical response. 5 cases did not complete initial therapy of 8 weeks. Out of 5 cases, 4 died in the process of chemotherapy, 1 patient abandoned for serious side effects but finally acquired remission following 4 cycles of CHOP regimen. 5 cases underwent the whole courses of initial therapy. Out of 5 cases, 3 patients acquired remission, and other 2 were not well controlled. Out of the 3 patients who had achieved remission, one died of relapse, and other 2 patients kept remission. Out of the 2 patients who were not well controlled, one patient died, but another patient acquired remission after being discharged. It is concluded that patients with infection-associated hemophagocytic syndrome (IAHS) have high rates of remission after receiving HLH-2004 regimen combining with effective antibiotics. However, patients with HLH secondary to EBV (EBV-HLH) or lymphoma (LAHS) have low rates of remission or are easy to get relapse after remission.
2208	Roles of old players in the suppression of a new player: networks for the transcriptional control of angiogenesis.	2117295419029143	During the formation of blood vessels, Id1, a member of the helix-loop-helix (HLH) family, and TAL1/SCL, a basic HLH (bHLH) transcription factor, play important roles in the activation of endothelial cells. Recent reports revealed that E2-2, another bHLH transcription factor, inhibits angiogenesis in vitro and in vivo by suppressing the expression of vascular endothelial growth factor receptor 2 (VEGFR2). Id1 and TAL1/SCL dimerize with E2-2 and relieve the E2-2-mediated down-regulation of VEGFR2 expression, leading to the activation of endothelial cells. These findings reveal a novel interplay between HLH transcription factors that regulate angiogenesis.
2209	Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes.	21152410	Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL.Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed.Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other.FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.
2210	Rickettsial infection with hemophagocytosis.	21149571	A five-year-old girl admitted with scrub typhus developed multiple organ dysfunction associated with hyperferritinaemia, hypofibrinogenaemia and hyperlipidaemia. Bone marrow aspiration studies confirmed haemophagocytic lymphohistiocytosis (HLH). HLH is a syndrome characterized by the uncontrolled activation and proliferation of macrophages and T-cells and can occur together with infections, connective tissue disorders, malignancies and genetic disorders.
2211	Olig1 and ID4 interactions in living cells visualized by bimolecular fluorescence complementation technique.	21132377	Olig1, a member of class B basic-helix-loop-helix (bHLH), plays key roles in early oligodendrocyte specification. Inhibitors of DNA binding (Id) is another sub-class of HLH proteins, act as dominant-negative regulators of bHLH proteins, which can form heterodimers with class A or B bHLH proteins, but lack the critical basic DNA binding domain. Id4 was recently found to interact with olig1 and inhibit oligodendrocyte differentiation. However, there still no direct evidence to reveal the spatial and temporal interaction of olig1 and ID4 in living cells. In this study, we performed bimolecular fluorescence complementation (BiFC) analysis to further characterize the distinct subcellular localization of olig1, ID4 and their dimer in living SW1116 cells. To examine the subcellular localization of olig1 and ID4 by themselves, the olig1-EGFP or ID4-DsRed2 fusion proteins were also expressed in SW1116 cells, respectively. As predicted, the olig1-EGFP fusion proteins were located in the nucleus, and ID4-DsRed2 fusion proteins were located in the cytoplasm. When olig1-EGFP and ID4-DsRed2 fusion proteins were co-expressed, the green and red signals were co-located in the cytoplasm. Using BiFC, the strong BiFC signals could be detected in pBiFC-olig1VN173 and pBiFC-ID4VC155 co-transfected cells and the fluorescence signal was located in the cytoplasm. These results collectively confirmed that olig1 and ID4 could interact and form dimer in living cells, and ID4 could block the transport of olig1 from cytoplasm to nucleus.
2212	Niemann - Pick disease associated with hemophagocytic syndrome.	27263747	Hemophagocytic lymphohistiocytosis (HLH) is a disease characterized by phagocytosis of blood cells by macrophages within the lymphoreticular tissue. It can develop secondary to some diseases or be familial as a result of genetic mutations. Niemann-Pick disease (NPD) is a very rare lipid storage disease. A three-month-old girl presented with high fever (39°C), abdominal distension and paleness. The parents were consanguineous. The liver and spleen were palpable 10 cm and 11 cm below the costal margins, respectively. Bicytopenia (Hb: 5.5 g/dl, platelet: 77000/mm3), hypertriglyceridemia (351 mg/dl), hyperferritinemia (>1500 ng/dl) and hypofibrinogenemia (120 mg/dl) were detected. Bone marrow aspiration demonstrated foam cells and hemophagocytosis by macrophages and Niemann-Pick cells. Lysosomal sphingomyelinase activity was 0.24 nmol/h/mg/protein (normal: 0.86-2.8). Due to the parents' refusal of further evaluation, the nature of HLH as primary or secondary could not be determined. To the best of our knowledge, this is the first case of NPD associated with HLH and the first demonstration of hemophagocytosis by Niemann-Pick cells. Hemofagositik lenfohistiositoz (HLH), lenforetiküler dokudaki makrofajların kan hücrelerini fagosite etmesiyle karakterize olan, genetik olarak mutasyonlara bağlı ailevi veya bazı hastalıklara ikincil gelişen bir hastalıktır. Nieman-Pick hastalığı ise bir lipid depo hastalığı olup son derece nadir görülmektedir. Üç aylık kız hasta yüksek ateş (39°C), karın şişliği ve solukluk nedeni ile başvurdu. Ebeveynler arasında ikinci derecede akrabalık vardı. Karaciğer 10 cm, dalak 11 cm ele geliyordu. Hastada bisitopeni (Hb: 5,5 gr/dl, trombosit: 77000/mm3), hipertrigliseridemi (351 mg/dl), hiperferritinemi (>1500 ng/dl), hipofibrinojenemi (120 mg/dl) tesbit edildi. Kemik iliği aspirasyonunda köpük hücresi, makrofajlar ve Niemann-Pick hücreleri tarafından yapılan hemofagositoz görüldü. Lizozomal sfingomyelinaz aktivitesi 0.24 nmol/hr/mg/ptn (normal: 0.86-2.8) bulundu. Hastadaki HLH’nin primer veya sekonder olduğu, ailenin tetkiki kabul etmemesi nedeni ile incelenemedi. Olgumuz literatürde Niemann-Pick ile birlikte HLH’nin saptandığı ilk vakadır ve Niemann-Pick hücresinin de hemofagositoz yaptığı ilk kez gösterilmiştir.
2213	Clinical and laboratory data of primary hemophagocytic lymphohistiocytosis: A retrospective review of the Turkish Histiocyte Study Group.	27263739	This study analyzes the clinical and laboratory findings of children with primary hemophagocytic lymphohistiocytosis (HLH) followed in various referral centers of Turkey.A simple three-page questionnaire prepared by the Turkish Histiocyte Study Group was used for documentation of patient data.Age at diagnosis varied from 0.6 to 78 months (median±SD, 16.5±26.1). Sex distribution was almost equal (F/M=10/12). The frequencies of parental consanguinity and sibling death in the family history were 100% and 81.1%, respectively. The most common clinical findings were hepatomegaly (100%) and fever (95%). The most common laboratory findings were anemia (100%), hyperferritinemia (100%) and thrombocytopenia (90.9%). Triglyceride and total bilirubin levels in the deceased versus surviving group appear to be high (triglyceride: 394±183 mg/dl, 289±7 mg/dl; total bilirubin: 2.7±6.9 mg/dl, 0.5±1.2 mg/dl, respectively).We concluded that fever, hepatosplenomegaly, anemia, thrombocytopenia, and hyperferritinemia are the most common clinical and laboratory findings in primary HLH. Increased triglyceride and total bilirubin level at the time of diagnosis might be an indicator of poor prognosis in HLH.AMAÇ: Primer hemofagositik lenfohistiositoz tanısı ile Türkiye’deki farklı merkezlerde takip edilen hastaların klinik ve laboratuar değerlerini değerlendirmektir. YÖNTEMLER: Hasta verilerinin değerlendirilmesi için Türk Histiosit Çalışma Grubu tarafından düzenlenmiş 3 sayfalık soru formu kullanıldı.Olguların tanı sırasındaki yaşları 0.6 ile 78 ay arasında idi (median±SD, 16.5±26.1). Cinsiyet dağılımı eşite yakındı (K/E=10/12). Anne-baba akrabalığı ve kardeş ölüm öyküsü oranları sırası ile %100 ve %81.1 idi. Ensık görülen klinik bulgular hepatomegali (%100), ateş (%95), labaratuvar bulguları ise anemi (%100), hiperferritinemi (%100) ve trombositopeniydi (%90.9). Ölen olguların trigliserid ve bilirubin düzeyleri yüksek gibi gözükmekteydi (trigliserid; 394±183 mg/dL, 289±7 mg/dL, total bilirubin; 2.7±6.9 mg/dL, 0.5±1.2 mg/dL) SONUÇ: Ateş, hepatosplenomegali, anemi, hiperferritinemi ve trombositopeni primer HLH'li hastalarda en sık görülen klinik ve labaratuvar bulgulardır. Tanı sırasındaki artmış trigliserid ve bilirubin düzeyleri kötü prognostik belirteç olabilir.
2214	Hemophagocytic syndrome in a child with severe Crohn's disease and familial Mediterranean fever.	21122524	Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal, severe condition of hyperinflammation caused by the uncontrolled proliferation of activated lymphocytes and histiocytes secreting high amounts of inflammatory cytokines. Here we report a fatal hemophagocytic syndrome in a 11-year-old boy with a diagnosis of both Crohn's disease receiving immunosuppressive therapy and familial Mediterranean fever. It is important to evaluate the patients with inflammatory bowel disease receiving immunosuppressive therapy presenting with unexplained fever, cytopenia, progression of organomegaly and biochemical changes for the investigation of HLH for diagnosis and treatment.
2215	Clinical similarities and differences of patients with X-linked lymphoproliferative syndrome type 1 (XLP-1/SAP deficiency) versus type 2 (XLP-2/XIAP deficiency).	21119115	X-linked lymphoproliferative syndromes (XLP) are primary immunodeficiencies characterized by a particular vulnerability toward Epstein-Barr virus infection, frequently resulting in hemophagocytic lymphohistiocytosis (HLH). XLP type 1 (XLP-1) is caused by mutations in the gene SH2D1A (also named SAP), whereas mutations in the gene XIAP underlie XLP type 2 (XLP-2). Here, a comparison of the clinical phenotypes associated with XLP-1 and XLP-2 was performed in cohorts of 33 and 30 patients, respectively. HLH (XLP-1, 55%; XLP-2, 76%) and hypogammaglobulinemia (XLP-1, 67%; XLP-2, 33%) occurred in both groups. Epstein-Barr virus infection in XLP-1 and XLP-2 was the common trigger of HLH (XLP-1, 92%; XLP-2, 83%). Survival rates and mean ages at the first HLH episode did not differ for both groups, but HLH was more severe with lethal outcome in XLP-1 (XLP-1, 61%; XLP-2, 23%). Although only XLP-1 patients developed lymphomas (30%), XLP-2 patients (17%) had chronic hemorrhagic colitis as documented by histopathology. Recurrent splenomegaly often associated with cytopenia and fever was preferentially observed in XLP-2 (XLP-1, 7%; XLP-2, 87%) and probably represents minimal forms of HLH as documented by histopathology. This first phenotypic comparison of XLP subtypes should help to improve the diagnosis and the care of patients with XLP conditions.
2216	Treatment of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH); update 2010.	21088619	The clinical features of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH) vary significantly, from mild to severe, at the time of the treatment decision. There are many reports of successful treatments, for example conservative treatment without etoposide, HLH-94/2004-type immunochemotherapy with etoposide, and hematopoietic stem cell transplantation. When considering the treatment of EBV-HLH, the most important factor is the finding that a survival benefit is obtained when etoposide-containing therapy is initiated within 4 weeks of diagnosis. This indicates that there may be a window for observation or conservative corticosteroid/cyclosporine A or intravenous immunoglobulin (IVIG) treatment; however, once the disease is defined as "high risk" and/or refractory to such therapy, prompt introduction of etoposide (ideally within 4 wk) is recommended. In deciding whether the disease is "high-risk," evaluation of clinical staging, EBV genome copy numbers in the serum, cellular EBV tropism, chromosome analysis, and screening for hereditary immuno-deficient diseases such as familial HLH, are required.
2217	Neonatal enteroviral sepsis/meningoencephalitis and hemophagocytic lymphohistiocytosis: diagnostic challenges.	21089006	We present the clinical course of three neonates with proven enteroviral infection and an initial clinical picture suggestive of hemophagocytic lymphohistiocytosis (HLH). After a complete workup, only one was treated for HLH. Of particular interest, the first newborn presented with hemophagocytic cells in the cerebrospinal fluid (CSF) and proved to have enteroviral meningoencephalitis but was ultimately not diagnosed with HLH. A fourth infant, who fulfilled the diagnostic criteria for HLH but did not have enteroviral infection, is included for comparison. We suggest that severe neonatal enteroviral infection and HLH are difficult to distinguish. Careful assessment is recommended, as prognosis and treatment differ between these two entities. Literature regarding neonatal enteroviral infection and HLH is reviewed, to demonstrate the continuum between the inflammation triggered by enteroviral infection and the occurrence of HLH, as well as their comparable CSF findings.
2218	Acute respiratory distress syndrome as an initial presentation of hemophagocytic lymphohistiocytosis after induction therapy for acute myeloid leukemia.	2108336221381868	A 7-month-old girl with acute myeloid leukemia (AML) developed acute respiratory distress syndrome (ARDS) during the pancytopenic period after induction chemotherapy. Respiratory failure did not improve despite intensive treatments. Eventually, hemophagocytic lymphohistiocytosis (HLH) was diagnosed based on hemophagocytosis in bone marrow, and high soluble interleukin-2 receptor (sIL-2R) and ferritin levels. Even after cyclosporin A was started against HLH, she did not recover. Autopsy showed macrophage proliferation in bone marrow and lymph nodes. HLH should be considered, even in the pancytopenic period after chemotherapy, when patients develop ARDS that does not respond to supportive therapies.
2219	Differential regulation of transcription through distinct Suppressor of Hairless DNA binding site architectures during Notch signaling in proneural clusters.	21041480	In Drosophila melanogaster, achaete (ac) and m8 are model basic helix-loop-helix activator (bHLH A) and repressor genes, respectively, that have the opposite cell expression pattern in proneural clusters during Notch signaling. Previous studies have shown that activation of m8 transcription in specific cells within proneural clusters by Notch signaling is programmed by a "combinatorial" and "architectural" DNA transcription code containing binding sites for the Su(H) and proneural bHLH A proteins. Here we show the novel result that the ac promoter contains a similar combinatorial code of Su(H) and bHLH A binding sites but contains a different Su(H) site architectural code that does not mediate activation during Notch signaling, thus programming a cell expression pattern opposite that of m8 in proneural clusters.
2220	Otx-dependent expression of proneural bHLH genes establishes a neuronal bilateral asymmetry in C. elegans.	21041366	Bilateral asymmetry in Caenorhabditis elegans arises in part from cell lineages that differ on the left and right sides of the animal. The unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage that generates the MI neuron on the right and the e3D epithelial cell on the left. We isolated mutations in three genes that caused left-right symmetry in this normally asymmetric cell lineage by transforming MI into an e3D-like cell. These genes encode the proneural bHLH proteins NGN-1 and HLH-2 and the Otx homeodomain protein CEH-36. We identified the precise precursor cells in which ceh-36 and ngn-1 act, and showed that CEH-36 protein is asymmetrically expressed and is present in an MI progenitor cell on the right but not in its bilateral counterpart. This asymmetric CEH-36 expression promotes asymmetric ngn-1 and hlh-2 expression, which in turn induces asymmetric MI neurogenesis. Our results indicate that this left-right asymmetry is specified within the two sister cells that first separate the left and right branches of the cell lineage. We conclude that the components of an evolutionarily conserved Otx/bHLH pathway act sequentially through multiple rounds of cell division on the right to relay an initial apparently cryptic asymmetry to the presumptive post-mitotic MI neuron, thereby creating an anatomical bilateral asymmetry in the C. elegans nervous system.
2221	[Use of recombinant human luteinizing hormone for ovulation stimulation in in vitro fertilization-embryo transfer].	21029591	To evaluate application of recombinant human luteinizing hormone (r-hLH) used in ovarian stimulation of assisted reproductive technique and impact on outcome of pregnancy.From Apr. to Jul. 2009, 123 patients with low LH level (< 1 U/L) at day 3 of menstruation and down-regulation of pituitary function undergoing in vitro fertilization-embryo transfer (IVF-ET) in Reproductive Medical Center, Provincial Hospital Affiliated to Shandong University were enrolled in this study, whom were classified into 66 cases treated by r-hLH in r-hLH group and 57 cases without r-hLH treatment in non-r-hLH group. In the mean time, 145 patients with normal level of serum LH (1 - 2 U/L) not given by r-hLH treatment and undergoing IVF-ET were matched as control group. Total amount of gonadotropin, estradiol levels and LH levels on the administration of human chorionic gonadotropin (hCG), number of oocytes retrieved, number of 2PN zygotes, rate of high quality embryos, the rates of implantation and clinical pregnancy were compared among these three groups.The level of serum LH on the day of hCG administration were (1.59 ± 0.77) U/L in r-hLH group, (0.54 ± 0.25) U/L in non-r-hLH group and (2.39 ± 1.01) U/L in control group, which reached statistical difference between every two groups (P < 0.05). The rates of high quality embryo were 59.36% in r-hLH group, 57.79% in non-r-hLH group, which were significantly lower than 65.94% in control group, respectively (P < 0.05). The rates of 2PN were 67.62% in r-hLH group and 68.32% in control group, which were significantly higher than 62.84% in non-r-hLH group, respectively (P < 0.05). The rates of implantation of 29.77% in r-hLH group were significantly higher than 18.26% in non-r-hLH group (P < 0.05). The total amount of gonadotropin, estradiol level on the day of hCG administration, the number of oocytes retrieved, and clinical pregnancy rate were not significantly different among those three groups (P > 0.05).The administration of recombinant human luteinizing hormone in patients who are profoundly suppressed after down-regulation with long protocol can get more quality embryos, the higher rates of 2PN and implantation.
2222	Haemophagocytic lymphohistiocytosis complicating Hodgkin's lymphoma in an HIV-positive individual.	20975096	We report the case of an HIV-positive man who presented with pyrexia of unknown origin. Histological specimens from an inguinal lymph node and liver biopsy gave a diagnosis of Epstein Barr virus (EBV)-positive Hodgkin's lymphoma and haemophagocytic lymphohistiocytosis (HLH), respectively. HLH is a condition characterized by proliferation of activated macrophages that phagocytose leukocytes, erythrocytes and platelets. Clinical features include splenomegaly, fever and pancytopenia, all of which have a wide differential diagnosis in HIV-positive patients. HLH can be caused by infections, malignancy, drugs or autoimmune conditions. There have been a number of reports of HLH in HIV-positive patients, and it can be seen at all stages of HIV infection. HIV, lymphomas, EBV infection and haemophagocytic syndrome have a complicated pathophysiology. Unfortunately, HLH in this setting has a particularly aggressive course, often with a poor outcome. This case highlights the need for awareness of the syndrome to ensure prompt diagnosis and instigation of appropriate treatment.
2223	Profile of hemophagocytic lymphohistiocytosis in children in a tertiary care hospital in India.	20972297	To describe the epidemiology, clinical features, laboratory findings, outcome and the difficulties in diagnosis and management of children with Hemo-phagocytic Lymphohistiocytosis (HLH) in a tertiary children's hospital in India.Retrospective analysis of case records of all the children with a diagnosis of HLH from December 2006 to December 2008.Tertiary care children's teaching hospital in Chennai, India.43 children had a diagnosis of hemo-phagocytosis, of who only 33 (19 male, mean age 46 months, range 50 days-14 years) met the inclusion criteria based on the HLH 2004 protocol of the Histiocyte Society. The predominant presenting features included prolonged fever and hepatosplenomegaly. CNS symptoms were present in 36%. Anemia (Hb <9 gm/dL), and thrombocytopenia (platelets <1,00,000/mm3) were present in 97% and 72%, respectively. Among the biochemical markers, hyperferritinemia was present in 97%, and hypofibrinogenemia and high LDH in 92%. Bone marrow examination showed hemophagocytosis in 84%. Infectious agents were identified in 42% children, with viruses accounting for 2/3 of them (5 Dengue virus, 3 EBV, 1 CMV, 1 TB and 5 bacterial agents). The mean duration between the onset of symptoms and the diagnosis was 16 days. Corticosteroids were the most commonly used immunomodulatory agents (67%), followed by IVI g (64%). Cyclosporine was used in 33% and Etoposide in 15%. Improvement of laboratory parameters was noticed within 5-7 days of starting treatment. Overall survival rate was 76%.HLH should be considered in the differential diagnosis of children with prolonged fever, hepatosplenomegaly and cytopenia. Prompt recognition and appropriate therapy may result in good outcome, particularly in Infection associated HLH.
2224	X-linked lymphoproliferative disease due to SAP/SH2D1A deficiency: a multicenter study on the manifestations, management and outcome of the disease.	20926771	X-linked lymphoproliferative disease (XLP1) is a rare immunodeficiency characterized by severe immune dysregulation and caused by mutations in the SH2D1A/SAP gene. Clinical manifestations are varied and include hemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinemia, often triggered by Epstein-Barr virus infection. Historical data published before improved treatment regimens shows very poor outcome. We describe a large cohort of 91 genetically defined XLP1 patients collected from centers worldwide and report characteristics and outcome data for 43 patients receiving hematopoietic stem cell transplant (HSCT) and 48 untransplanted patients. The advent of better treatment strategies for HLH and malignancy has greatly reduced mortality for these patients, but HLH still remains the most severe feature of XLP1. Survival after allogeneic HSCT is 81.4% with good immune reconstitution in the large majority of patients and little evidence of posttransplant lymphoproliferative disease. However, survival falls to 50% in patients with HLH as a feature of disease. Untransplanted patients have an overall survival of 62.5% with the majority on immunoglobulin replacement therapy, but the outcome for those untransplanted after HLH is extremely poor (18.8%). HSCT should be undertaken in all patients with HLH, because outcome without transplant is extremely poor. The outcome of HSCT for other manifestations of XLP1 is very good, and if HSCT is not undertaken immediately, patients must be monitored closely for evidence of disease progression.
2225	Antitumor activity of glycosylated molluscan hemocyanins via Guerin ascites tumor.	20923331	As observed in most molluscan hemocyanins, high-mannose type glycans were identified in hemocyanins from Rapana venosa (RvH), Helix lucorum (HlH) and keyhole limpet (Megatura crenulata). In addition, a glycan with a branching structure containing xylose, fucose and terminal methyl hexose was identified in β-HlH. We have examined the immuno-adjuvant properties of hemocyanins, their derivatives and conjugates associated with the cell mediated immunity in experimental tumor-bearing animals with ascites tumor of Guerin. After immunization of the animals with the experimental vaccine preparations, the highest values of splenic lymphocytes were observed in groups immunized with the conjugates RvH-TAg, β-HlH-TAg and KLH-TAg (42.3%; 40.8% and 40.58%, respectively) than with the native hemocyanins (36.5%; 35.1% and 32.4%, respectively). The immunization of rats with the hemocyanins β-HlH, RvH and KLH and their conjugates, prolonged the median survival time of tumor-bearing animals compared with non-immunized animals (39, 33, 31 and 7 days, respectively). Both hemocyanins β-HlH and RvH activate the immune system of the experimental animals and therefore could be a good alternative for KLH. For this reason they could be included into the composition of non-specific anti-tumor vaccines to enhance their effectiveness.
2226	[Treatment of severe hemophagocytic syndrome associated with visceral leishmaniasis].	20880678	The association of hemophagocytic syndrome (HS) and visceral leishmaniasis is a frequent disorder during infancy in endemic areas such as Tunisia. The range of severity of HS secondary to visceral leishmaniasis includes both pure biological forms that resolve with antimicrobial therapy and life-threatening emergencies that require specific treatment. We describe 2 cases of severe HS secondary to visceral leishmaniasis. The diagnosis of HS was based on the HLH-2004 diagnostic criteria. Therapy involved pentavalent antimonial (Glucantime) in both cases. The combination of corticosteroids with immunoglobulins, used in the 1st case, but introduced late, led to an unfavorable course and death. In the 2nd case, the specific treatment of HS was based on immunochemotherapy including etoposide and corticosteroids. Progression was favorable with a follow-up of 24 months. Etoposide containing therapeutic regimens can be proposed in severe forms of HS associated with visceral leishmaniasis.
2227	Myelodysplastic syndrome (MDS) as a late stage of subclinical hemophagocytic lymphohistiocytosis (HLH): a putative role for Leptospira infection. A hypothesis.	20870590	It is proposed that hemophagocytic lymphohistiocytosis (HLH) and myelodysplastic syndromes (MDS) may be temporally distinct phases of pathophysiologically related disease processes. A significant subgroup of MDS may develop from subclinical HLH. In that case, HLH-like disease would chronically proceed with little disease activity or under occasional flares only, until it first becomes clinically apparent at the MDS stage. At the MDS stage, however, HLH activity may be easily overlooked by histological or cytogenetic means, since hemophagocytosis has fallen already largely silent. Current treatment options for HLH, like high-dose intravenous immunoglobulins (IVIG), may turn out to be helpful in MDS patients as well. In rare and extreme cases, Leptospira infection causes severe and life-threatening HLH. Thus, this proposal also implies that an insufficient, dysfunctional or misdirected immunological response to Leptospira infection may lead to MDS in the long run in a significant number of cases, which have not been recognized as Leptospira -triggered events in the first place. Infections by agents other than Leptospira may lead to subclinical HLH-like disease with MDS as a late stage as well.
2228	Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin.	20863484	Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex(0-9) HexNAc(2-4) Hex(0-3) Pent(0-3) Fuc(0-3). A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(β1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.
2229	The transcriptional repressor ID2 can interact with the canonical clock components CLOCK and BMAL1 and mediate inhibitory effects on mPer1 expression.	20861012	ID2 is a rhythmically expressed HLH transcriptional repressor. Deletion of Id2 in mice results in circadian phenotypes, highlighted by disrupted locomotor activity rhythms and an enhanced photoentrainment response. ID2 can suppress the transactivation potential of the positive elements of the clock, CLOCK-BMAL1, on mPer1 and clock-controlled gene (CCG) activity. Misregulation of CCGs is observed in Id2(-/-) liver, and mutant mice exhibit associated alterations in lipid homeostasis. These data suggest that ID2 contributes to both input and output components of the clock and that this may be via interaction with the bHLH clock proteins CLOCK and BMAL1. The aim of the present study was to explore this potential interaction. Coimmunoprecipitation analysis revealed the capability of ID2 to complex with both CLOCK and BMAL1, and mammalian two-hybrid analysis revealed direct interactions of ID2, ID1 and ID3 with CLOCK and BMAL1. Deletion of the ID2 HLH domain rendered ID2 ineffective at inhibiting CLOCK-BMAL1 transactivation, suggesting that interaction between the proteins is via the HLH region. Immunofluorescence analysis revealed overlapping localization of ID2 with CLOCK and BMAL1 in the cytoplasm. Overexpression of CLOCK and BMAL1 in the presence of ID2 resulted in a significant reduction in their nuclear localization, revealing that ID2 can sequester CLOCK and BMAL1 to the cytoplasm. Serum stimulation of Id2(-/-) mouse embryonic fibroblasts resulted in an enhanced induction of mPer1 expression. These data provide the basis for a molecular mechanism through which ID2 could regulate aspects of both clock input and output through a time-of-day specific interaction with CLOCK and BMAL1.
2230	Reduced-intensity conditioning significantly improves survival of patients with hemophagocytic lymphohistiocytosis undergoing allogeneic hematopoietic cell transplantation.	20855862	Recent experience suggests that reduced-intensity conditioning (RIC) regimens can improve the outcomes of patients with hemophagocytic lymphohistiocytosis (HLH) undergoing allogeneic hematopoietic cell transplantation (HCT). However, studies directly comparing RIC to myeloablative conditioning (MAC) regimens are lacking. Forty patients with HLH underwent allogeneic HCT between 2003-2009 at Cincinnati Children's Hospital. Fourteen patients received MAC consisting of busulfan, cyclophosphamide, and antithymocyte globulin plus or minus etoposide. Twenty-six patients received RIC consisting of fludarabine, melphalan, and alemtuzumab. All patients engrafted. Acute graft-versus-host disease grades II to III occurred in 14% of MAC patients and 8% of RIC patients (P = .3171). Posttransplantation mixed donor/recipient chimerism developed in 18% of MAC patients and 65% of RIC patients (P = .0110). The majority of patients with mixed chimerism received intervention with reduction of immune suppression plus or minus donor lymphocyte infusion or stem cell boost, which stabilized or increased donor contribution to hematopoiesis and prevented relapse of HLH in all but 1 patient. Grade II to III graft-versus-host disease occurred in 5 of 14 RIC patients after donor lymphocyte infusion. The overall estimated 3-year survival after HCT was 43% (confidence interval = ± 26%) for MAC patients and 92% (confidence interval = ± 11%) for RIC patients (P = .0001). We conclude that RIC significantly improves the outcome of patients with HLH undergoing allogeneic HCT.
2231	Initial audit of a basic and emergency neurosurgical training program in rural Tanzania.	20849781	As of 2006, only three formally trained neurosurgeons are licensed in Tanzania. Recently, efforts have increased toward training local Tanzanian physicians and assistant medical officers (AMOs) to meet the basic neurosurgical needs of nonurban areas. Between January and July 2006, an initial attempt at such an apprenticeship was undertaken with a locally trained AMO already performing general surgery at Haydom Lutheran Hospital, Tanzania.Fifty-one neurosurgical patients were identified and their patient charts were requested from the medical records office. Records were not available for 4 of the 51 patients for undeterminable reasons.The neurosurgical infrastructure at HLH is basic but adequate for a number of procedures. Cases performed included ventriculoperitoneal shunts, repair of myelomeningoceles, and burr holes and craniotomies for trauma and biopsies. Of 51 patients initially identified, 14 (27%) were confirmed deceased and 20 (39%) confirmed living. The remaining 17 (33%) were lost to follow-up. There were no significant differences in the mortality rates of patients receiving care from the American-trained neurosurgeon and those receiving care from the Tanzanian AMO trained and mentored by the American neurosurgeon.This initial audit provides support for the development of limited neurosurgery programs in underserved communities. Combined utilization of available neurosurgeons and continued training for available local clinicians may help to meet this need.
2232	Rate of decline of ferritin in patients with hemophagocytic lymphohistiocytosis as a prognostic variable for mortality.	20842751	Hemophagocytic lymphohistiocytosis (HLH) is difficult to diagnose and treat. Highly elevated ferritin is strongly associated with HLH and levels may provide a prognostic marker. A comprehensive review of ferritin data from our patients during treatment was analyzed with respect to mortality. A patient was 17 times more likely to die when percent ferritin decrease was less than 50% as compared to a 96% or greater decrease as indicated with multivariate logistic modeling. Higher maximum ferritin levels in the first 3 weeks also contributed to the odds of death (OR = 5.6; 90% CI = 1.2-24.9). Regular ferritin measurements may be useful predicting outcomes in HLH patients.
2233	Ventriculocoronary artery connections with the hypoplastic left heart: a 4-year prospective study: incidence, echocardiographic and clinical features.	20820769	Ventriculocoronary connections (VCCs), also called sinusoids, occur with hypoplastic left heart (HLH). Previous reports are limited to case reports, pathologic series, and surgical series with limited detail, which may underestimate the incidence and overestimate the severity of VCCs in HLH. A study was conducted to determine the incidence VCCs in HLH, their effect on survival, and their echocardiographic and clinical features. The echocardiograms and medical records of 100 consecutive neonatal HLH cases were analyzed. All had an aortic and a mitral valve diameter and a left ventricular (LV) volume less than Z-3. For palliation, Norwood, Sano, or hybrid procedures were used, and if the patient was alive, subsequent bidirectional Glenn and extracardiac Fontan procedures were applied. Cases were classified as manifesting mitral and aortic atresia (MAAA), mitral and aortic stenosis (MSAS), or mitral stenosis and aortic atresia (MSAA). All other diagnoses or any case with additional cardiac anomalies were excluded from the study. Overall, VCCs were found in 15% of the cases. They occurred in 56% of the MSAA subtype cases and were not statistically associated with a high mortality rate. However, in one case, large and multiple VCCs definitely caused or contributed to early death. All VCCs had a transmyocardial course, a turbulent color-Doppler flow, and a dominant usually retrograde systolic coronary artery flow pattern. The VCCs were associated (p < 0.05) with MSAA, endocardial fibroelastosis, and ascending aortic size less than 2 mm. As shown by the findings, 15% of the HLH patients had MSAA with VCCs. Unless the VCCs were large or extensive, they did not contribute to mortality. Detailed echocardiographic analysis of VCCs in HLH was feasible. Recent reports emphasize more severe cases.
2234	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis: a retrospective study of 78 pediatric cases in mainland of China.	20819601	The clinical characteristics of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) are largely unreported in the pediatric patients in mainland of China. The main aim of this study was to recognize the clinical features of EBV-HLH in children and to explore its prognosis and risk factors.A retrospective study was performed on 78 pediatric patients with EBV-HLH who were admitted to Beijing Children's Hospital between 2003 and 2008. All patients' medical records were reviewed and analyzed. For each patient, demographic, clinical, laboratory and outcome information was collected. Statistical analysis was conducted via multivariate and univariate analysis.The age of onset peaked between 1 - 2 years and boys were more likely developed EBV-HLH. EBV-HLH occurred mainly in the serological pattern with EBV nuclear antigen (EBNA) positive (70.5%). The overall fatality of the disease was 56.7%. Twelve of the 39 fatalities (30.8%) died rapidly within 2 months after diagnosis. Multivariate analysis revealed that not receiving chemotherapy (P = 0.002), > or = 4 weeks of illness prior to diagnosis (P = 0.004), and albumin levels < 20 g/L (P = 0.045) significantly predicted an increased fatality risk.EBV-HLH is a severe disease with a high fatality rate that occurs mainly in the serological pattern with EBNA positive. Early initiation of chemotherapy and timely diagnosis significantly improves survival rate. Practical strategies should focus on reducing the likelihood of early death.
2235	Using flow cytometry to screen patients for X-linked lymphoproliferative disease due to SAP deficiency and XIAP deficiency.	20816973	X-linked lymphoproliferative disease is a rare congenital immunodeficiency that is most often caused by mutations in SH2D1A, the gene encoding signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). XLP caused by SAP deficiency is most often characterized by fulminant mononucleosis/EBV- associated hemophagocytic lymphohistiocytosis (HLH), lymphoma, and dysgammaglobulinemia. XLP has also been found to be caused by mutations in BIRC4, the gene encoding X-linked inhibitor of apoptosis (XIAP). Patients with XIAP deficiency often present with HLH or recurrent HLH, which may or may not be associated with EBV. XLP is prematurely lethal in the majority of cases. While genetic sequencing can provide a genetic diagnosis of XLP, a more rapid means of diagnosis of XLP is desirable. Rapid diagnosis is especially important in the setting of HLH, as this may hasten the initiation of life-saving medical treatments and expedite preparations for allogeneic hematopoietic cell transplantation (HCT). Flow cytometry offers a means to quickly screen patients for XLP. Flow cytometry can be used to measure lymphocyte SAP or XIAP protein expression, and can also be used to observe lymphocyte phenotypes and functional defects that are unique to XLP. This review will give a brief overview of the clinical manifestations and molecular basis of SAP deficiency and XIAP deficiency, and will focus on the use of flow cytometry for diagnosis of XLP.
2236	Secondary hemophagocytic syndrome in adults: a case series of 18 patients in a single institution and a review of literature.	20809477	Hemophagocytic lymphohistiocytosis (HLH) is rare in adults and is usually fatal without treatment. We present a consecutive series of 18 adults with HLH diagnosed at our institution between 2004 and 2009. All diagnoses were confirmed by pathology. The median age at diagnosis was 56 years (range: 18-73 years), with a male: female ratio of 2:1. Patients uniformly presented with fever. Fifty-five per cent of the patients presented with evidence of hepatomegaly or splenomegaly. All of the patients had at least a bi- or trilineage cytopenia. Elevated liver enzymes, hyperferritinemia, hypertriglyceridemia and hyperfibrinogenemia were seen in 50, 100, 40 and 50% of patients, respectively. The presumed causes were as follows; haematological malignancies (n = 4), post-autologous stem cell transplant (n = 2), infection (n = 2), rheumatologic illness (n = 2), sickle cell disease (n = 1), post-orthotopic liver transplant (n = 1) and idiopathic (n = 3). The median time from suspicion to diagnosis was 5 days (1-27 days). Corticosteroids and/or cyclosporine were the most frequently used treatment regimen. Other agents used were etoposide, IVIG, cyclophosphamide and chemotherapy. The mortality rate was 72%, with multi-system organ failure being the most common cause of death. Median survival time from diagnosis was 35 days. Six patients are alive to date. In a univariate analysis, the presence of fever was the only factor that was statistically significant for predicting a poor prognosis (early mortality) (p = 0.05). In conclusion, a high index of suspicion is the critical factor for early diagnosis. Early treatment with immunosuppressant is warranted, and a thorough diagnostic evaluation to identify the underlying cause should be undertaken.
2237	Spontaneous resolution of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	20806367	Secondary hemophagocytic lymphohistiocytosis (sHLH) is a reactive, proliferative disorder of the immune system resulting in lymphohistiocytic proliferation, hemophagocytosis, and cytokine dysregulation. The most common infectious trigger in sHLH is Epstein-Barr virus (EBV-HLH). Current treatment protocols for EBV-HLH have a cure rate of approximately 75%; however, there are significant toxicities associated with these therapies. We present two patients with EBV-HLH who experienced spontaneous resolution of their disease prior to the initiation of therapy, suggesting there may be a subgroup of patients with EBV-HLH who do well with conservative management and can avoid potentially toxic therapies.
2238	STXBP2 mutations in children with familial haemophagocytic lymphohistiocytosis type 5.	20798128	Familial haemophagocytic lymphohistiocytosis (FHL) is a rare immune deficiency with uncontrolled inflammation; the clinical course usually starts within the first years of life, and is usually fatal unless promptly treated and then cured with haematopoietic stem cell transplant. FHL is caused by genetic mutations resulting in defective cell cytotoxicity; three disease related genes have been identified to date: perforin, Munc13-4 and syntaxin-11. A fourth gene, STXBP2, has been identified very recently as responsible for a defect in Munc18-2 in FHL-5.To describe the result of the screening of families with HLH and previously unassigned genetic defects.Patients with HLH diagnosed according to current diagnostic criteria, and who lacked mutations in the PRF1, Munc13-4, and STX11 genes were sequenced for mutations in STXBP2. Functional study was performed when material was available.Among the 28 families investigated, 4 (14%) with biallelic STXBP2 mutations were identified. They originated from Italy, England, Kuwait and Pakistan. The p.Pro477Leu resulting from c.1430C>T, and p.Arg405Gln resulting from the single c.1214G>A nucleotide change are known, while we contribute two novel mutations: p.Glu132Ala resulting from c.395A>C, and p.Gly541Ser, resulting from c.1621G>A. The detrimental effect of the p.Gly541Ser mutation was documented biochemically and functionally in NK and CD8 cells. Additional polymorphisms are also described.These data expand current knowledge on the genetic heterogeneity of FHL and suggest that patients with FHL5 may have different results in degranulation assays under different conditions.
2239	Familial hemophagocytic lymphohistiocytosis in a 6-week-old male infant.	20698142	Familial hemophagocytic lymphohistiocytosis (FLH) is an autosomal recessively inherited multisystem disease. This defect in cellular cytotoxicity is a life threatening condition characterized by fever, rash, splenomegaly, cytopenias and neurologic manifestations. PRF1, UNC13D and STX11 gene defects underlie in about 40-50% of primary cases. Chemoimmunotherapy followed by hematopoietic stem cell transplantation improved disease outcome. We report a case of a 6-week-old boy who presented with a fever, diffuse rash, disseminated intravascular coagulation, hypofibrinogenemia, hypertrigliceridemia, hepatosplenomegaly, leukocytosis with 90% of lymphocytes, granulocytopenia, anemia, trombocytopenia, hyperferritinemia and pathological findings in cerebrospinal fluid. The patient had decreased frequency of NK cells and low NK cell activity in peripheral blood. Bone marrow aspiration analysis showed degenerative changes of histocyte cells, with preserved cytophages (lymphophages and erythrophages) consistent with hematophagocytic syndrome. Given that the molecular diagnosis of the known mutations in genes PRF1 and UNC13D showed a mutation in UNC13D, the diagnosis of familial hemophagocytic lymphohistiocytosis subtype 3 was established. HLH-2004 chemotherapy protocol was performed and partial remission with residual central nervous system disease was achieved. Hematopoietic stem cell transplantation was successfully performed with an unrelated HLA-matched donor. Familiar HLH is generally a progressive and fatal disease. Early diagnosis with molecular genetic analysis and chemoimmunotherapy followed by hematopoietic stem-cell transplantation is the best approach.
2240	Identification of specific protein/E-box-containing DNA complexes: lessons from the ubiquitously expressed USF transcription factors of the b-HLH-LZ super family.	20694681	In order to determine how gene expression is regulated in response to environmental cues, it is necessary to identify the specific interaction between transcription factors and their cognate cis-regulatory DNA elements. Here we have out-lined electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) protocols to define in vitro and in vivo USFs specific interacting sequences. The proposed procedures have been optimized for the USFs transcription factor family, allowing the identification of USF-specific targets.
2241	poky/chuk/ikk1 is required for differentiation of the zebrafish embryonic epidermis.	20692251	An epidermis surrounds all vertebrates, forming a water barrier between the external environment and the internal space of the organism. In the zebrafish, the embryonic epidermis consists of an outer enveloping layer (EVL) and an inner basal layer that have distinct embryonic origins. Differentiation of the EVL requires the maternal effect gene poky/ikk1 in EVL cells prior to establishment of the basal layer. This requirement is transient and maternal Ikk1 is sufficient to allow establishment of the EVL and formation of normal skin in adults. Similar to the requirement for Ikk1 in mouse epidermis, EVL cells in poky mutants fail to exit the cell cycle or express specific markers of differentiation. In spite of the similarity in phenotype, the molecular requirement for Ikk1 is different between mouse and zebrafish. Unlike the mouse, EVL differentiation requires functioning Poky/Ikk1 kinase activity but does not require the HLH domain. Previous work suggested that the EVL was a transient embryonic structure, and that maturation of the epidermis required replacement of the EVL with cells from the basal layer. We show here that the EVL is not lost during embryogenesis but persists to larval stages. Our results show that while the requirement for poky/ikk1 is conserved, the differences in molecular activity indicate that diversification of an epithelial differentiation program has allowed at least two developmental modes of establishing a multilayered epidermis in vertebrates.
2242	C. elegans twist gene expression in differentiated cell types is controlled by autoregulation through intron elements.	20691175	The temporospatial regulation of genes encoding transcription factors is important during development. The hlh-8 gene encodes the C. elegans mesodermal transcription factor CeTwist. Elements in the hlh-8 promoter restrict gene expression to predominantly undifferentiated cells of the M lineage. We have discovered that hlh-8 expression in differentiated mesodermal cells is controlled by two well-conserved E box elements in the large first intron. Additionally, we found that these elements are bound in vitro by CeTwist and its transcription factor partner, CeE/DA. The E box driven expression is eliminated or diminished in an hlh-8 null allele or in hlh-2 (CeE/DA) RNAi, respectively. Expression of hlh-8 is also diminished in animals harboring an hlh-8 intron deletion allele. Altogether, our results support a model in which hlh-8 is initially expressed in the undifferentiated M lineage cells via promoter elements and then the CeTwist activates its own expression further (autoregulation) in differentiated cells derived from the M lineage via the intron elements. This model provides a mechanism for how a transcription factor may regulate distinct target genes in cells both before and after initiating the differentiation program. The findings could also be relevant to understanding human Twist gene regulation, which is currently not well understood.
2243	Cytophagic histiocytic panniculitis and hemophagocytic lymphohistiocytosis: an overview.	20666826	Cytophagic histiocytic panniculitis (CHP) is a rare panniculitis that is associated with systemic features including fevers, hepatosplenomegaly, lymphadenopathy, pancytopenia, hepatic abnormalities, hypertriglyceridemia, and coagulopathy without an elevated erythrocyte sedimentation rate. The panniculitis lesions show adipose tissue lymphocytic and histiocytic infiltration along with hemophagocytosis, which may also appear in bone marrow, spleen, lymph nodes, and liver. Patients may have a rapidly fatal disease course, a longer disease course with intermittent remissions and exacerbations for many years prior to death, or a nonfatal acute or intermittent course responsive to treatment. The cytophagocytic disorder in these patients is a hemophagocytic lymphohistiocytosis (HLH), similar to the infection-activated reaction associated with perforin mutations found in familial hemophagocytic lymphohistiocytosis. HLH is a group of autoinflammatory disorders, which include macrophage activation syndrome and infection-associated hemophagocytic syndrome, which if not treated rapidly, can be fatal. The relationship of CHP and HLH is discussed. CHP associated diseases include: subcutaneous panniculitis-like T cell lymphomas; infections, connective tissue diseases, other malignancies, and the molecular disorders that cause HLH. Treatment of CHP includes: glucocorticoids in combination with cyclosporine, combined chemotherapeutic medications and most recently, anakinra, an Interleukin-1 receptor antagonist; along with supportive care, search for underlying malignancies and treatment thereof, and control of associated infections.
2244	X-linked lymphoproliferative syndromes: brothers or distant cousins?	20660790	X-linked lymphoproliferative disease (XLP1), described in the mid-1970s and molecularly defined in 1998, and XLP2, reported in 2006, are prematurely lethal genetic immunodeficiencies that share susceptibility to overwhelming inflammatory responses to certain infectious triggers. Signaling lymphocytic activation molecule-associated protein (SAP; encoded by SH2D1A) is mutated in XLP1, and X-linked inhibitor of apoptosis (XIAP; encoded by BIRC4) is mutated in XLP2. XLP1 is a disease with multiple and variable clinical consequences, including fatal hemophagocytic lymphohistiocytosis (HLH) triggered predominantly by Epstein-Barr virus, lymphomas, antibody deficiency, and rarer consequences of immune dysregulation. To date, XLP2 has been found to cause HLH with and without exposure to Epstein-Barr virus, and HLH is commonly recurrent in these patients. For both forms of XLP, the only curative therapy at present is allogeneic hematopoietic cell transplantation. Beyond their common X-linked locus and their requirement for normal immune responses to certain viral infections, SAP and XIAP demonstrate no obvious structural or functional similarity, are not coordinately regulated with respect to their expression, and do not appear to directly interact. In this review, we describe the genetic, clinical, and immunopathologic features of these 2 disorders and discuss current diagnostic and therapeutic strategies.
2245	Clinical significance of clonality and Epstein-Barr virus infection in adult patients with hemophagocytic lymphohistiocytosis.	20652965	We assessed the clinical significance of T or B cell clonality and Epstein-Barr virus (EBV) infection in adult patients with hemophagocytic lymphohistiocytosis (HLH) to identify factors related to prognosis. A total of 30 adult patients with diagnosed HLH were included in the study. In all patients, EBV-DNA in peripheral blood was examined by quantitative real-time polymerase chain reaction and bone marrow cells were examined for clonal rearrangement of T cell receptor gamma(TCRG) and immunoglobulin heavy chain (IGH) genes. TCRG clones were detected in 10 patients (33.3%) and IGH clones were detected in 8 patients (26.7%). We found no correlation between clonality and patient outcome. The patients less than 1,000 copies (mL)21 of EBVDNA showed a significantly higher clinical response (P 5 0.008) and longer overall survival (P 5 0.01) than those with high viral load of EBV-DNA. Our results suggest that TCRG and IGH rearrangement do not have any clinical significance in adult patients with HLH, but that high viral load of EBV-DNA may be a risk factor for poor outcomes. In HLH, high viral load of EBV-DNA should thus suggest a prompt approach with aggressive therapeutic interventions.
2246	Beta-blocker therapy and hemophagocytic lymphohistiocytosis: a case report.	20634935	Objective. The aim of this paper is to describe a fatal case of hemophagocytic lymphohistiocytosis (HLH) in a patient with severe heart failure, who was treated with low-dose propranolol. Patient and Interventions. We report on a 7-month-old boy with Downs syndrome who was born with an unbalanced, left dominant atrioventricular septal defect and aortic coarctation. Despite coarctation repair and pulmonary artery banding he developed intractable heart failure and fever of unknown origin. Since he remained in heart failure he received a trial of low-dose propranolol to stabilize his cardiopulmonary status, which resulted in unexpected immunomodulatory effects. Measurements and Main Result. Immunoactivation was evidenced by high concentrations of procalcitonin, soluble CD 25, tumor necrosis factor alpha, and interleukin 6 and 8. Propranolol resulting in hepatic compromise as indicated by high lactate dehydrogenase and alanine aminotransferase levels. A therapeutic switch from propranolol to the beta(1)-receptor blocker metoprolol appeared to be instrumental in hemodynamic improvement and allowed discharge from hospital. However, the infant ultimately died from secondary inflammatory reactivation and intractable pulmonary obstructive disease. The autopsy results revealed HLH. Conclusion. Our case describes HLH secondary to heart failure and Downs syndrome. In this highly activated inflammatory state the beneficial hemodynamic effects of propranolol may be accompanied by immunomodulatory effects and the risk of acute liver failure. HLH occurs with a distinct pathophysiology, and specific treatment might be mandatory to increase the chance of survival.
2247	Early and rapid detection of X-linked lymphoproliferative syndrome with SH2D1A mutations by flow cytometry.	20632414	X-linked lymphoproliferative syndrome (XLP) is a rare immunodeficiency with extreme vulnerability to Epstein-Barr virus (EBV) infection. It presents with fatal infectious mononucleosis, lymphoproliferative disorder, or dysgammaglobulinemia. The majority of affected males have mutations in the SH2D1A/SLAM-associated protein (SAP) gene. We previously generated an antihuman SAP monoclonal antibody (KST-3) for a flow cytometric assay and described the activation of T cells to be necessary for the flow cytometric assessment of the SAP expression using an FITC-conjugated secondary antibody.Between 2005 and 2008, we recruited 23 male patients with suspected XLP, including mainly EBV-associated hemophagocytic lymphohistiocytosis (HLH), and attempted to evaluate SAP expression in fresh lymphoid cells using Alexa Fluor 488-conjugated secondary antibody instead of an FITC-conjugated one.The method demonstrated that SAP was intensely expressed in CD8(+) T cells and NK cells in normal fresh blood samples, thus suggesting the possible rapid identification of individuals with SAP deficiency. SH2D1A mutations were identified in six patients with SAP deficiency, but not in patients with normal SAP expression.The outcomes from this trial were verified by a flow cytometric assay using KST-3 and Alexa Fluor 488 secondary antibody. Based on the demonstration SAP deficiency in patients with suspected XLP, including mainly EBV-associated HLH, this approach could serve as a method for the early and rapid detection of patients with XLP-1.
2248	Local insulin-like growth factor I expression is essential for Purkinje neuron survival at birth.	20596079	IGF1, an anabolic and neuroprotective factor, promotes neuronal survival by blocking apoptosis. It is released into the bloodstream by the liver, or synthesized locally by muscles and neural cells, acting in an autocrine or paracrine fashion. Intriguingly, genetic studies conducted in invertebrate and murine models also suggest that an excess of IGF1 signaling may trigger neurodegeneration. This emphasizes the importance of gaining a better understanding of the mechanisms controlling IGF1 regulation and gene transcription. In the cerebellum, Igf1 expression is activated just before birth in a subset of Purkinje cells (PCs). Mice carrying a null mutation for HLH transcription factor EBF2 feature PC apoptosis at birth. We show that Igf1 is sharply downregulated in Ebf2 null PCs starting before the onset of PC death. In vitro, EBF2 binds a conserved distal Igf1 promoter region. The pro-survival PI3K signaling pathway is strongly inhibited in mutant cerebella. Finally, Ebf2 null organotypic cultures respond to IGF1 treatment by inhibiting PC apoptosis. Consistently, wild type slices treated with an IGF1 competitor feature a sharp increase in PC death. Our findings reveal that IGF1 is required for PC survival in the neonatal cerebellum, and identify a new mechanism regulating its local production in the CNS.
2249	Development of secondary T-cell acute lymphoblastic leukemia in a child with hemophagocytic lymphohistiocytosis.	20589661	Hemophagocytic lymphohistiocytosis (HLH) is a severe life-threatening disorder, characterized by hyperactivation of macrophages. A 12-year-old female was referred to our center; the diagnosis of HLH was made for the patient and immunosuppressive regimen was started. After a 2-year follow-up, the patient developed secondary T-cell acute lymphoblastic leukemia (T-ALL), confirmed by flow cytometric studies. Treatment was started based on T-ALL protocol, but the patient died because of relapse and sepsis. This case highlights the issue of secondary malignancy following HLH and demonstrates the need for continued follow-up in such patients.
2250	Hidden in plain sight: macrophage activation syndrome complicating Adult Onset Still's Disease.	23875527	Hemophagocytic Lymphystiocytosis is a rare and fatal complication of rheumatic diseases, particularly Adult Onset Still's Disease (AOSD). It may be precipitated with immunosuppressive drugs and with viral and bacterial infections. A diagnosis depends on a high index of suspicion associated to certain clinical manifestations (fever, rash, Splemomegaly, any cytology blood dyscrasia, hipertrigliceridemia, hiperfibrinogenemia, and others), as well as pathologic evidence of hemophagocitosis from bone marrow biopsy or tissue samples of affected organs. Therapy consists of high dose corticosteroids and immunosuppressive drugs. We present a 42 year old woman with AOSD in remission who developed HLH in spite of receiving therapy with high dose steroids and immunosuppressive drugs. She had 2 negative bone marrow aspirates. Evidence of Hemophagocytosis was detected in both bone marrow biopsies. Timely evaluation and recognition of the signs and symptoms of HLH is crucial for the prompt management and a decrease in the mortality associated with this disease.
2251	Review of haemophagocytic lymphohistiocytosis.	20584844	Haemophagocytic lymphohistiocytosis (HLH) describes a clinical syndrome of hyperinflammation resulting in an uncontrolled and ineffective immune response. It may develop subsequent to a number of recognised genetic mutations or in association with infection, malignancy, autoinflammatory or metabolic conditions. Even with the published diagnostic criteria it can be difficult to make the diagnosis of HLH. Patients presenting acutely to the general paediatrician or paediatric intensivist with a clinical picture of likely sepsis, ie fever, laboratory evidence of inflammatory response, coagulopathy and thrombocytopaenia should be appropriately investigated and managed for sepsis, but the possible diagnosis of HLH should be borne in mind, particularly in the child who deteriorates despite maximal therapy. This review discusses current knowledge on the classification, diagnosis and management of primary and secondary HLH, and suggests a pathway of investigation for the paediatrician faced with a potential case.
2252	Upregulation of Id1 by Epstein-Barr virus-encoded LMP1 confers resistance to TGFbeta-mediated growth inhibition.	20565867	Epstein-Barr virus (EBV)-encoded LMP1 protein is commonly expressed in nasopharyngeal carcinoma (NPC). LMP1 is a prime candidate for driving tumourigenesis given its ability to activate multiple signalling pathways and to alter the expression and activity of variety of downstream targets. Resistance to TGFbeta-mediated cytostasis is one of the growth transforming effects of LMP1. Of the downstream targets manipulated by LMP1, the induction of Id1 and inactivation of Foxo3a appear particularly relevant to LMP1-mediated effects. Id1, a HLH protein is implicated in cell transformation and plays a role in cell proliferation, whilst Foxo3a, a transcription factor controls cell integrity and homeostasis by regulating apoptosis. The mechanism(s) by which LMP1 induces these effects have not been fully characterised.In this study, we demonstrate that the ability of LMP1 to induce the phosphorylation and inactivation of Foxo3a is linked to the upregulation of Id1. Furthermore, we show that the induction of Id1 is essential for the transforming function of LMP1 as over-expression of Id1 increases cell proliferation, attenuates TGFbeta-SMAD-mediated transcription and renders cells refractory to TGFbeta-mediated cytostasis. Id1 silencing in LMP1-expressing epithelial cells abolishes the inhibitory effect of LMP1 on TGFbeta-mediated cell growth arrest and reduces the ability of LMP1 to attenuate SMAD transcriptional activity. In response to TGFbeta stimulation, LMP1 does not abolish SMAD phosphorylation but inhibits p21 protein expression. In addition, we found the induction of Id1 in LMP1-expressing cells upon stimulation by TGFbeta. We provide evidence that LMP1 suppresses the transcriptional repressor ATF3, possibly leading to the TGFbeta-induced Id1 upregulation.The current data provide novel information regarding the mechanisms by which LMP1 suppresses TGFbeta-induced cytostasis, highlighting the importance of Id1 in LMP1 mediated cell transformation.
2253	Spectrum of clinical presentations in familial hemophagocytic lymphohistiocytosis type 5 patients with mutations in STXBP2.	20558610	Hemophagocytic lymphohistiocytosis (HLH) is an often-fatal hyperinflammatory syndrome characterized by fever, hepatosplenomegaly, cytopenia, and in some cases hemophagocytosis. Here, we describe the mutation analysis, clinical presentation, and functional analysis of natural killer (NK) cells in patients with mutations in STXBP2 encoding Munc18-2, recently associated with familial HLH type 5. The disease severity among 11 persons studied here was highly variable and, accordingly, age at diagnosis ranged from 2 months to 17 years. Remarkably, in addition to typical manifestations of familial HLH (FHL), the clinical findings included colitis, bleeding disorders, and hypogammaglobulinemia in approximately one-third of the patients. Laboratory analysis revealed impairment of NK-cell degranulation and cytotoxic capacity. Interleukin-2 stimulation of lymphocytes in vitro rescued the NK cell-associated functional defects. In conclusion, familial HLH type 5 is associated with a spectrum of clinical symptoms, which may be a reflection of impaired expression and function of Munc18-2 also in cells other than cytotoxic lymphocytes. Mutations in STXBP2 should thus also be considered in patients with clinical manifestations other than those typically associated with HLH.
2254	Inherited defects in lymphocyte cytotoxic activity.	20536552	The granule-dependent cytotoxic activity of lymphocytes plays a critical role in the defense against virally infected cells and tumor cells. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytic syndrome (HLH) that occurs in mice and humans with genetically determined impaired lymphocyte cytotoxic function. HLH manifests as the occurrence of uncontrolled activation of T lymphocytes and macrophages infiltrating multiple organs. In this review, we focus on recent advances in the characterization of effectors regulating the release of cytotoxic granules, and on the role of this cytotoxic pathway in lymphocyte homeostasis and immune surveillance. Analysis of the mechanisms leading to the occurrence of hemophagocytic syndrome designates gamma-interferon as an attractive therapeutic target to downregulate uncontrolled macrophage activation, which sustains clinical and biological features of HLH.
2255	Critical care management of patients with hemophagocytic lymphohistiocytosis.	20532477	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition associated with multiple organ dysfunction. We sought to describe ICU management and outcomes in HLH patients meeting HLH-2004 criteria and to identify determinants of mortality.Retrospective study between January 1998 and January 2009.Medical ICU of a teaching hospital.Among the 72 patients fulfilling the HLH-2004 criteria, we report the 56 patients with complete follow-up and no missing data.None.Clinical and laboratory data were abstracted from the medical records. Median SOFA score at admission was 6.5 (IQR, 4-8). At ICU admission, the number of HLH-2004 criteria was 6 (5-7). Sixty-six precipitating factors were found in 52 patients and consisted of 43 tumoral causes (8 Castleman's diseases, 18 B cell lymphoma and 17 various malignancies), 13 non-viral infections and 10 viral infections. Underlying immune deficiency was present in 38 (67.8%) patients. Etoposide was used in 45 patients, corticosteroids in 31 and intravenous immunoglobulins in 3. Mechanical ventilation was required in 32 patients, vasoactive agents in 30 and renal replacement therapy in 19. Hospital mortality was 29/56 patients. By multivariate analysis, factors associated with increased hospital death were shock at ICU admission [OR, 4.33; 95% confidence interval (95% CI), 1.11-16.90; P = 0.03] and platelet count <30 g/l (OR, 4.75; 95% CI, 1.20-18.81; P = 0.02). B cell lymphoma [odds ratio (OR), 0.17; 95% CI, 0.04-0.80; P = 0.02] and Castleman's disease (OR, 0.11; 95% CI, 0.02-0.90; P = 0.04) were associated with increased hospital survival.Aggressive supportive care combined with specific treatment of the precipitating factor can produce meaningful survival in patients with HLH responsible for multiple organ failures. Survival is highest in patients with HLH related to Castleman's disease or B cell lymphoma.
2256	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in adults characterized by high viral genome load within circulating natural killer cells.	20504238	Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) is a rare and aggressive disease usually encountered in the context of primary EBV infection. In most analyzed cases, EBV has been found predominantly in T cells. We describe the novel finding of high EBV genome numbers within circulating natural killer cells in adult patients with EBV-HLH.
2257	XIAP deficiency: a unique primary immunodeficiency best classified as X-linked familial hemophagocytic lymphohistiocytosis and not as X-linked lymphoproliferative disease.	20489057	X-linked inhibitor of apoptosis (XIAP) deficiency, caused by BIRC4 mutations, is described to cause X-linked lymphoproliferative disease (XLP) phenotypes. However, compared with XLP caused by SLAM-Associated Protein deficiency (SH2D1A mutation), XIAP deficiency was originally observed to be associated with a high incidence of hemophagocytic lymphohistiocytosis (HLH) and a lack of lymphoma, suggesting that classification of XIAP deficiency as a cause of XLP may not be entirely accurate. To further characterize XIAP deficiency, we reviewed our experience with 10 patients from 8 unrelated families with BIRC4 mutations. Nine of 10 patients developed HLH by 8 years of age. Most patients presented in infancy, and recurrent HLH was common. There were no cases of lymphoma. Lymphocyte defects thought to contribute to HLH development in SLAM-Associated Protein deficiency were not observed in XIAP deficiency. We conclude that XIAP deficiency is a unique primary immunodeficiency that is more appropriately classified as X-linked familial hemophagocytic lymphohistiocytosis.
2258	STX11 mutations and clinical phenotypes of familial hemophagocytic lymphohistiocytosis in North America.	20486178	Mutations in STX11 are responsible for Familial Hemophagocytic Lymphohistiocytosis (FHLH) type 4, a rare primary immunodeficiency which has previously been observed only in patients of Kurdish, Turkish, and Lebanese ethnic background.We reviewed our experience with STX11 mutations among North American patients and studied the impact of patient mutations upon syntaxin 11 expression and NK cell function.Between 2007 and 2008, 243 patients with HLH (lacking disease-causing mutations in PRF1 and UNC13D) were referred for STX11 mutational analysis. We observed 1 novel homozygous nonsense mutation, 73 G > T (E25X), occurring in Hispanic siblings, and 2 novel biallelic heterozygous missense mutations, 106G > C (E36Q) and 616G > A (E206K), occurring in 1 Caucasian patient. The N-terminal nonsense mutation resulted in absence of detectable syntaxin 11 and abrogation of in vitro NK cell degranulation and function, while the biallelic heterozygous missense mutations resulted in detectable mutated syntaxin 11 and preservation of in vitro NK cell degranulation and cytotoxicity. The two sibling patients with the nonsense mutations presented with HLH during infancy, whereas the patient with biallelic heterozygous missense mutations presented at 5 years of age.We conclude that mutations in STX11 are responsible for HLH in approximately 1% of North American patients and can cause variable defects in syntaxin 11 expression and function with resultant impact on clinical phenotype.
2259	Synthesis and conformation of an analog of the helix-loop-helix domain of the Id1 protein containing the O-acyl iso-prolyl-seryl switch motif.	20474042	Synthetic peptides reproducing the helix-loop-helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self-association. Recently, we have shown that the replacement of the dipeptide Val-Ser at the loop-helix-2 junction with the corresponding O-acyl iso-dipeptide leads to a completely unfolded state that only refolds after intramolecular O --> N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro-Ser motif at the helix-1-loop junction with the corresponding O-acyl iso-dipeptide. This analog has been successfully synthesized by solid-phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O-acyl iso-peptide before its conversion into the native form by O --> N acyl shift. These results show that the loop-helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O-acyl iso-Pro-Ser/Thr units are synthetically accessible by Fmoc chemistry.
2260	Distinct cytokine profiles of systemic-onset juvenile idiopathic arthritis-associated macrophage activation syndrome with particular emphasis on the role of interleukin-18 in its pathogenesis.	20472718	To compare the pro-inflammatory cytokine profiles and the cytokine kinetics in patients with secondary macrophage activation syndrome (MAS) due to systemic-onset juvenile idiopathic arthritis (s-JIA) and in both active and inactive disease states of s-JIA (but no MAS), with those demonstrated in EBV-induced haemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD), and to investigate the significance of IL-18 in the pathogenesis of s-JIA.Five patients with MAS complicating s-JIA (MAS/s-JIA), 10 with HLH due to EBV infection (EBV-HLH), 22 with KD and 28 healthy controls were analysed. Cytokine concentrations (IL-18, IL-6, neopterin and TNF-alpha receptor Types I and II) were quantified in serum by ELISA. Results were compared with clinical features of MAS/s-JIA, including ferritin concentrations.Serum IL-18 concentrations in MAS/s-JIA patients were significantly higher than those in EBV-HLH or KD patients (P < 0.05). Serum IL-6 concentrations in KD patients were significantly higher than those in EBV-HLH or MAS/s-JIA patients. Serum neopterin concentrations in EBV-HLH patients were significantly higher than those in MAS/s-JIA or KD patients. Serum IL-18 correlated positively with the following measurements of disease activity: CRP, ferritin, lactate dehydrogenase and other cytokines (P < 0.05). Serum concentrations of IL-18 in s-JIA patients remained elevated in the inactive phase of disease, whereas clinical parameters and other cytokines normalized.IL-18 may be an important mediator in s-JIA. Although serum Il-18 concentrations correlated with markers of the disease activity, IL-18 concentrations remained elevated even when other markers of disease activity normalized. Serum IL-18 concentration may be a promising indicator of the disease activity. The cytokine release pattern in MAS/HLH is different among patients with different aetiologies. Monitoring the cytokine profile, including IL-18, may be useful for differentiation of MAS/HLH and evaluation of disease activity in s-JIA.
2261	[Aggressive NK-cell leukemia with sustained relapse-free survival after allogeneic peripheral blood stem cell transplantation].	20467222	A 46-year-old Japanese man was admitted to our hospital because of prolonged fever. Laboratory examination demonstrated leukopenia, thrombocytopenia, marked liver dysfunction, and elevation of serum ferritin. A bone marrow examination showed several hemophagocytic macrophages, and a diagnosis of hemophagocytic syndrome was made. He was treated using HLH-94 protocol, and his clinical symptoms and laboratory data were rapidly improved. After 5 weeks, fever and liver dysfunction reappeared. A repeat bone marrow examination demonstrated that 28.4% of marrow nucleated cells were atypical lymphocytes, which were positive for CD2, CD7, CD16, CD56, and HLA-DR. Clonality of these proliferating NK cells was confirmed by an analysis of EB virus terminal repeat sequence and cytogenetic analysis, and final diagnosis of aggressive NK-cell leukemia was made. After induction chemotherapy consisting of dexamethasone, etoposide, ifosfamide, and L-asparaginase, the patient achieved partial remission. He received allogeneic peripheral blood stem cell transplantation from his one locus mismatched son, and is alive with no evidence of disease 20 months after transplantation.
2262	Characterization of variants in the promoter of BZLF1 gene of EBV in nonmalignant EBV-associated diseases in Chinese children.	20459737	Diseases associated with Epstein-Barr virus (EBV) infections, such as infectious mononucleosis (IM), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV) are not rare in Chinese children. The association of type 1 or type 2 EBV and variants of the EBV BZLF1 promoter zone (Zp) with these diseases is unclear.The objective of this study was to investigate the relationship between EBV genotypes (Zp variants and EBV type 1 and 2) and the clinical phenotypes of EBV-associated diseases in Chinese children. The Zp region was directly sequenced in 206 EBV-positive DNA samples from the blood of patients with IM, EBV-HLH, CAEBV, and healthy controls. Type 1 or type 2 EBV was examined by PCR for EBNA2 and EBNA3C subtypes. Four polymorphic Zp variants were identified: Zp-P, Zp-V3, Zp-P4 and Zp-V1, a new variant. The Zp-V3 variant was significantly associated with CAEBV (P <or= 0.01). The frequency of co-infection with Zp variants was higher in patients with CAEBV and EBV-HLH, compared with IM and healthy controls, mostly as Zp-P+V3 co-infection. Type 1 EBV was predominant in all categories (81.3-95%) and there was no significant difference in the frequency of the EBV types 1 and 2 in different categories (P > 0.05).Type 1 EBV and BZLF1 Zp-P of EBV were the predominant genotypes in nonmalignant EBV associated diseases in Chinese children and Zp-V3 variant may correlates with the developing of severe EBV infection diseases, such as CAEBV and EBV-HLH.
2263	Angeborene hämophagozytische Lymphohistiozytose (HLH).	20458667	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune disorder characterized by uncontrolled lymphocyte- and macrophage-activation. The resulting hypercytokinemia and cell infiltration of organs lead to the clinical and laboratory features of HLH. Viral infections and other triggers can induce both, inherited and acquired forms of HLH. Disease-causing mutations in the genes encoding perforin (PRF1, FHL2), munc13-4 (UNC13D, FHL3), syntaxin 11 (STX11, FHL4), and munc18-2 (UNC18-2/STXBP2, FHL5) have been previously identified in Familial Hemophagocyic Lymphohistiocytosis (FHL), whereas mutation in RAB27A and LYST account for Griscelli syndome type 2 and Chediak-Higashi syndrome, respectively. These genes all encode proteins which are involved in the cytotoxic activity of lymphocytes. The inability of activated cytotoxic cells to clear antigen-presenting targets results in sustained immune stimulation, likely accounting for the unremitting polyclonal CD8 T-cell activation and hyperimmune reaction which characterizes FHL. Treatment of HLH consists of elimination of the trigger and immunosuppressive treatment in order to induce remission from the uncontrolled inflammation. Allogeneic hematopoietic stem cell transplantation can be indicated in the inherited forms of HLH.
2264	Evolution of a genomic regulatory domain: the role of gene co-option and gene duplication in the Enhancer of split complex.	20458100	The Drosophila Enhancer of split complex [E(spl)-C] is a remarkable complex of genes many of which are effectors or modulators of Notch signaling. The complex contains different classes of genes including four bearded genes and seven basic helix-loop-helix (bHLH) genes. We examined the evolution of this unusual complex by identifying bearded and bHLH genes in the genome sequences of Arthropods. We find that a four-gene E(spl)-C, containing three bHLH genes and one bearded gene, is an ancient component of the genomes of Crustacea and Insects. The complex is well conserved in insects but is highly modified in Drosophila, where two of the ancestral genes of the complex are missing, and the remaining two have been duplicated multiple times. Through examining the expression of E(spl)-C genes in honeybees, aphids, and Drosophila, we determined that the complex ancestrally had a role in Notch signaling. The expression patterns of genes found inserted into the complex in some insects, or that of ancestral E(spl)-C genes that have moved out of the complex, imply that the E(spl)-C is a genomic domain regulated as a whole by Notch signaling. We hypothesize that the E(spl)-C is a Notch-regulated genomic domain conserved in Arthropod genomes for around 420 million years. We discuss the consequence of this conserved domain for the recruitment of novel genes into the Notch signaling cascade.
2265	Clinical significance of cloned expansion and CD5 down-regulation in Epstein-Barr Virus (EBV)-infected CD8+ T lymphocytes in EBV-associated hemophagocytic lymphohistiocytosis.	20443735	Epstein-Barr virus (EBV) is the pathogen that most commonly triggers infection-associated hemophagocytic lymphohistiocytosis (HLH) and ectopically infects CD8(+) T cells in EBV-associated HLH (EBV-HLH). We recently described an EBV-HLH patient who had a clonally expanded population of EBV-infected CD8(+) T cells with CD5 down-regulation. To determine whether this finding could serve as a useful marker for EBV-HLH, we investigated 5 additional patients. We found a significant increase in the subpopulation of CD8(+) T cells with CD5 down-regulation and bright human leukocyte antigen (HLA)-DR expression in all patients with EBV-HLH but not in patients with infectious mononucleosis or in control subjects. Such T cells were frequently found to be larger cells that stained positive for a specific T cell receptor VB. We also demonstrated that those cells were the major cellular target of EBV, and their numbers progressively declined in parallel with the serum ferritin levels. All together, our findings reveal the immunophenotypic characteristics of EBV-infected CD8(+) T cells and may provide a valuable tool for the diagnosis of EBV-HLH.
2266	Structure of the EF-hand domain of polycystin-2 suggests a mechanism for Ca2+-dependent regulation of polycystin-2 channel activity.	20439752	The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca(2+)-permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720-797), a flexible linker (798-827), and an oligomeric coiled coil domain (828-895). We found that PC2-EF binds Ca(2+) at a single site and undergoes Ca(2+)-dependent conformational changes, suggesting it is an essential element of Ca(2+)-sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca(2+)-bound PC2-EF. Human PC2-EF contains a divergent non-Ca(2+)-binding helix-loop-helix (HLH) motif packed against a canonical Ca(2+)-binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R(1) and R(2) relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca(2+)-bound states, consistent with a role for PC2-EF as a Ca(2+)-sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca(2+)-dependent protein interactions. We propose that Ca(2+)-sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca(2+) dependence of PC2 channel activity by PC2-EF.
2267	Structure of hemocyanin from garden snail Helix lucorum.	20433940	Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as beta-HlH, alpha(D)-HlH and alpha(N)-HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (beta-HlH) and 1079 kDa (alpha(D)-HlH, and alpha(N)-HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl(2). All isoforms were reassociated into didecamers and tubules with different length, but in contrast to alpha(D)-HlH isoform, longer tubules were observed in beta-HlH. Moreover the structure of beta-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes.
2268	Neonatal hemophagocytic lymphohistiocytosis--case report.	20432763	Hemophagocytic lymphohystiocytosis (HLH) represents a severe hyperinflammatory condition with the cardinal symptoms prolonged fever, hepatosplenomegaly, and cytopenias. The most prominent histopathological feature of HLH is an accumulation of activated T lymphocytes and macrophages predominantly in lymphoid tissues. Although it can occur in all age groups, neonatal-onset HLH is very rare. We report on a case of HLH presenting with anemia and respiratory distress at birth. Several weeks prior to diagnosis the symptoms were attributed to a systemic infection. The child developed typical clinical and laboratory findings, and was diagnosed with HLH according to HLH-2004 guidelines. Chemo-immunotherapy was initiated, but after a temporary control of the disease the patient succumbed to rapidly progressive HLH. Post-mortem, extensive hemophagocytosis was found in multiple organs. No specific genetic defect was identified. HLH is potentially fatal childhood disease. It is important for pediatricians to be able to early identify this disorder and commence the therapy before overwhelming disease activity develops.
2269	[Clinical analysis and follow-up study of Epstein-Barr virus associated-hemophagocytic lymphohistiocytosis in childhood].	20426937	To identify the clinical characteristics of and to explore the prognostic factors influencing mortality in children with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH).A retrospective study was conducted on 62 pediatric patients with EBV-HLH who were admitted to our hospital between 2003 and 2008. All their medical records were reviewed and analyzed. For each patient, demographic, clinical and laboratory data, genetic findings and outcome information were collected. The patients were divided into two groups: deceased or survived based on the follow-up results. Comparative analysis of the data was done by using independent-samples t test and Logistic multiple and univariate regression.(1) Among the 62 EBV-HLH patients, 36 were male and 26 were female. The age of onset ranged from 2 months to 14 years and most of the patients were between 1 and 3 years of age. EBV-HLH occurred mainly in the setting of reactivation (61.3%). (2) All patients exhibited persistent or intermittent fever and cytopenia >/= 2 cell lines. Most of the patients presented with hepatomegaly (83.9%), splenomegaly (72.6%) and lymphadenopathy (69.4%). The main laboratory features showed an elevation of serum ferritin and aminotransferase levels. A reduction in serum albumin was observed and exhibited coagulopathy with hypofibrinogenemia and hypertriglyceridemia in most of the patients. Forty-eight of patients had hemophagocytosis in bone marrow at diagnosis of EBV-HLH. The serum EBV DNA level in 14 of 31 patients with EBV-HLH was in the range of 5.12 x 10(2) - 7.69 x 10(7) copies/ml with a mean value of 10(3.9) copies/ml. (3) Three heterozygous mutations in coding region were found, which resulted in amino acid change (C102F, S108N and T450M) in 3 patients. One patient had compound heterozygous mutations (S108N and T450M) in the PRF1 gene as the background defect and documented familial HLH type 2 (FHL2). (4) During the observational period, 35 of 57 patients (61.4%) died 3 months to 3 years after the onset, while 21 of whom died despite aggressive polychemotherapy, 15 of whom died within 2 months after hospitalization. The deceased patients were more likely to have lower albumin level and more prolonged activated partial thromboplastin time than the survived patients (P < 0.05 for all comparisons). Multivariate Logistic regression analysis revealed that duration of illness >/= 1 month, non-chemotherapy, albumin level < / = 25 microg/L and internal organs hemorrhage were related with the prognosis significantly (P < 0.05 for all comparisons).This study revealed that EBV-HLH infection in pediatric patients had severe clinical courses and prognosis was poor and the majority of cases underwent EBV reactivation. The early diagnosis, prompt and proper chemotherapy can improve the survival rate. The duration of illness >/= 1 month, non-chemotherapy, decreases in albumin and internal organs hemorrhage were the risk factors related to mortality in children with EBV-HLH.
2270	Myc interacts with Max and Miz1 to repress C/EBPdelta promoter activity and gene expression.	20426839	"Loss of function" alterations in CCAAT/Enhancer Binding Proteindelta (C/EBPdelta) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPdelta gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPdelta exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing. We previously reported that C/EBPdelta expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPalpha, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPdelta expression.Myc represses C/EBPdelta promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPdelta promoter in proliferating cells, when C/EBPdelta expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPdelta promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPdelta promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPdelta promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPdelta promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPdelta expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPdelta promoter activity.Myc represses C/EBPdelta expression by associating with the C/EBPdelta proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPdelta promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPdelta, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.
2271	[Clinical analysis on 28 patients with hemophagocytic lymphohistiocytosis syndrome].	20416189	In order to profoundly understand the clinical and laboratorial characteristics and inducing factors of hemophagocytic lymphohistiocytosis syndrome (HLH), 28 HLH patients received from 2004 to 2009 years in our hospital were analyzed retrospectively. The results indicated that all of the patients had a history with prolonged fever (more than 1 week), pancytopenia, hepatosplenomegaly, elevated ferritin level, hypofibrinogen, and hemophagocytosis in bone marrow. HLH was the first characteristic sign of malignant lymphoma in 9 patients; 1 patient had a clinical manifestation similar to fulminant hepatic failure; severe psycho-abnormality occurred in 1 HLH patient and pronounced hemophagocytosis were detected in his cerebrospinal fluid; 1 patient was eventually diagnosed as having HLH by the findings in a lymph node biopsy showing obvious hemophagocytosis. Additionally, the analysis of underlying factors in 28 patients with HLH indicated 11 patients with EB virus-associated HLH, 11 with lymphoma-associated HLH, 2 with Leishmania-associated HLH, and 3 with autoimmune disease-associated HLH. It is concluded that HLH disease is characterised with high heterogenicity in both clinical features and inducing factors; in addition, the patients from a pasturing area should be paid attention to parasite infection such as leishmania.
2272	Recombinant human follicular stimulating hormone and recombinant human luteinizing hormone in a 2:1 ratio combination. Pharmacological characteristics and clinical applications.	20415593	A new fixed combination of recombinant human follicle stimulating hormone (r-hFSH) and recombinant human luteinizing hormone (r-hLH) at a 2:1 ratio has been recently developed to induce ovulation in patients with hypogonadotrophic hypogonadism. Whether or not this compound is useful for controlled ovarian stimulation for in vitro fertilization is still a matter of debate.Description of pharmacological and clinical aspects of this new product, through the analysis of the Phase I, II and II trials and post-marketing clinical randomized trials, performed since the initial assays in 1998 to nowadays.After reading this review the reader will understand the pharmacological aspects of this new compound, in terms of efficacy and safety, and will have an update of the potential role of r-LH administration in controlled ovarian stimulation for in vitro fertilization.The 2:1 combination of r-hFSH and r-hLH has been seen as an optimum preparation in terms of safety and clinical efficacy in hypogonatrophic hypogonadism patients. Its use in ovarian stimulation for IVF remains controversial, as the target population that may receive a benefit of it is not well defined.
2273	The spatial dynamics of tissue-specific promoters during C. elegans development.	20395364	To understand whether the spatial organization of the genome reflects the cell's differentiated state, we examined whether genes assume specific subnuclear positions during Caenorhabditis elegans development. Monitoring the radial position of developmentally controlled promoters in embryos and larval tissues, we found that small integrated arrays bearing three different tissue-specific promoters have no preferential position in nuclei of undifferentiated embryos. However, in differentiated cells, they shifted stably toward the nuclear lumen when activated, or to the nuclear envelope when silent. In contrast, large integrated arrays bearing the same promoters became heterochromatic and nuclear envelope-bound in embryos. Tissue-specific activation of promoters in these large arrays in larvae overrode the perinuclear anchorage. For transgenes that carry both active and inactive promoters, the inward shift of the active promoter was dominant. Finally, induction of master regulator HLH-1 prematurely induced internalization of a muscle-specific promoter array in embryos. Fluorescence in situ hybridization confirmed analogous results for the endogenous endoderm-determining gene pha-4. We propose that, in differentiated cells, subnuclear organization arises from the selective positioning of active and inactive developmentally regulated promoters. We characterize two forces that lead to tissue-specific subnuclear organization of the worm genome: large repeat-induced heterochromatin, which associates with the nuclear envelope like repressed genes in differentiated cells, and tissue-specific promoters that shift inward in a dominant fashion over silent promoters, when they are activated.
2274	Analysis of human luteinizing hormone and human chorionic gonadotropin preparations of different origins by reversed-phase high-performance liquid chromatography.	20395104	Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their alpha- and beta-subunits migrated with significantly different retention times (t(R)) in the following order of increasing hydrophobicity: alpha-hCG<alpha-hLH<hCG<hLH<beta-hCG<beta-hLH. Under these conditions, the main peak of three hCG preparations ran about 4% faster than the average t(R) (38.35+/-0.42 min; RSD=1.1%) of four hLH preparations. Four heterogeneous urinary products were also analyzed, hLH, hFSH and hCG peaks being identified. Quantitative analysis was validated for the homogeneous preparations and a highly linear dose-response curve (r=0.99998; p<0.0001; n=20) used to assess the accuracy, precision and sensitivity of the analysis. Quantification of the different gonadotropins in the heterogeneous preparations was also carried out, but with limitations in accuracy.
2275	Primary and secondary hemophagocytic lymphohistiocytosis: clinical features, pathogenesis and therapy.	20383897	Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyperinflammatory syndrome with prolonged high fever, hepatosplenomegaly and characteristic laboratory findings. HLH may be inherited (primary) or may be secondary to any severe infection, malignancy or rheumatologic condition. The last several years have witnessed an explosion in our understanding of HLH. Of the inherited causes for which the underlying genetic cause is known, most involve abnormalities of proteins important in the exocytosis cytolytic pathway, whereby perforin and granzymes are delivered to a target cell to induce apoptosis. The exact mechanisms underlying this process remain unclear. However, when a known genetic defect is not present, the diagnosis of HLH is still made on a constellation of clinical features and good clinical judgment. Rapid diagnosis is crucial, as early therapy with immunosuppressive agents and/or proapoptotic chemotherapy can be life-saving. This article examines recent advances in our understanding of the pathophysiology, clinical features, diagnosis, etiology and treatment of HLH, as well as the challenges that lie ahead.
2276	Microbiological findings and treatment of EBV-associated hemophagocytic lymphohistiocytosis: a case report.	20383798	Epstein-Barr virus (EBV) is the major triggering factor for hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH). In patients with EBV-HLH, the EBV-infected T cells or natural killer cells are mostly mono- or oligoclonally proliferating, whereby hypercytokinemia plays a major role and causes hemophagocytosis, cellular damage, and dysfunction of various organs. This report describes the detection and treatment of EBV-associated HLH in the case of a 17-year-old male. Serum samples and skin swabs were tested for the presence of viral DNA using real-time PCR techniques. To confirm the molecular biological tests, electron microscopy was also performed. EBV DNA was detected with real-time PCR in both blood samples and skin swabs. The level of viral DNA constantly decreased during the applied therapy. The presence of the virus in the skin was confirmed by the appearance of herpes virus-like particles detected by electron microscopy in fluid taken from skin ulcerations. The results show that in terms of treatment, special therapeutic measures are required to control the cytokine storm generated by EBV and to suppress proliferating EBV genome-containing cells because the clinical course is often fulminate and results in a poor outcome. Therefore the potential of chemotherapy with a combination of steroids, etoposide, and cyclosporine to control HLH was assessed in the adolescent, who met the stringent diagnostic criteria for this reactive disorder of the mononuclear phagocyte system.
2277	Visceral leishmaniasis and macrophagic activation syndrome in a patient with rheumatoid arthritis under treatment with adalimumab.	20378385	Visceral leishmaniasis is a protozoan infection usually asymptomatic, but can progress to fatal disease in immunocompromised hosts, especially in HIV patients. Visceral leishmaniasis is rare among patients under immunosuppressive therapies, and even more among patients under anti-TNF-alpha treatment, where only four cases have been described.1) To describe a patient with rheumatoid arthritis receiving adalimumab who developed fever, pancytopenia, splenomegaly, and extreme hyperferritinemia. 2) To perform a review of the published cases of visceral leishmaniasis and anti-TNF-alpha therapy, and cases of coexisting leishmaniasis and macrophagic activation syndrome by search in PubMed (period 1991-2008).Visceral leishmaniasis was established by bone marrow aspiration, and although there was no histological confirmation, according to HLH-2004 criteria, a secondary macrophagic activation syndrome was established. The patient had a favourable outcome.We report herein the fifth case of visceral leishmaniasis in a patient under TNF-alpha therapy, and the first one, to our knowledge, presenting a consequent secondary macrophagic activation syndrome.
2278	Two novel mutations identified in an african-american child with chediak-higashi syndrome.	20368792	Background. Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder characterized by oculocutaneous albinism, immunodeficiency, coagulopathy and late-onset, progressive neurological dysfunction. It also has an "accelerated phase" characterized by hemophagocytic lymphohistiocytosis (HLH). The disease is caused by mutations in the CHS1/LYST gene located on chromosome 1, which affects lysosome morphology and function. We report the case of an African-American child with CHS in Case. This 16-month old African-American girl presented with fever and lethargy. The proband had pale skin compared to her parents, with light brown eyes, silvery hair and massive hepatosplenomegaly. Her laboratory evaluation was remarkable for pancytopenia, high serum ferritin and an elevated LDH. Bone marrow aspirate revealed large inclusions in granulocytes and erythrophagocytosis consistent with HLH. Genetic evaluation revealed two novel nonsense mutations in the CHS1 gene: c.3622C > T (p.Q1208X) and c.11002G > T (p.E3668X). Conclusions. Our patient is one of the few cases of CHS reported in the African American population. We identified 2 nonsense mutations in the CHS1 gene, the first mutation analysis published of an African-American child with Chediak-Higashi Syndrome. These two mutations predict a severe phenotype and thus identification of these mutations has an important clinical significance in CHS.
2279	Hemophagocytic lymphohistiocytosis at initiation of kawasaki disease and their differential diagnosis.	20367269	The authors report a case of hemophagocytic lymphohistiocytosis that became apparent and was confirmed by a specific Th1/Th2 cytokine pattern at the initiation of Kawasaki disease in an 18-month-old child. His condition deteriorated fast and produced no response to intravenous immunoglobulin and dexamethasone. A standard HLH-2004 regimen was started. But he developed respiratory failure, seizure, and cardiac arrest, and died. This case is unique for developing hemophagocytic lymphohistiocytosis at the initiation of Kawasaki disease, with fatal outcome. This case may indicate a yet unknown mechanism triggers these 2 diseases; a specific Th1/Th2 cytokine pattern helps rapid differential diagnosis between these 2 diseases.
2280	Acquired hemophagocytic lymphohistiocytosis associated with multiple myeloma.	20358309	Acquired or secondary hemophagocytic lymphohistiocytosis (sHLH) is a life-threatening hyperinflammation syndrome caused mostly by various infectious agents, autoimmune disorders or malignancy. So far, only anecdotal cases of sHLH associated with multiple myeloma have been published. We provide a review of all these reports and include a previously not published case of myeloma-associated sHLH in a 59-year-old male with complex partial epilepsy. Due to aggressive course of sHLH, increased awareness is indicated in all patients with malignancies which develop unremitting fever, cytopenia and splenomegaly. Early diagnosis and immediate introduction of adequate therapy is crucial for the outcome of HLH.
2281	Affinity of synthetic peptide fragments of MyoD for Id1 protein and their biological effects in several cancer cells.	20235117	MyoD is a DNA-binding protein capable of specific interactions that involve the helix-loop-helix (HLH) domain. The HLH motif of MyoD can form oligomers with the HLH motif of Id1 (the inhibitor of DNA-binding proteins) that folds into a highly stable helical conformation stabilized by the self-association. The Id family consists of four related proteins that contain a highly conserved dimerization motif known as the HLH domain. In signaling pathways, Id proteins act as dominant negative antagonists of the basic helix-loop-helix (bHLH) family of transcription factors which play important roles in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by binding as dominant negative HLH proteins to form high-affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation-associated genes. The goal of this study is to design and synthesize peptide fragments of MyoD with high affinity for Id1 to interrupt the interactions among Id1, MyoD, and other bHLH DNA-binding proteins and to inhibit the proliferation of cancer cells. Affinity of each peptide for Id1 was determined by surface plasmon resonance (SPR) technology. The secondary structure of each peptide was studied by circular dichroism (CD) spectroscopy. Biological effects of each peptide in several cancer cells such as breast and colon cancer cells were analyzed. Results demonstrated that the peptide 3C (H-Tyr-Ile-Glu-Gly-Leu-Gln-Ala-Leu-Leu-Arg-Asp-Gln-NH(2)) not only showed high affinity for Id1 but also exhibited antiproliferative effects in HT-29 and MCF-7 cancer cells; the IC(50) value of 3C was determined as 25 microM in both cells. The percentage of sub-G1 in the cell cycle of the cancer cells treated with 5 microM of 3C was increased, indicating the induced apoptosis of cancer cells by 3C. Taken together, the peptide 3C is a promising lead compound for the development of antiproliferative agents.
2282	Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis.	20231428	E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2-mediated effects on luciferase reporter activities. Interestingly, injection of E2-2-expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2-mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect.
2283	[Significance of hyperprolactinemia for cytomorphologic features of breast secretions].	20225634	Nipple discharge syndrome is a clinical entity capable of presenting various disorders such is mammary infection (nonpuerperal and puerperal mastitis), intraductal papillomas, fibrodenoma, breast cancer and hyperprolactinemia syndrome. The aim of the study was to determine differences in cytological features of mammary secretion in patients with hyperprolactinemia and those with normal serum prolactin levels and to define the role of growth hormone, follicle-stimulating hormone, luteinizing hormone and thyroid-stimulating hormone in creating cellular profile of breast secretion.The study included 50 patients with nipple discharge syndrome. The patients were devided into the clinical group (27 patients with hyperprolactinemia and nipple discharge) and the control group I (23 patients with normal serum prolactin and nipple discharge). The control group II included the patients of the clinical group achieving normalised serum prolactin levels after the treatment of hyperprolactinemia. Serum prolactin, follicle-stimulating hormone and luteinizing hormone levels were assessed by RIA using commercial kits IRMA hPRL, hLH and hFSH, (INEP, Zemun, Serbia) while serum growth hormone and thyroid-stimulating hormone levels were assessed by RIA using commercial kits LKB-wallac. Cytologic evaluation of samples, taken from all the patients with mammary secretion, was done using standard techniques of staining Haemathoxilin-eozine and May-Grünwald/Giemsa.Our results showed a significantly higher presence of lipid and protein material in clinical group, in comparison with the control group I (p < 0.01). Also, our data demonstrated significantly higher number of ductal epithelial cells (p < 0.05) and ductal histiocities (p < 0.001) in the clinical group, compared with the control group I. Macrophagies frequency was proportionally higher in clinical group (44.44%) compared the control group I (17.39%). Erythrocites were significantly lower in the clinical group (p < 0.001) than in the control group I. Significantly decreased mammary secretion (p < 0.01), lower lipid (p < 0.01) and protein synthesis (p < 0.01), and less presence of all cellular categories (p < 0.01) were obtained after normalization of serum prolactin levels.Growth hormone, follicle-stimulating hormone, luteinizing hormone and thyroid-stimulating hormone did not show significant influence on creating cytological features of mammary secretion. The most expressive role, hyperprolactinemia demonstrated in the domain of mammary ductal secretory activity, making mammary secretion reach in lipid and protein material and simultaneously increasing number of ductal epithelial cells, ductal histiocytes and "foam cells"--macrophages. These cytological findings indicate that hyperprolactinemia promote periductal and intraductal steril inflammation which withdraws after serum prolactin normalization.
2284	Hemophagocytic lymphohistiocytosis secondary to Ehrlichia chaffeensis infection: a case report.	20224442	Hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially life-threatening condition that is characterized by fever, splenomegaly, and cytopenia in 2 or more peripheral blood lineages, hypertriglyceridemia, hypofibrinogenemia, and hemophagocytosis. HLH may be primary or may be triggered by numerous etiologies, including infections. Identification of underlying etiology of HLH is important as proper treatment can completely resolve the disease process. We present a patient whose clinical presentation fulfilled the diagnostic criteria for HLH but whose illness was caused by infection with Ehrlichia chaffeensis, emphasizing the need to explore all possible etiologies during evaluation of patients presenting with illnesses consistent with HLH.
2285	Aplastic anemia presenting as hemophagocytic lymphohistiocytosis.	27265797	Two unusual cases of hemophagocytic lymphohistiocytosis (HLH) complicating aplastic anemia (AA) are described. Each patient had a history of preexisting acute hepatitis of unknown cause at the time of HLH diagnosis and infection-associated secondary HLH. They developed high fever and pancytopenia. Hemophagocytes were seen in the bone marrow. With steroid (in combination with etoposide and CyA in 1 patient), high fever disappeared and the patients' liver function gradually recovered. As severe pancytopenia persisted, bone marrow became acellular and AA was diagnosed. Since HLH is known to be able to cause an aplastic bone marrow if untreated for a prolonged time, it is therefore in line that hepatitis-associated AA may also be associated with HLH. Aplastic anemia-associated HLH has been reported rarely, and problems in the diagnostic procedure are discussed. Bu makalede hemofagositik sendrom (HLH) tanısı alıp daha sonra aplastik anemi (AA) gelişen 2 hasta sunulmuştur. Her 2 hastada da HLH tanısından önce etyolojisi net olarak konulmamış akut hepatit öyküsü vardı ve bu enfeksiyon nedeni ile HLH’leri enfeksiyon ilişkili HLH olarak değerlendirilmişti. İki olguda da ateş ve pansitopeni vardı. Hemofagositoz iki olguda da kemik iliği aspirasyon materyalinde gösterilmişti. Bir olguda steroid diğerinde steroide ek olarak etoposid ve siklosporin (CyA) ile yüksek ateş ve karaciğer fonksiyonlarında düzelme saptanmaya başlanmıştı. Tedaviye rağmen ağır sitopeninin devam etmesi üzerine kemik iliğinin tekrar değerlendirilmesi sonucunda hastalara aplastik anemi tanısı konuldu. Hemofagositik sendrom uzun süreli tedavi edilmediğinde kemik iliğinde aplaziye neden olabilir, bu nedenle hepatite bağlı gelişen aplastik anemilerin gelişim sürecinde HLH’ da olabilir.
2286	Assessment of clinical and laboratory presentations of familial hemophagocytic lymphohistiocytosis patients with homozygous W374X mutation.	20197201	Homozygous W374X mutation was identified in unrelated 13 patients (6M/7F) from consanguineous families, 62% of which had history of deceased sibling. Haplotype analysis provided evidence for the probable existence of a founder effect. Age at disease onset ranged from 1 day to 5.5 months (median 2 months). Hepatic dysfunction was observed in 69%, ascite 62%, hypertriglyceridemia 77%, each hyperferritinemia and hypofibrinogenemia 85%, CNS involvement 46% of patients while birth weights were in normal range. Those with very high ferritin (>20,000ng/ml) had extremely low fibrinogen levels. Two-thirds of patients receiving HLH protocol died within 20 days of therapy.
2287	Chronic murine typhoid fever is a natural model of secondary hemophagocytic lymphohistiocytosis.	20195482	Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory clinical syndrome associated with neoplastic disorders especially lymphoma, autoimmune conditions, and infectious agents including bacteria, viruses, protozoa and fungi. In both human and veterinary medicine, hemophagocytic histiocytic disorders are clinically important and frequently fatal. HLH in humans can be a primary (familial, autosomal recessive) or secondary (acquired) condition, with both types generally precipitated by an infectious agent. Previously, no mouse model for secondary HLH has been reported. Using Salmonella enterica serotype Typhimurium by oral gavage to mimic naturally-occurring infection in Sv129S6 mice, we characterized the clinical, hematologic and morphologic host responses to disease thereby describing an animal model with the clinico-pathologic features of secondary HLH as set forth by the Histiocyte Society: fever, splenomegaly, cytopenias (anemia, thrombocytopenia), hemophagocytosis in bone marrow and spleen, hyperferritinemia, and hypofibrinogenemia. Disease severity correlates with high splenic and hepatic bacterial load, and we show disease course can be monitored and tracked in live animals. Whereby secondary HLH is known to occur in human patients with typhoid fever and other infectious diseases, our characterization of a viable natural disease model of secondary HLH offers an important means to elucidate pathogenesis of poorly understood mechanisms of secondary HLH and investigation of novel therapies. We characterize previously unreported secondary HLH in a chronic mouse model of typhoid fever, and novel changes in hematology including decreased tissue ferric iron storage that differs from classically described anemia of chronic disease. Our studies demonstrate S. Typhimurium infection of mice is a natural infectious disease model of secondary HLH that may have utility for elucidating disease pathogenesis and developing novel therapies.
2288	Silencing Id-1 with RNA interference inhibits adenoid cystic carcinoma in mice.	20189601	The helix-loop-helix (HLH) protein Id-1 (inhibitor of DNA binding/differentiation) has been demonstrated to play an important role in tumor development. Our previous in vitro research has shown that Id-1 is a potential target in the treatment of human adenoid cystic carcinoma (ACCM). The purpose of this study was to analyze the influence of Id inhibition on ACCM in mice.To suppress the expression of Id-1 gene, we used lentivirus-mediated RNA interference to silence the Id-1 gene post-transcriptionally in ACCM models that stably express GFP in mice. Tumor development was evaluated by size measurement. Effects of Id-1 siRNA on mRNA and protein expression of Id-1 were analyzed using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting respectively. Ki-67 expression was measured by immunohistochemistry. In vitro studies of Hoechst staining for cell apoptosis, Boyden-chamber assay for cell invasion, and MTT-tests for cell growth were performed as well.Id-1 knockdown resulted in inhibition of tumor growth in mice. Id-1 siRNA significantly decreased not only Id-1 in mRNA and protein level, but also Ki-67 expression. In addition, apoptosis was induced and cell proliferation activity and invasion were significantly reduced.Lentivirus-mediated gene knockdown by silencing Id-1 constitute a valid methodological approach, which may represent an attractive, potent and specific therapeutic tool for the treatment of ACCM.
2289	Two percent of patients suspected of having Angelman syndrome have TCF4 mutations.	20184619	The TCF4 gene encodes a basic helix-loop-helix (bHLH) transcription factor which belongs to the family of E-proteins. E-proteins form homo- and heterodimers with other members of the HLH family and bind to the common DNA sequence called E-box. Haploinsufficiency of the TCF4 gene has been found to be associated with the Pitt-Hopkins syndrome (PTHS). PTHS is characterized by severe mental retardation, a wide mouth plus other distinctive facial features (fleshy lips, beaked nose, broad nasal bridge) and breathing abnormalities. Because of some phenotypical overlap with Angelman syndrome (AS), it has been suggested that PTHS be considered in its differential diagnosis. To explore this possibility, we screened 86 patients who were suspected of having AS. All the patients were negative for UBE3A testing, and 53 were known to be negative for methylation analysis. We identified two TCF4 mutations in this cohort. The p.S384Tfsx7 mutation lacks the bHLH domain. The p.R582P mutation lies within the bHLH domain in which seven other missense mutations have been reported. Both mutations most likely affect the critical function of the bHLH domain of the TCF4 protein. In summary, we found two TCF4 mutations in 86 patients (2%) suspected to have AS. Screening for mutations in this gene should be considered in patients who present with findings of AS but who have been negative for methylation and UBE3A testing.
2290	Kinetic analysis of the interaction of b/HLH/Z transcription factors Myc, Max, and Mad with cognate DNA.	20170194	Myc, Mad, and Max proteins belong to the basic helix-loop-helix leucine zipper family of transcription factors. They bind to a specific hexanucleotide element of DNA, the E-box (CACGTG). To be biologically active, Myc and Mad require dimerization with Max. For the route of complex assembly of these dimers, there are two proposed pathways. In the monomer pathway, two monomers bind DNA sequentially and assemble their dimerization interface while bound to DNA. In the dimer pathway, two monomers form a dimer first prior to association with DNA. The monomer pathway is kinetically favored. In this report, stopped-flow polarization was utilized to determine the rates and temperature dependence of all of the individual steps for both assembly pathways. Myc.Max dimerization had a rate constant approximately 5- and approximately 2-fold higher than those of Max.Max and Mad.Max dimerization, respectively. The protein dimerization rates as well as the dimer-DNA rates were found to be independent of concentration, suggesting conformational changes were rate-limiting. The Arrhenius activation energies for the dimerization of Myc, Mad, and Max with Max were 20.4 +/- 0.8, 29 +/- 0.6, and 40 +/- 0.2 kJ/mol, respectively. Further, rate constants for Max.Max homodimer DNA binding are significantly higher than for Myc.Max and Mad.Max heterodimers binding to DNA. Monomer-DNA binding showed a faster rate than dimer-DNA binding. These studies show the rate-limiting step for the dimer pathway is the formation of protein dimers, and this reaction is slower than formation of protein dimers on the DNA interface, kinetically favoring the monomer pathway.
2291	Juvenile myelomonocytic leukemia presenting with features of neonatal hemophagocytic lymphohistiocytosis and cutaneous juvenile xanthogranulomata and successfully treated with allogeneic hemopoietic stem cell transplant.	20168243	Juvenile xanthogranuloma (JXG) is rarely associated with either hemophagocytic lymphohistiocytosis (HLH) or juvenile myelomonocytic leukemia (JMML) and when in association with the latter there is usually neurofibromatosis type 1. We report a child who presented with JXG and HLH during the neonatal period and who subsequently developed JMML during early infancy in whom there is no evidence of neurofibromatosis type 1. The patient was refractory to standard HLH therapy but he is well and is now 42 months after mismatched unrelated donor hemopoietic stem cell transplant without evidence of HLH or JMML. His JXG lesions show involution, in keeping with the expected natural history of this disorder.
2292	Transcriptional control of stem cell maintenance in the Drosophila intestine.	20147375	Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. Here we find that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and identify genes of the Enhancer of split complex [E(spl)-C] as the major targets of this repression. In addition, we find that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. We propose that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation.
2293	[Research advance on hemophagocytic lymphohistiocytosis].	20137160	Hemophagocytic lymphohistiocytosis (HLH) is named as hemophagocytic syndrome (HPS) and is a complicated disease with reactive hyperplasia of mononuclear/macrophagocytic system. This disease characterised by release of massive cytokines and severe functional destruction of visceral organs, which results from immune function disturbance causing by various pathogenic factors. The cardinal clinical symptoms of HLH are prolonged fever, hepatosplenomegaly, cytopenia, elevated ferritin and triglycerides, low fibrinogen, symptom in nerve system and so on. Nevertheless, impaired function of natural killer cells and cytotoxic T-cell is characteristic for HLH. HLH has of two different types that may be difficult to distinguish from one another: a primary and a secondary form. The combined immunochemotherapy of dexamethasone, etoposide and cyclosporin A and hematopoietic stem cell transplantation are considered as the effective therapies for HLH. In this article, the recent advance in research on the etiological factors, pathogenesis, clinical manifestations, laboratory examination, diagnosis as well as recommended therapy of HLH were reviewed.
2294	Kawasaki disease preceding haemophagocytic lymphohistiocytosis: challenges for developing world practitioners.	2013570220658642	Kawasaki disease (KD) is a recognised precipitant of haemophagocytic lymphohistiocytosis (HLH). Although KD has been previously described in the developing world, there are no reported cases of KD preceding HLH. We report a case of a child with a persistent rash and unremitting fever consistent with the diagnosis of KD, who was found to have HLH, after intravenous gamma globulin failed to produce a clinical response. The diagnosis was made using the revised diagnostic criteria for HLH from the Histiocyte Society (1994). She fulfilled six of the eight clinical and laboratory criteria needed to make the diagnosis.
2295	[Five adult cases of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis].	20134144	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is more common in children, and is characterized by pancytopenia, liver dysfunction and coagulopathy caused by interactions between EBV-infected T cells and activated macrophages. We describe here five adults with EBV-HLH. The median age was 17 years (range 16 approximately 40). HLH developed in 4 patients within 2 months after the primary infection, and in the other one during the reactivation. All patients had a high EBV viral load in peripheral blood (2 x 10(2)-3 x 10(6) copies/ml) and monoclonal proliferation of EBV-infected T cells. All patients received immunosuppressive therapy with or without etoposide, and two patients required plasmapheresis due to the severity. Three patients are alive in complete remission (follow up periods; 13, 19, 30 months), while two patients became refractory to chemo-immunotherapy and died despite multidrug chemotherapy. EBV-HLH should be more widely recognized in adults in order to achieve early diagnosis and appropriate treatment.
2296	Viral infections associated with haemophagocytic syndrome.	20127750	Haemophagocytic syndrome (HPS) or haemophagocytic lymphohistiocytosis (HLH) is a rare disease caused by a dysfunction of cytotoxic T cells and NK cells. This T cell/NK cell dysregulation causes an aberrant cytokine release, resulting in proliferation/activation of histiocytes with subsequent haemophagocytosis. Histiocytic infiltration of the reticuloendothelial system results in hepatomegaly, splenomegaly, lymphadenopathy and pancytopenia ultimately leading to multiple organ dysfunctions. Common clinical features include high fevers despite broad spectrum antimicrobials, maculopapular rash, neurological symptoms, coagulopathy and abnormal liver function tests. Haemophagocytic syndrome can be either primary, i.e. due to an underlying genetic defect or secondary, associated with malignancies, autoimmune diseases (also called macrophage activation syndrome) or infections. Infectious triggers are most commonly due to viral infections mainly of the herpes group, with EBV being the most common cause. HPS can be fatal if untreated. Early recognition of the clinical presentation and laboratory abnormalities associated with HPS and prompt initiation of treatment can be life saving. HPS triggered by viral infections generally does not respond to specific antiviral therapy but may be treated with immunosuppressive/immunomodulatory agents and, in refractory cases, with bone marrow transplantation.
2297	Disseminated histoplasmosis associated with hemophagocytic lymphohistiocytosis in kidney transplant recipients.	20121728	Transplant patients are susceptible to infectious complications due to chronic immunosuppression. We present two cases of persistent fever, weight loss and pancytopenia in kidney transplant recipients (originally concerning for posttransplant lymphoproliferative disease) that were later diagnosed with disseminated histoplasmosis on bone marrow and lymph node biopsy. In both patients, pancytopenia was due to hemophagocytic lymphohistiocytosis (HLH) which has rarely been described in association with histoplasmosis and not previously reported in kidney transplant recipients with this fungal infection. The diagnosis of histoplasmosis can be complex due to nonspecific symptomatology, delays in isolating histoplasma by fungal culture and false-negative antibody titers in immunocompromised patients. A review of the literature including the clinical features of histoplasmosis in immunosuppressed patients (prevalence, current diagnostic testing and treatment options) as well as the association of HLH in immunocompromised states are discussed.
2298	The outcome of hematopoietic stem cell transplantation in Korean children with hemophagocytic lymphohistiocytosis.	20113424	Chemoimmunotherapy-based treatments have improved the survival of patients with HLH, but outcomes of the patients are still unsatisfactory. We report here the outcome of Korean children with HLH who underwent HSCT, which was analyzed from the data of a nation-wide HLH registry. Retrospective nation-wide data recruitment for the pediatric HLH patients diagnosed between 1996 and 2008 was carried out by the Histiocytosis Working Party of the Korean Society of Hematology. Nineteen patients who received HSCT among the total of 148 enrolled children with HLH were analyzed for the transplant-related variables and events. The probability of five-yr survival after HSCT was 73.3% with a median follow-up of 57. Two months compared to 54.3% for the patients who were treated with chemoimmunotherapy only (p = 0.05). The reasons for HSCT were active disease after eight wk of initial treatment (n = 9), relapsed disease (n = 5), and FHL (n = 5). Fourteen patients are currently alive without disease after HSCT, four patients died of treatment-related events (infection in two and graft failure in two) at early post-transplant period, and one patient died of relapse at one yr post transplantation. The survival of patients who were transplanted because of active disease after eight wk of initial treatment was worse compared to those patients who had inactive state at that time (60.6% vs. 100%, respectively, p = 0.06). Of the four patients who received transplants using cord blood, three died of graft failure (n = 2) and relapse (n = 1). The five-yr probability of survival after HSCT according to the donor type was 85.7% for the MRDs (n = 6), 87.5% for the MUDs (n = 8), and 40% for the MMUDs (n = 5) (p = 0.03). Other variables such as age, CNS involvement at the time of diagnosis, the etiology of HLH (familial or secondary), and the conditioning regimens had no influence on the five-yr OS of the HLH patients who underwent HSCT. HSCT improved the survival of the patients who had familial, relapsed, or severe and persistent SHLH in the Korean nation-wide HLH registry. Although numbers were small, these results are similar to other reports in the literature. The disease state after initial treatment, the stem cell source of the transplant, and the donor type were the important prognostic factors that affected the OS of the HLH patients who underwent HSCT.
2299	Mutations in the perforin gene in children with hemophagocytic lymphohistiocytosis.	20092789	Recent studies have reported germline mutations in the perforin gene (PRF1) in some types of hemophagocytic lymphohistiocytosis (HLH). However, the prevalence of PRF1 mutations in HLH in Chinese pediatric patients has not been extensively studied. The aim of this study was to investigate the prevalence of mutations and sequence variations in the PRF1 gene in Chinese pediatric patients with HLH.Polymerase chain reaction (PCR) was performed with five pairs of primers for the coding exons and the flanking intron sequences of PRF1. Sequencing of PCR products was subsequently applied in 30 pediatric patients with HLH and in 50 controls.Three heterozygous mutations in a coding region were found, which resulted in amino acid changes (C102F, S108N and T450M) in three patients. These mutations were not detected in control subjects. One patient had compound heterozygous mutations (S108N and T450M) in PRF1 as the background defect, and documented familial HLH type 2 (FHL2). One synonymous sequence variant (Q540Q) was observed in one patient but not in the controls. Two SNPs (A274A, H300H) in the coding region were detected in HLH patients and controls, but without differences in the heterozygosity rate between the two groups (P > 0.05 for all comparisons).We have identified three patients with three heterozygous missense mutations in PRF1; two of those three mutations (C102F and S108N) have so far been found only from Chinese patients. These findings are useful in evaluating the prevalence of PRF1 mutations in Chinese pediatric patients with HLH, and to correlate their genotype with phenotype. Some patients without familial history probably have primary HLH, which should be suspected even beyond the usual age range.
2300	Reengineering natural design by rational design and in vivo library selection: the HLH subdomain in bHLHZ proteins is a unique requirement for DNA-binding function.	20086039	To explore the role of the HLH subdomain in bHLHZ proteins, we designed sets of minimalist proteins based on bHLHZ protein Max, bHLH/PAS protein Arnt and bZIP protein C/EBP. In the first, the Max bHLH and C/EBP leucine zipper were fused such that the leucine heptad repeats were not in register; therefore, the protein dimerization interface was disrupted. Max1bHLH-C/EBP showed little ability to activate transcription from the E-box (5'-CACGTG) in the yeast one-hybrid assay, and no E-box binding by quantitative fluorescence anisotropy. Max1bHLH-C/EBP's activity was significantly improved after library selection (three amino acids randomized between HLH and leucine zipper), despite the Max bHLH and C/EBP zipper still being out of register: a representative mutant gave a high nanomolar K(d) value for E-box binding. Thus, selection proved to be a powerful tool for salvaging the flawed Max1bHLH-C/EBP, although the out-of-register mutants still did not achieve the strong DNA-binding affinities displayed by their in-register counterparts. ArntbHLH-C/EBP hybrids further demonstrated the importance of maintaining register, as out-of-register mutants showed no E-box-responsive activity, whereas the in-register hybrid showed moderate activity. In another design, we eliminated the HLH altogether and fused the Max basic region to the C/EBP zipper to generate bZIP-like hybrids. Despite numerous designs and selections, these hybrids possessed no E-box-responsive activity. Finally, we tested the importance of the loop sequence in MaxbHLHZ by fluorescence and circular dichroism. In one mutant, the loop was shortened by two residues; in the other, the Lys57:DNA-backbone interaction was abolished by mutation to Gly57. Both showed markedly decreased E-box-binding relative to MaxbHLHZ. Our results suggest that, in contrast to the more rigid bZIP, the HLH is capable of significant conformational adaptation to enable gene-regulatory function and is required for protein dimerization and positioning the basic region for DNA recognition.
2301	[Hemophagocytosis associated with cytomegalovirus infection and azathioprine treatment for inflammatory bowel disease].	20056098	Haemophagocytic lymphohistiocytosis (HLH) is a rare syndrome characterized by fever and cytopenia due to phagocytosis of erythrocytes, leukocytes, platelets, and their precursors in bone marrow and other tissues. HLH can be primary (genetic) or associated with neoplastic diseases, autoimmune diseases or infections. HLH is a rare syndrome but can cause very severe illness, and if unrecognized/untreated the condition is associated with a high mortality. We present two cases of cytomegalovirus infection-associated HLH in patients with inflammatory bowel disease during azathioprin treatment.
2302	Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice.	20049711	Hereditary haemophagocytic lymphohistiocytosis (HLH) is a fatal inflammatory disease and treatments currently may lead to serious side effects. There is a pressing need for effective, less toxic treatments for this disease. Previous reports have suggested that interferon gamma (IFNgamma) has a role in the pathogenesis of HLH. Here, we report that blocking IFNgamma had a therapeutic effect in two different murine models of human hereditary HLH (perforin-deficient and Rab27a-deficient mice, both infected with lymphocytic choriomeningitis virus). Therapeutic administration of an anti-IFNgamma antibody induced recovery from haemophagocytosis in both genetic models, as evidenced by increased survival in perforin-deficient mice and correction of blood cytopenia, moderation of body temperature changes, decreased cytokinaemia, restoration of splenic architecture and reduced haemophagocytosis in the liver of both murine models. Involvement of the central nervous system in Rab27a-deficient mice was prevented by anti-IFNgamma therapy. Hepatic T-cell infiltrates and virus persisted, with no detectable harm during the time course of these studies. These data strongly suggest that neutralization of IFNgamma could be used in humans to safely alleviate the clinical manifestations of haemophagocytosis.
2303	Familial haemophagocytic lymphohistiocytosis: two case reports.	22736603	Haemophagocytic lymphohistiocytosis (HLH) is a life threatening inflammatory syndrome, which presents a highly stimulated but ineffective immune response with severe hypercytokinaemia. HLH, primary or secondary, is characterised by prolonged fever and hepatosplenomegaly associated with pancytopenia, hypertriglyceridaemia and hypofibrinogenaemia. However, the hallmark of HLH is impaired or absent function of natural killer cells and cytotoxic T lymphocytes. HLH presents major diagnostic difficulties, since it may have an incomplete and/or late onset and with many conditions leading to the same clinical picture. When untreated, it is fatal in all primary cases and in a high percentage of acquired cases. Awareness of the clinical picture and diagnostic criteria is thus important to start life saving treatment. We describe two cases of primary HLH, with significant differences in their clinical presentation and evolution.
2304	Mid-follicular LH supplementation in women aged 35-39 years undergoing ICSI cycles: a randomized controlled study.	2003103221575849	This single-centre, randomized, parallel group, comparative study aimed to identify potential benefits of mid-follicular recombinant human LH (r-HLH) supplementation in women aged 35-39 years undergoing ovarian stimulation for intracytoplasmic sperm injection (ICSI). The main endpoint was the number of metaphase II oocytes retrieved. After pituitary suppression with a gonadotrophin-releasing hormone agonist, ovarian stimulation was initiated with recombinant human FSH (r-HFSH; 300-450 IU/day). On stimulation day 6, patients were randomized to receive r-HFSH alone or r-HFSH + r-HLH (r-HLH 150 IU/day) for the remainder of the stimulation period. Final follicular maturation was triggered with 250 mug of recombinant human chorionic gonadotrophin. After assessing oocyte nuclear maturity, oocyte were fertilized by ICSI and afterwards embryo quality was analyzed. Of the 131 women enrolled, 68 were allocated to r-HFSH alone and 63 to r-HFSH + r-HLH. No significant differences were observed in markers of either oocyte or embryo quality or quantity. However, higher rates of implantation and live birth per started cycle were observed with r-HLH supplementation than with r-HFSH alone. Although additional large studies are required to further investigate these findings, r-HLH supplementation for women aged 35-39 years undergoing ICSI is recommended as it may have a beneficial action on implantation.
2305	[Application of measuring human peripheral NK cell activity with flow cytometry in diagnosis for hemophagocytic lymphohistiocytosis].	20030934	The aim of study was to establish an accurate and stable technique for the detection of NK cell activity in the diagnosis of hemophagocytic lymphohistiocytosis (HLH). 21 suspected acquired HLH patients and 20 healthy subjects as controls were enrolled in the study, and the suspected HLH patients were divided into confirmedly diagnosed group and excluded group according to HLH-2004 diagnostic criteria. The plasmid pEGFP-N1 was transfected into K562 cells. After scanned with G4l8 and monoclone, the EGFP-K562 cell line stably expressing enhanced green fluorescent protein was obtained. PBMNC and EGFP-K562 were mixed at the effector to target ratio of 10:1. After incubation for of 2 hours, propidium iodine (PI) was added to stain dead cells, and then cytotoxic activity was analyzed by using flow cytometry. Meanwhile, the cytotoxic activity of NK cells in peripheral blood on K562 cells was detected by LDH release assay, and was compared with results detected by flow cytometry. The results showed that a K562 cell line stably expressing EGFP was constructed and were used to measure NK cytotoxicity against the target cells by flow cytometry without pre-staining or pre-labeling target cells. There was significant difference in killing rate of NK cells between the diagnostic group and the control group. This new technique correlated strongly with the results by LDH release assay. In conclusion, this study provides a novel, simple, rapid, repeatable and reproducible method to measure NK activity by flow cytometry using EGFP-K562 without pre-staining or pre-labeling target cells. It may be widely used in the diagnosis of hemophagocytic lymphohistiocytosis.
2306	UNC13D is the predominant causative gene with recurrent splicing mutations in Korean patients with familial hemophagocytic lymphohistiocytosis.	2001588820378576	Familial hemophagocytic lymphohistiocytosis is a fatal disease characterized by immune dysregulation from defective function of cytotoxic lymphocytes. Three causative genes have been identified for this autosomal recessive disorder (PRF1, UNC13D, and STX11). We investigated the molecular genetics of familial hemophagocytic lymphohistiocytosis in Korea.Pediatric patients who fulfilled the HLH-2004 criteria were recruited from the Korean Registry for Histiocytosis. Molecular genetic studies were performed on the patients' DNA samples by direct sequencing of all coding exons and flanking sequences of PRF1, UNC13D, and STX11.Forty patients were studied and familial hemophagocytic lymphohistiocytosis mutations were identified in nine; eight patients had UNC13D mutations (89%) and one had a mutation in PRF1. No patient had a STX11 mutation. Notably, four patients had only one UNC13D mutant allele, suggesting that the other mutation was missed by conventional direct sequencing. All UNC13D mutations were deleterious in nature. One known splicing mutation, c.754-1G>C, was recurrent, accounting for 58% of all the mutant alleles (7/12). Five UNC13D mutations were novel (p.Gln98X, p.Glu565SerfsX7, c.1993-2A>G, c.2367+1G>A, and c.2954+5G>A). The one patient with PRF1 mutation was homozygous for a frameshift mutation (p.Leu364GlufsX93), which was previously reported to be the most frequent PRF1 mutation in Japan.This is the first investigation on the molecular genetics of familial hemophagocytic lymphohistiocytosis in Korea. The data showed that UNC13D is the predominant causative gene in the Korean population. The identification of mutations missed by conventional sequencing would better delineate the mutation spectrum and help to establish the optimal molecular diagnostic strategy for familial hemophagocytic lymphohistiocytosis in Korea, which might need an RNA-based screening strategy.
2307	Evidence that the C-terminal domain (CtD) autoinhibits neural repression by Drosophila E(spl)M8.	20014344	Analysis of the retinal defects of a CK2 phosphomimetic variant of E(spl)M8 (M8S(159)D) and the truncated protein M8* encoded by the E(spl)D allele, suggest that the nonphosphorylated CtD "autoinhibits" repression. We have investigated this model by testing for inhibition (in "trans") by the CtD fragment in its nonphosphorylated (M8-CtD) and phosphomimetic (M8SD-CtD) states. In N(+) flies, ectopic M8-CtD compromises lateral inhibition, i.e., elicits supernumerary bristles as with loss of N signaling. This antimorphic activity of M8-CtD strongly rescues the reduced eye and/or bristle loss phenotypes that are elicited by ectopic M8SD or wild type M8. Additionally, the severely reduced eye of N(spl)/Y; E(spl)D/+ flies is also rescued by M8-CtD. Rescue is specific to the time and place, the morphogenetic furrow, where "founding" R8 photoreceptors are specified. In contrast, the phosphomimetic M8SD-CtD that is predicted to be deficient for autoinhibition, exhibits significantly attenuated or negligible activity. These studies provide evidence that autoinhibition by the CtD regulates M8 activity in a phosphorylation-dependent manner.
2308	Dermatopathic lymphadenitis with generalized erythroderma in a patient with epstein-barr virus-associated hemophagocytic lymphohistiocytosis.	20010284	Here, we describe a patient with Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) who simultaneously presented with generalized erythroderma and dermatopathic lymphadenitis (DL). A 63-year-old Korean woman presented at our hospital with fever, hepatosplenomegaly, axillary lymphadenopathy, and generalized erythrodermic eruption. The bone marrow biopsy findings were consistent with the diagnosis of HLH, and EBV DNA was detected using the polymerase chain reaction. Based on serologic tests that indicated a primary EBV infection, the patient was diagnosed with EBV-associated HLH. Histopathologic analysis of enlarged lymph nodes was consistent with DL, and EBV-encoded small nuclear RNA-positive mononuclear cells were detected. We assume that activated histiocytes, lymphocytes, and proinflammatory cytokines in HLH may have important roles in the development of generalized erythroderma and DL. Disrupted epidermal/dermal junctions owing to erythroderma may also be involved in the development of DL.
2309	Hemophagocytic lymphohistiocytosis in the premature neonate: a case study.	20010143	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease resulting from an abnormal proliferation of histiocytes within the body's tissues leading to an ineffective immune response. Typically, HLH is characterized by fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia, and hemophagocytosis. However, the premature infant with HLH may present differently making diagnosis of the disease cumbersome. If an infant is born with ascites, cytopenias, hypofibrinogenemia, and hepatosplenomegaly, a diagnosis of HLH cannot be ruled out. In addition, premature infants oftentimes will not present with fever because they are kept normothermic from ambient sources. Reports of premature infants with HLH in the literature are rare. This is a case presentation of a 27-week-gestation female with a family history of HLH.
2310	Hemophagocytic lymphohistiocytosis in the premature neonate.	20010142	Hemophagocytic lymphohistiocytosis (HLH), a rare disease, results in pathological findings secondary to an abnormal proliferation of activated lymphocytes and histiocytes (tissue macrophages) and is lethal unless identified and adequately treated. Clinical features of HLH include fever, hepatosplenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, elevated blood levels of ferritin, lymphadenopathy, skin rash, jaundice, and edema. Often, the symptoms of HLH are misinterpreted as infection, resulting in inadequate treatment and death. Several case studies of premature neonates with HLH have recently been published. Therapeutic guidelines for HLH exist and, when identified, HLH in the premature infant can be successfully treated resulting in resolution of symptoms.
2311	Antagonistic HLH/bHLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis.	20009022	In rice (Oryza sativa), brassinosteroids (BRs) induce cell elongation at the adaxial side of the lamina joint to promote leaf bending. We identified a rice mutant (ili1-D) showing an increased lamina inclination phenotype similar to that caused by BR treatment. The ili1-D mutant overexpresses an HLH protein homologous to Arabidopsis thaliana Paclobutrazol Resistance1 (PRE1) and the human Inhibitor of DNA binding proteins. Overexpression and RNA interference suppression of ILI1 increase and reduce, respectively, rice laminar inclination, confirming a positive role of ILI1 in leaf bending. ILI1 and PRE1 interact with basic helix-loop-helix (bHLH) protein IBH1 (ILI1 binding bHLH), whose overexpression causes erect leaf in rice and dwarfism in Arabidopsis. Overexpression of ILI1 or PRE1 increases cell elongation and suppresses dwarf phenotypes caused by overexpression of IBH1 in Arabidopsis. Thus, ILI1 and PRE1 may inactivate inhibitory bHLH transcription factors through heterodimerization. BR increases the RNA levels of ILI1 and PRE1 but represses IBH1 through the transcription factor BZR1. The spatial and temporal expression patterns support roles of ILI1 in laminar joint bending and PRE1/At IBH1 in the transition from growth of young organs to growth arrest. These results demonstrate a conserved mechanism of BR regulation of plant development through a pair of antagonizing HLH/bHLH transcription factors that act downstream of BZR1 in Arabidopsis and rice.
2312	New tools for investigating the comparative biology of Caenorhabditis briggsae and C. elegans.	20008572	Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.
2313	Hemophagocytic lymphohistiocytosis (HLH) and related disorders.	20008190	Hemophagocytic lymphohistiocytosis (HLH), which has many genetic causes, is characterized by multi-system inflammation. HLH is a reactive process resulting from prolonged and excessive activation of antigen presenting cells (macrophages, histiocytes) and CD8(+) T cells. Hemophagocytosis, which is mediated through the CD163 heme-scavenging receptor, is a hallmark of activated macrophages/histiocytes and is the characteristic finding for which the disorder was named. The majority of genetic causes identified to date affect the cytotoxic function of NK and T cells, crippling immunologic mechanisms that mediate natural immune contraction. The predominant clinical findings of HLH are fevers (often hectic and persistent), cytopenias, hepatitis and splenomegaly. Due to the life-threatening implications of the diagnosis of genetically determined HLH, antiinflammatory therapy, often consisting of steroids, etoposide or antithymocyte globulin (ATG), should be instituted promptly, followed by curative hematopoietic cell transplantation. Secondary HLH, associated with autoimmune disorders or viral infections in teens and adults, also carries a significant mortality rate and should be managed in consultation with specialists familiar with the diagnosis and treatment of such disorders.
2314	Novel syntaxin 11 gene (STX11) mutation in three Argentinean patients with hemophagocytic lymphohistiocytosis.	19967551	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease with major diagnostic and therapeutic difficulties, basically comprising two different conditions: primary and secondary forms. Recent advances regarding molecular diagnosis may be useful to distinguish from one another, especially in sporadic cases starting in early infancy.In this report, we evaluated three Argentinean patients with clinical suspicion of HLH, but without family history. We excluded mutations in the perforin gene but identified in the three patients a novel homozygous deletion (c. 581_584delTGCC; p.Leu194ProfsX2) in the gene-encoding syntaxin 11 (STX11), causing a premature termination codon.Each parent from the three unrelated families resulted heterozygous for this deletion confirming the diagnosis of familial hemophagocytic lymphohistiocytosis type 4. Patients shared the same single-nucleotide polymorphism profile in STX11 gene, and genotyping at ten microsatellites surrounding this gene support the presence of a single-haplotype block carrying the novel mutation.
2315	Breastfeeding facilitates acceptance of a novel dietary flavour compound.	19962799	Infants may learn to accept flavours via exposure to flavour in mother's milk. This study examines whether acceptance of a flavour compound develops over 10 exposures through mother's milk (part 1), and how such acceptance evolves after 10 exposures, on alternate days, to the same flavour in solid food (part 2).Three groups of 5-8 month-old infants participated. Breast-fed infants were randomised into two groups: a non-exposed group (n=20) and a group exposed to caraway flavour (d-carvone) via mother's milk (n=20). Mothers in the second group consumed a caraway-flavoured food. The third group was composed of formula-fed infants whose mothers consumed the caraway-flavoured food (n=8). Infants' acceptance of caraway-flavour was tested after both exposure periods by evaluating, on separate days, intake parameters and mother's judgement of liking of plain and caraway-flavoured purées.Part 1 showed that exposed and non-exposed breast-fed infants had higher initial acceptance of the caraway-flavoured purée than formula-fed infants. Part 2 results showed no evolution in acceptance of caraway-flavoured purée among breast-fed infants. In contrast formula-fed infants' acceptance of caraway-flavoured purée increased, corresponding to flavour-specific acceptance learning.This study suggests that breastfeeding facilitates acceptance of novel flavours. This effect is not necessarily due to exposure to specific flavour compounds.
2316	Hemophagocytic lymphohistiocytosis in Texas: observations on ethnicity and race.	19953651	Early recognition and aggressive treatment of hemophagocytic lymphohistiocytosis (HLH) has changed a uniformly fatal disease to one 55% survive. We examined the diagnosis and treatment of pediatric patients with HLH from the three largest academic medical centers in Texas for information on modern non-study treatment and survival. In contrast with previously reported series, the racial and ethnic composition of Texas provided a unique opportunity to evaluate the impact of race and ethnicity on survival with HLH.A retrospective chart review of local oncology and pathology databases identified 70 patients with HLH from 1992 to 2007. Median age was 1.8 years (range 0.1-16.5 years) and 43% were Latino.We identified 70 patients with an overall survival of 67% after a median follow-up of 3 months (range 1-139 months). Twenty patients (29%) underwent stem cell transplant (SCT). Seven patients (18%) had mutations in the Perforin, Munc 13-4, or Syntaxin-11 genes, consistent with primary disease. Calculated cross-sectional prevalence of HLH in Texas from our study is 1 in 100,000 children. The effect of Latino ethnicity on survival was not statistically significant.HLH is a rare but potentially treatable illness with modern aggressive therapy. Though treatment is more standardized for HLH, the role of race and ethnicity as risk factors for development of disease and impact on outcome may warrant further investigation.
2317	Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations.	19953648	Griscelli syndrome type 2 (GS2) is an autosomal-recessive immunodeficiency caused by mutations in RAB27A, clinically characterized by partial albinism and haemophagocytic lymphohistocytosis (HLH). We evaluated the frequency of RAB27A mutations in 21 unrelated patients with haemophagocytic syndromes without mutations in familial HLH (FHL) causing genes or an established diagnosis of GS2. In addition, we report three patients with known GS2. Moreover, neurological involvement and RAB27A mutations in previously published patients with genetically verified GS2 are reviewed.Mutation analysis of RAB27A was performed by direct DNA sequencing. NK cell activity was evaluated and microscopy of the hair was performed to confirm the diagnosis.RAB27A mutations were found in 1 of the 21 families. This Swedish family had three affected children with heterozygous compound mutations consisting of a novel splice error mutation, [c.239G>C], and a nonsense mutation, [c.550C>T], p.R184X. The three additional children all carried homozygous RAB27A mutations, one of which is a novel splice error mutation, [c.240-2A>C]. Of note, five of the six patients displayed neurological symptoms, while three out of six patients displayed NK cell activity within normal reference values, albeit low. A literature review revealed that 67% of GS2 patients have been reported with neurological manifestations.Identification of RAB27A mutations can facilitate prompt diagnosis and treatment, and aid genetic counselling and prenatal diagnosis. Since five of six patients studied herein initially were diagnosed as having FHL, we conclude that the diagnosis of GS2 may be overlooked, particularly in fair-haired patients with haemophagocytic syndromes.
2318	Doppler indices of the middle cerebral artery in fetuses with cardiac defects theoretically associated with impaired cerebral oxygen delivery in utero: is there a brain-sparing effect?	19953563	To assess changes in the Doppler flow profiles of the middle cerebral artery in fetuses with cardiac defects theoretically associated with impaired cerebral oxygen delivery in utero.Z-scores were calculated for pulsatility and resistance indices (PI and RI, respectively) of the middle cerebral artery (MCA) and the cerebroplacental ratio (CPR) between 19 and 41 weeks' gestation, and for head circumference at birth (HC), in 113 fetuses with the following isolated cardiac defects: transposition of the great arteries (TGA; n = 18), hypoplastic left heart (HLH; n = 46), severe aortic stenosis (n = 17), pulmonary atresia (n = 18) and tetralogy of Fallot (TOF; n = 14). Pregnancies with uteroplacental dysfunction (indicated by increased uterine and/or umbilical Doppler indices), growth restriction, extracardiac malformations, chromosomal anomalies as well as multiple pregnancies were excluded to avoid any additional hypoxemic effect as strictly as possible. The results were compared with 1378 normal controls.Fetuses with pulmonary atresia, severe aortic stenosis and TOF had no significant alterations of Doppler parameters or HC at birth. In fetuses with TGA, mean Z-scores of HC at birth were significantly smaller compared with controls (mean +/- SD, -0.73 +/- 1.25; P < 0.05), but there was no significant difference in the Doppler parameters. Fetuses with HLH had significantly lower MCA-PI (-0.57 +/- 0.74; P < 0.05), MCA-RI (-0.73 +/- 0.85; P < 0.05), CPR (-1.44 +/- 1.05; P < 0.05) and HC (-0.50 +/- 1.24; P < 0.05) Z-scores compared with controls.Fetuses with cardiac defects theoretically associated with markedly impaired cerebral oxygen delivery in utero (TGA and HLH) have smaller HCs at birth. However, only fetuses with HLH have cerebrovascular alterations that are detectable by evaluation of the Doppler indices MCA-PI, MCA-RI and CPR.
2319	Fatal Epstein-Barr virus infection in a case of familial hemophagocytic lymphohistiocytosis with syntaxin-11 mutation.	19950846	Familial hemophagocytic lymphohistiocytosis (FHL) is a fatal disease of early infancy caused by defective natural killer cell activity and is characterized by fever, organomegaly, pancytopenia, and coagulopathy. Disease-causing mutations have been found in perforin, Munc 13-4 and syntaxin-11 genes. We herein describe a case of late-onset FHL with syntaxin-11 mutation in a six-year-old boy in whom only partial response was obtained by immunochemotherapy (HLH-94 protocol) and who died with persistent Epstein-Barr virus (EBV) infection. The role of EBV infection in the prognosis of FHL is discussed.
2320	Histiocytic disorders: recent insights into pathophysiology and practical guidelines.	19932759	The Histiocytoses are defined as non-malignant disorders due to abnormal accumulation and behavior of cells of the mononuclear phagocytic system. The best known histiocytoses, Langerhans cell histiocytosis (LCH), and hemophagocytic lymphohistiocytosis (HLH), each with an estimated incidence of 1/50,000 to 1/150,000, are sufficiently "common," complex and costly, to constitute an important problem in medical practice. At the same time, LCH, HLH and an array of other and more rare histiocytoses are sufficiently uncommon that most physicians lack the experience to diagnose, let alone care for patients with these conditions. The pathophysiology of most of the histiocytoses is unknown and, in the case of the widely-disseminated and potentially fatal forms, treatments to date have been variably effective and sometimes highly toxic. MAS has been reported to occur in association with almost any rheumatic disease, it is by far most common in the systemic form of Juvenile Idiopthic Arthritis (SoJIA). It is now recognized that MAS bears a close resemblance to Hemophagocytic Lymphohistiocytosis or HLH, and MAS is recognized as the major fatal complication of soJIA.
2321	Frequency and development of CNS involvement in Chinese children with hemophagocytic lymphohistiocytosis.	19908295	We investigated the characteristics, frequency, and prognosis of central nervous system (CNS) involvement in patients with hemophagocytic lymphohistiocytosis (HLH).Neurological manifestations were prospectively assessed in 92 children with HLH treated from January 2004 to August 2008 at our center; 82 (89%) had associated viral infections (69 Epstein-Barr virus), one empyema, while no associated disease was identified in the remaining nine. Prior to treatment, all underwent cerebrospinal fluid (CSF) evaluation, brain computer tomography (CT) and/or magnetic resonance imaging (MRI).At diagnosis, 43 (47%) children had CNS involvement. Twelve patients (13%) had neurological symptoms, including seizures, ataxia, coma, cranial nerve palsy, and hemiplegia. All patients improved after 8 weeks of therapy, but one later developed progressive neurological symptoms and six discontinued therapy due to progressive systemic symptoms and/or other reasons. Fifteen patients had CSF abnormalities that all normalized completely after 6 weeks of treatment. Thirty-six patients (39%) had neuroradiological abnormalities; with 5 still under treatment, 15 lost to follow-up, and 16 followed after completion of therapy. Of these 16, 12 improved, 3 were unchanged, and 1 progressed. Among all 21 children with CNS involvement followed after completion of therapy, 10 recovered completely, 10 improved (3 had remaining neuroradiological abnormalities), and 1 progressed clinically and neuroradiologically.Most patients reported here suffered from secondary HLH and since CNS involvement is frequent in HLH, brain MRI at diagnosis is recommended in all HLH patients. Clinical and CSF abnormalities often improved within 8 weeks of therapy, but CT/MRI abnormalities normalized more slowly and less frequently.
2322	Genetic evidence for pax-3 function in myogenesis in the nematode Pristionchus pacificus.	19878288	PAX3 is a member of the PAX3/7 subfamily of the paired box proteins. In vertebrates, Pax3 is essential for skeletal myogenesis by activating a cascade of transcriptional events that are necessary and sufficient for skeletal myogenesis. Four related basic helix-loop-helix transcription factors, MyoD, Myf5, Mrf4, and Myogenin, are targets of PAX3 and serve as myogenic regulatory factors. Although the role of Pax3 in myogenesis is well studied in vertebrates, little is known about invertebrate PAX-3 proteins and myogenesis. Here, we took advantage of viable alleles of pax-3 in the nematode satellite model organism Pristionchus pacificus to investigate the function of PAX-3 in myogenesis. Two strong reduction-of-function alleles of Ppa-pax-3 show severe muscle-derived abnormalities and phalloidin staining indicates a disruption of body wall muscle patterning. Furthermore, we identified a myogenic regulatory factor-related gene Ppa-hlh-1/MyoD and a serum response factor-related gene Ppa-unc-120. Expression of both genes in Ppa-pax-3 mutant animals is down regulated suggesting that Ppa-pax-3 acts upstream in the regulatory network. Together, our results provide the first genetic evidence for a conserved function of PAX-3 in myogenesis between vertebrates and nematodes.
2323	Comprehensive analyses and characterization of haemophagocytic lymphohistiocytosis in Vietnamese children.	19863536	Haemophagocytic lymphohistiocytosis (HLH) is a fatal haematological disorder with diverse aetiology. This prospective study was undertaken to characterize HLH cases in Vietnamese children. Clinical and laboratory data, genetic analyses and outcome of the HLH patients were analysed. A total of 33 patients were enrolled from March 2007 to December 2008, with a median age of 3 years. Mutations of the SH2D1A (SAP) and PRF1 genes were detected in one patient, respectively. The virus association was high, up to 63.6% (21/33), including Epstein-Barr virus (19/33), cytomegalovirus (2/33) and dengue virus (2/33). Five patients had malignant lymphoma and two had autoimmune diseases. Twenty-eight patients were treated according to the HLH-2004 protocol. The first response rate was 64.3% (18/28), with an early death rate of 35.7% (10/28). High levels of interferon-gamma, interleukin-10, MIG and interferon-inducible protein-10 (IP-10) were associated with early mortality (P < 0.05). Reactivation among the responders was high (9/18) and the uneventful resolution was low (3/18) after a median follow-up of 35 weeks. In conclusion, the majority of HLH cases are associated with virus infections in Vietnamese children. Familial HLH is rare. The frequent reactivation and high mortality demands a more appropriate therapeutic regimen in tropical areas like Vietnam.
2324	Identification and characterization of a putative basic helix-loop-helix (bHLH) transcription factor interacting with calcineurin in C. elegans.	19855932	Calcineurin is a Ca(2+)/Calmodulin activated Ser/Thr phosphatase that is well conserved from yeast to human. It is composed of catalytic subunit A (CnA) and regulatory subunit B (CnB). C. elegans homolog of CnA and CnB has been annotated to tax-6 and cnb-1, respectively and in vivo function of both genes has been intensively studied. In C. elegans, calcineurin play roles in various signaling pathways such as fertility, movement, body size regulation and serotonin-mediated egg laying. In order to understand additional signaling pathway(s) in which calcineurin functions, we screened for binding proteins of TAX-6 and found a novel binding protein, HLH-11. The HLH-11, a member of basic helix-loop-helix (bHLH) proteins, is a putative counterpart of human AP4 transcription factor. Previously bHLH transcription factors have been implicated to regulate many developmental processes such as cell proliferation and differentiation, sex determination and myogenesis. However, the in vivo function of hlh-11 is largely unknown. Here, we show that hlh-11 is expressed in pharynx, intestine, nerve cords, anal depressor and vuvla muscles where calcineurin is also expressed. Mutant analyses reveal that hlh-11 may have role(s) in regulating body size and reproduction. More interestingly, genetic epistasis suggests that hlh-11 may function to regulate serotonin-mediated egg laying at the downstream of tax-6.
2325	[Preliminary clinical analysis of the hepatic dysfunction in patients with acquired hemophagocytic lymphohistiocytosis].	19840483	The aim of this study was to investigate the clinical features of acquired hemophagocytic lymphohistiocytosis (HLH) complicated with hepatic dysfunction. 18 cases of acquired HLH were analyzed. The characteristics of hepatic dysfunction, the relationship between hepatic dysfunction and the cause, as well as prognosis of the acquired HLH were preliminarily analysed. The results indicated that characteristics of hepatic dysfunction in acquired HLH patients were hypoproteinemia, jaundice and increase of L-aspirate aminotransferase (AST) and lactate dehydrogenase (LDH) levels. The level of AST and direct bilirubin (DBil) in the non-malignancy associated hemophagocytic lymphohistiocytosis group were higher than that in malignancy-associated hemophagocytic lymphohistiocytosis group (p<0.05). And the increase of LDH and AST levels indicated poor prognosis (p<0.05). In conclusion, liver damage is a common organ functional disorder in patients with acquired HLH, which may be correlated to the cause and the prognosis of acquired HLH.
2326	Hematopoietic stem cell transplantation for familial hemophagocytic lymphohistiocytosis and Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Japan.	19827139	Post-transplant outcomes of hemophagocytic lymphohistiocytosis (HLH) patients were analyzed in Japan where Epstein-Barr virus (EBV)-associated severe forms are problematic.Fifty-seven patients (43 familial HLH [12 FHL2, 11 FHL3, 20 undefined], 14 EBV-HLH) who underwent stem cell transplantation (SCT) between 1995 and 2005 were enrolled based on the nationwide registration.Fifty-seven patients underwent 61 SCTs, including 4 consecutive SCTs. SCTs were employed using allogeneic donors in 93% of cases (allo 53, twin 1, auto 3). Unrelated donor cord blood transplantation (UCBT) was employed in half of cases (21 FHL, 7 EBV-HLH). Reduced intensity conditioning was used in 26% of cases. The 10-year overall survival rates (median +/- SE%) were 65.0 +/- 7.9% in FHL and 85.7 +/- 9.4% in EBV-HLH patients, respectively. The survival of UCBT recipients was >65% in both FHL and EBV-HLH patients. Three out of four patients were alive with successful engraftment after second UCBT. FHL patients showed a poorer outcome due to early treatment-related deaths (<100 days, seven patients) and a higher incidence of sequelae than EBV-HLH patients (P = 0.02). The risk of death for FHL patients having received an unrelated donor bone marrow transplant was marginally higher than that for a related donor SCT (P = 0.05) and that for UCBT (P = 0.07).EBV-HLH patients had a better prognosis after SCT than FHL patients. FHL patients showed either an equal or better outcome even after UCBT compared with the recent reports. UCB might therefore be acceptable as an alternate SCT source for HLH patients, although the optimal conditioning remains to be determined.
2327	Malignancy-associated hemophagocytic lymphohistiocytosis in pediatric cases: a multicenter study from Turkey.	19817262	This study evaluates the clinical and laboratory data of children with secondary hemophagocytic lymphohistiocytosis (sHLH) related to malignancy. Charts of patients who met the diagnostic criteria for sHLH associated with malignancy between January 2000-2006 at six different hospitals in Turkey were reviewed retrospectively. The diagnosis of HLH had been established by bone marrow aspiration in 27 patients, cerebrospinal fluid and bone marrow aspiration in one patient and lung-liver biopsy in another. Twenty-nine children were diagnosed as having sHLH related to malignancy. Twenty cases (18 ALL and 2 AML) with acute leukemia (10 girls/10 boys, median age: 8 years [3-14 years]) were found to have sHLH. Five patients with acute leukemia had HLH at the time of diagnosis (Group 1a), and 15 patients with acute leukemia were diagnosed as having sHLH during therapy (Group 1b), namely reactive sHLH associated with the chemotherapy. Nine patients, including two cases each of rhabdomyosarcoma, neuroblastoma, Hodgkin disease, and non-Hodgkin lymphoma (NHL) and one case with Langerhans cell histiocytosis, were diagnosed as having concomitant hemophagocytosis at the initial evaluation of the tumor (Group 2). Fever, anemia, and hypertriglyceridemia were present in all sHLH cases of all three groups. Hepatomegaly was detected in 60.0%, 73.3%, and 88.8% of the three groups, respectively. Splenomegaly was more frequent in patients of Groups 1a (60.0%) and 2 (88.8%) than in those of Group 1b, the reactive ones (13.3%). Hypofibrinogenemia was detected in all patients of Group 1a and Group 2. Low level of fibrinogen was present in 91.6% of patients in Group 1b. All patients in Group 1b (100%) had neutropenia and thrombocytopenia. Neutropenia was found at rates of 60.0% and 55.5% in Group 1a and Group 2, respectively. Thrombocytopenia was detected in 80.0% of patients in Group 1a and 77.7% in Group 2. The overall mortality rate was 34.4% (10 cases) in our series of 29 children with sHLH; 50% of deaths were directly attributable to HLH. Pediatric malignancy-associated HLH patients have been commonly described as case presentations or in a review of the literature. We believe that our cohort, compiling 29 children regarding the association between malignancy and HLH, will be useful for pediatricians who are interested in this still mysterious topic.
2328	Hemophagocytic lymphohistiocytosis in a patient with x-linked lymphoproliferative disease.	19772767	X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency affecting approximately 1 to 3 per million live male births. Patients are generally healthy until facing a viral infection such as Epstein-Barr Virus and then may develop fulminant infectious mononucleosis and die. XLP patients are also at increased risk of hemophagocytic lymphohistiocytosis (HLH), which may be triggered by assorted viruses. Here we report a novel case of HLH in a patient with XLP. Significant to his presentation is a paradoxical increase in natural killer (NK) cell activity. We hypothesize that this indicates that Parvovirus B19 activates NK cells via a signaling lymphocytic activation molecule-associated protein (SAP)-independent mechanism. Our case demonstrates an important etiology to consider in the differential diagnosis of XLP patients with nonfocal findings and febrile illnesses.
2329	Expression and importance of inhibitor of DNA binding helix-loop-helix protein in salivary adenoid cystic carcinoma.	19766362	Studies have reported that Id-1 (inhibitor of DNA binding, or differentiation 1) protein, helix-loop-helix (HLH) transcription factor, has important roles in the development of several carcinomas. However, the role of Id-1 protein in salivary adenoid cystic carcinoma (ACC) is not clear. The expression of Id-1 protein was assessed in formalin-fixed, paraffin-embedded surgical specimens of ACCs by immunohistochemical staining. Thirty-nine of the 54 specimens of ACC stained for Id-1expression, which was significantly more than was found in pleomorphic adenomas (4/12) and normal salivary gland tissues (1/10, p<0.05). There were also significant differences between stages I and II (7/14) and III-IV (32/40) (p<0.05). Of tumours with metastases, 31/38 stained for Id-1, which was significantly higher than among those without metastases (8/16) (p<0.05). Our results showed that Id-1 may have important roles in the development of salivary ACC. It might also be a useful therapeutic tool to help prevent salivary ACC or to inhibit its malignant progression.
2330	Id proteins facilitate self-renewal and proliferation of neural stem cells.	19757990	Members of helix-loop-helix (HLH) protein family of Id (inhibitor of differentiation) dimerize with bHLH transcription factors and function as negative regulators of differentiation during development. Most of inhibitory roles of Id proteins have been demonstrated in non-neural tissues, and their roles in the developing nervous system are not clearly demonstrated. In this study, we show that Id1, Id2, and Id3 increase self-renewing and proliferation potential of cortical neural stem cells (NSCs) while inhibiting neuronal differentiation. In electrophoretic mobility gel shift and luciferase assays, Id proteins interfered with binding of NeuroD/E47 complexes to the E-box sequences and inhibited E-box-mediated gene expression. Overexpression of Id proteins in NSCs increased both the number and the size of neurospheres in colony-forming assays. Expression of Hes1 and Hes5 was not increased by overexpression of Id proteins under the condition in which Nestin expression was increased. In utero electroporation of Id yielded higher numbers of Ki67-positive and Sox2-positive cells in the mouse embryonic brain. The study suggests Id proteins play independent roles in the maintenance of neural stem properties.
2331	Sequential transplants for respective relapse of Hodgkin disease and hemophagocytic lymphohistiocytosis: a treatment dilemma.	19755921	The association of hemophagocytic lymphohistiocytosis (HLH) with Hodgkin disease (HD) seems to be uncommon. A 9-year-old boy, diagnosed with HD-HLH, received chemotherapy and remained in complete remission for 21 months. Then the patient received the first autologous transplant and unfortunately died due to multiorgan failure after the second allogeneic transplant because of sequentially respective relapse of HD and HLH. The recurrence of HLH with a high Epstein-Barr virus load but without relapse of HD in the patient suggests that Epstein-Barr virus may have a major pathogenetic role in the specific disorder HLH-HD. Detailed reports relating to HLH-HD were also reviewed.
2332	Design and fabrication of white light emitting diodes with an omnidirectional reflector.	19745858	This study investigates white light emitting diodes (LEDs), packaged with an omnidirectional reflector (ODR) and UV LED of 378 nm. The current work designs an ODR to solve the problems of UV leak and low phosphors conversion efficiency with a thin film structure of S/(HLH)n/air modified from a quarter-wave stack. Since an ODR is a perfect dielectric mirror for UV light, the UV leak can be eliminated and recycled to enhance phosphors conversion efficiency. By measuring the emission spectrum of a white LED with an ODR, the orientation distribution of the color properties, and the radiation power, the current study did not detect any UV leak. The average luminous intensity enhancement is 15%.
2333	Hypertrichosis: the possible side effect of cyclosporin in an infant with hemophagocytic lymphohistiocytosis receiving HLH-2004 chemotherapy protocol.	27265500	Hemophagocytic lymphohistiocytosis is a life-threatening condition of severe hyperinflammation that results from an uncontrolled proliferation of activated lymphocytes and histiocytes secreting high amounts of inflammatory cytokines. The immediate treatment strategies include immune suppressive therapy such as corticosteriod, etaposide and cyclosporin A. Herein, we present a 13- month-old infant, who developed severe hypertrichosis after the administration of HLH-2004 treatment protocol and discuss the various hypotheses regarding the causal relationship between cyclosporine A and hypertrichosis, emphasizing the importance of patient follow up. Hemofagositik lenfohistiositoz yüksek düzeylerde enflamatuvar sitokin salgılayan, kontrol edilemeyen aktif lenfosit ve histiosit proliferasyonu ile seyreden ağır enflamasyonun görüldüğü hayatı tehdit eden bir hastalıktır. Tedavi stratejileri arasında korikosteroid, etoposid, ve siklosporin gibi immünsupresif tedaviler yer almaktadır. Bu raporda hemofagositik lenfohistiositoz tanısı alan ve HLH-2004 protokolüne uygun tedavi sonrası ağır hipertrikoz gelişen 13 aylık bir hasta anlatılmıştır. Siklosporin A ve hipertrikoz arasındaki nedensel hipotez tartışılmış ve hasta takibinin önemi anlatılmıştır.
2334	Nationwide survey of single-system single site Langerhans cell histiocytosis in Japan.	19728332	Since neither a standard treatment nor a protocol study for single-system single site (SS-s)-type Langerhans cell histiocytosis (LCH) exists, we conducted a nationwide survey in Japan to clarify the epidemiology and clinical outcome of this subtype.Questionnaires regarding the clinical course of children with SS-s-type LCH diagnosed between 1995 and 2006 were sent to all members of the Japanese Society of Pediatric Hematology.One hundred forty-six children with histologically proven SS-s LCH were evaluable. The most frequently affected organ was bone (82%), followed by skin (12%). Few patients (14%) had a CNS-RISK lesion defined by the Histiocyte Society. Patients with a skin lesion were diagnosed at a significantly younger age than patients with a bone lesion (median: 6 months vs. 5 years 11 months, P < 0.001). The treatment regimen varied, but one-third of the patients in total and 71% of patients with a CNS-RISK lesion received chemotherapy that did not include etoposide. All but one patient attained remission. Ten patients (7%) showed reactivation. Of these, all eight with an initial bone lesion only exhibited reactivation in the bone(s). One patient with an initial skin lesion exhibited reactivation in the thymus. None of the patients died from disease progression or treatment complications.Our retrospective study, in which a relatively large proportion of the patients received chemotherapy, reveals that patients with SS-s LCH have a good prognosis. A prospective study should be conducted to confirm this and to identify the most effective and least toxic therapy for SS-s LCH.
2335	Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates signaling from Toll-like receptor 3.	19720839	Epstein-Barr virus-encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by intermolecular base-pairing, giving rise to double-stranded RNA (dsRNA)-like molecules, and exists abundantly in EBV-infected cells. Here, we report that EBER induces signaling from the Toll-like receptor 3 (TLR3), which is a sensor of viral double-stranded RNA (dsRNA) and induces type I IFN and proinflammatory cytokines. A substantial amount of EBER, which was sufficient to induce signaling from TLR3, was released from EBV-infected cells, and the majority of the released EBER existed as a complex with a cellular EBER-binding protein La, suggesting that EBER was released from the cells by active secretion of La. Sera from patients with infectious mononucleosis (IM), chronic active EBV infection (CAEBV), and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), whose general symptoms are caused by proinflammatory cytokines contained EBER, and addition of RNA purified from the sera into culture medium induced signaling from TLR3 in EBV-transformed lymphocytes and peripheral mononuclear cells. Furthermore, DCs treated with EBER showed mature phenotype and antigen presentation capacity. These findings suggest that EBER, which is released from EBV-infected cells, is responsible for immune activation by EBV, inducing type I IFN and proinflammatory cytokines. EBER-induced activation of innate immunity would account for immunopathologic diseases caused by active EBV infection.
2336	Hemophagocytic lymphohistiocytosis: when the immune system runs amok.	19707989	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome in which an exaggerated but ineffective immune response leads to severe hyperinflammation. Key players in HLH are activated lymphocytes and histiocytes which infiltrate all organs and secrete large amounts of cytokines. Cardinal symptoms are prolonged fever, hepatosplenomegaly, cytopenias, and hemophagocytosis. Biochemical markers include elevated ferritin, triglycerides, and low fibrinogen. HLH occurs on the basis of various inherited and acquired immune defects. Impaired function of natural killer cells and cytotoxic T cells is shared by all forms of HLH. Nearly all genetic defects identified in inherited cases of HLH are either mutations in the perforin gene or in genes important for the exocytosis of cytotoxic granules. Cytotoxic granules contain perforin and granzymes which induce apoptosis upon entering the target cell. Additionally perforin is important for the down-regulation of the immune response. Acquired forms of HLH are found in association with infectious agents, in patients with autoimmune diseases, in malignant diseases, and in patients receiving immune suppression or after organ transplantation. - HLH is still difficult to diagnose and may be overlooked since initially it may masquerade as a normal infection. HLH should be considered when symptoms are more pronounced than usual and in case of progression. Suppression of the severe hyperinflammation can be achieved with immunosuppressive and immunomodulatory agents and cytostatic drugs. Patients with genetic HLH have to undergo stem cell transplantation for cure.
2337	[Significance of hemophagocytosis in diagnosis of hemophagocytic lymphohistiocytosis].	19698261	The study was aimed to explore the diagnostic significance of hemophagocytosis in the patients with hemophagocytic lymphohistiocytosis (HLH). 61 suspected HLH patients from June 2005 to October 2008 were enrolled in the study. The suspected HLH patients were divided into confirmed group (43 out of 61) and excluded group (18 out of 61) according to HLH-2004 diagnostic criteria. The incidences of hemophagocytosis in bone marrow, spleen or lymph nodes were compared in all groups. The results indicated that the hemophagocytosis in bone marrow, spleen or lymph nodes was found in 33 patients of confirmed group, while the hemophagocytosis was found in 4 patients of excluded group. The sensitivity of hemophagocytosis for the diagnosis of HLH was 76.7%, and its specificity was 77.8%. In conclusion, hemophagocytosis is a helpful marker for the diagnosis of most but not all HLH patients, however, the lack of hemophagocytosis does not mean to exclude the diagnosis of HLH.
2338	The Notch-responsive transcription factor Hes-1 attenuates osteocalcin promoter activity in osteoblastic cells.	19670267	Notch signaling plays a key role in osteoblast differentiation. A major transcriptional downstream regulator of this pathway is the helix-loop-helix (HLH) transcription factor Hairy/Enhancer of Split 1 (Hes-1). Here we investigated the function of Hes-1 in osteoblastic cells. Endogenous Hes-1 gene expression decreases during progression of bone cell phenotype development in MC3T3-E1 osteoblasts suggesting that it is a negative regulator of osteoblast differentiation. Forced expression of Hes-1 inhibits osteocalcin (OC) mRNA levels, and luciferase assays indicate that Hes-1 directly represses OC promoter activity. In vitro and in vivo protein/DNA interaction assays reveal that recombinant Hes-1 binds specifically to an E-box in the proximal promoter of the OC gene. Deletion of the Hes-1 WRPW domain (MHes-1) that recruits the co-repressor Groucho abrogates repression of OC promoter activity by Hes-1, but also blocks Hes-1 binding to the promoter. The latter result suggests that exogenous Hes-1 may be recruited to the OC promoter by both protein/DNA and protein/protein interactions. We conclude that the Notch-responsive Hes-1 protein is capable of repressing OC gene transcription in osteoblastic cells through an E-box in the proximal promoter. Hes-1 may contribute to osteoblast growth and differentiation by controlling basal bone-specific transcription directly through interactions with transcriptional regulators that are known to bind to the OC gene promoter.
2339	Prolonged neurologic course of familial hemophagocytic lymphohistiocytosis.	19664539	Familial hemophagocytic lymphohistiocytosis is a very unusual cause for demyelination and the clinician would do well to be aware of the condition, especially when children present with atypical findings on magnetic resonance imaging associated with fever, pancytopenia, and hepatosplenomegaly. This is a rare autosomal recessive, multisystem inflammatory disorder characterized by widespread organ infiltration by macrophages and activated lymphocytes. It is usually diagnosed in the first 2 years of life and is rapidly fatal if untreated. Reported here is the case of a 12-year-old boy, from a poor family, with a 6-year history of visual loss and fever for 5 months, and transient hemiparesis with hepatosplenomegaly and pancytopenia. Cranial magnetic resonance imaging showed multiple areas of hyperintense signal, predominantly involving white matter. The boy also had elevated triglycerides and ferritin, with low fibrinogen level. Bone marrow aspiration revealed hemophagocytosis. He was diagnosed as having familial hemophagocytic lymphohistiocytosis and treated with the HLH 2004 protocol. A sibling also had evidence of hemophagocytosis. Remission was achieved, but his parents could not afford the cost of hematopoietic stem cell transplantation. He relapsed after 8 months and later died.
2340	Hemophagocytosis: the cause of anemia and thrombocytopenia in congenital syphilis.	19657997	Congenital syphilis is a rare, serious disease that continues to be a major health-care problem. The infected neonate may be asymptomatic or multiple-organ system involvement may occur. Anemia and thrombocytopenia are common hematological findings. Hemophagocytic lymphohistiocytosis (HLH) is a clinicopathological condition characterized by activation and uncontrolled nonmalignant proliferation of T lymphocytes and macrophages. The authors report an infant with congenital syphilis as a very rare cause of hemophagocytic syndrome. The reason for anemia and thrombocytopenia in congenital syphilis is not clear. The authors suggest that hemophagocytosis may play role in pathogenesis of cytopenia, particularly thrombocytopenia in patients with congenital syphilis.
2341	Early diagnostic value of low percentage of glycosylated ferritin in secondary hemophagocytic lymphohistiocytosis.	19655220	Elevated levels of serum ferritin and a low percentage of glycosylated ferritin have been reported in adult-onset Still's disease (AOSD) as well as in hemophagocytic lymphohistiocytosis (HLH). The objective of the current study was to investigate total ferritin levels and the percentage of glycosylated ferritin in patients with secondary HLH. From October 2007 to October 2008, 29 patients with suspected HLH older than 14 years of age treated at Beijing Friendship Hospital were enrolled. Twenty-five healthy volunteers were recruited as controls. The suspected HLH patients were further divided into secondary HLH-confirmed (confirmed) (22 out of 29) and HLH-unconfirmed (unconfirmed) (7 out of 29) groups based on HLH-2004 diagnostic criteria. Total serum ferritin levels and the percentage of glycosylated ferritin were determined in subjects at the time of admission. Significantly higher levels of total serum ferritin were observed in confirmed (2,897.6 +/- 1,837.2 microg/L) compared with unconfirmed (653.1 +/- 249.1 microg/L) patients (P < 0.01) or controls (414.6 +/- 212.6 microg/L) (P < 0.01). A significantly lower percentage of glycosylated ferritin was observed in the confirmed (20.5 +/- 10.1%) compared with the unconfirmed (48.7 +/- 12.1%) group (P < 0.01) or the control group (53.6 +/- 13.3%) (P < 0.01). In addition, a low percentage of glycosylated ferritin was observed in HLH patients. Finally, regarding the diagnosis of HLH based on a low percentage of glycosylated ferritin, the sensitivity and specificity, as well as positive and negative predictive values were 0.86, 0.71, 0.91, and 0.63, respectively. For the diagnosis of HLH based on hyperferritinemia, the sensitivity, specificity, positive, and negative predictive values were 0.82, 0.43, 0.82, and 0.43, respectively. The results of this study suggest that a low percentage of glycosylated ferritin is associated with HLH. On comparison with hyperferritinemia, a low percentage of glycosylated ferritin appeared to be more specific, sensitive, and predictive of HLH. In conclusion, a low percentage of glycosylated ferritin may be a useful marker for the early diagnosis of secondary HLH.
2342	Severe course of community-acquired pneumonia in an adult patient who is heterozygous for Q481P in the perforin gene: are carriers of the mutation free of risk?	19639728	Most cases of autosomal recessive hemophagocytic lymphohistiocytosis (HLH) are associated with over 50 mutations in the perforin gene. Some of these mutations have no clear functional association. Only homozygous patients display a full-blown syndrome, whereas no severe disease has been described in heterozygous carriers of these mutations despite the presence of functional and phenotypic alterations in cytotoxic cells. We study the family of a child who died from HLH at 6 months of age due to a Q481P mutation in the perforin gene. The study is particularly interesting because the patient's heterozygous father experienced severe community-acquired pneumonia that could be attributed to deficient in vitro NK cell activity despite normal perforin expression. This case report suggests that impaired NK cell activity in a heterozygote can result in poorer initial control of infections with severe clinical expression.
2343	NMR-assignments of a cytosolic domain of the C-terminus of polycystin-2.	19636966	Mutations in the PKD2 gene lead to the development of polycystic kidney disease (PKD). The PKD2 gene codes for polycystin-2, a cation channel with unknown function. The cytoplasmic, C-terminal domain interacts with a large number of proteins including mDia1, alpha-actinin, PIGEA-14, troponin, and tropomyosin. The C-terminal fragment polycystin-2 (680-796) consisting of 117 amino acids contains a putative calcium binding EF-hand. It was produced in Escherichia coli and enriched uniformly with (13)C and (15)N. The backbone and side chain resonances were assigned by multidimensional NMR methods, the obtained chemical shifts are typical for a partially folded protein. The chemical shifts obtained are in line with the existence of two paired helix-loop-helix (HLH) motifs.
2344	Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis.	19614711	Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK-cell activity in 63 healthy Korean individuals using a flow-cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate-labeled K562 cells as target cells. NK-cell activity was calculated using the following equation: NK-cell activity (%) = (test lysis - spontaneous lysis) x 100/(maximum lysis - spontaneous lysis). NK-cell activity was analyzed in 13 known HLH patients and 16 suspected non-HLH patients using a flow-cytometric assay. The mean (+/-SD) cytotoxicity of PBMCs from healthy individuals was 20.9 +/- 5.3% and the reference interval was 11.8-31.9%. The mean NK-cell activity of HLH patients (8.3 +/- 8.9%) was significantly lower (P = 0.001) than that of non-HLH patients (20.1 +/- 7.8%). The sequential changes in NK-cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK-cell activity test for use in the clinical laboratory and obtained a reference interval of NK-cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.
2345	Phospho-ablated Id2 is growth suppressive and pro-apoptotic in proliferating myoblasts.	19609365	Inhibitor of differentiation protein-2 (Id2) is a dominant negative helix-loop-helix (HLH) protein, and a positive regulator of proliferation, in various cells. The N-terminal region of Id2 contains a consensus cdk2 phosphorylation sequence SPVR, which may be involved with the induction of apoptosis, at least in myeloid 32d.3 cells. However, the role of Id2 phosphorylation at serine 5 in skeletal muscle cells is unknown. The objective of this study was to determine if the phosphorylation of Id2 at serine 5 alters its cellular localization and its role in apoptosis in C2C12 myoblasts. Overexpression of wild type Id2 decreased MyoD protein expression, which corresponded to the increased binding of Id2 to basic HLH proteins E47 and E12. Bromodeoxyuridine incorporation was significantly decreased by the overexpression of phospho-ablated Id2 (S5A); conversely, overexpression of wild type Id2 increased cellular proliferation. The subcellular localization of Id2 and phospho-mimicking Id2 (S5D) were predominantly nuclear compared to S5A. The decreased nuclear localization of S5A corresponded to a decrease in cellular proliferation, and an increase in apoptosis. These data suggest that unphosphorylated Id2 is primarily localized in the cytosol, where it is growth suppressive and potentially pro-apoptotic. These results imply that reducing unphosphorylated Id2 may improve the pool of myoblasts available for differentiation by increasing proliferation and inhibiting apoptosis.
2346	Single- and multi-analyte determination of gonadotropic hormones in urine by Surface Plasmon Resonance immunoassay.	19591706	Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL(-1), which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL(-1) of hLH and 14 mIU mL(-1) of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities < 6%, chip-to-chip variabilities < 5%, recoveries in the range of 80-120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.
2347	Regulation of the Drosophila Enhancer of split and invected-engrailed gene complexes by sister chromatid cohesion proteins.	19587787	The cohesin protein complex was first recognized for holding sister chromatids together and ensuring proper chromosome segregation. Cohesin also regulates gene expression, but the mechanisms are unknown. Cohesin associates preferentially with active genes, and is generally absent from regions in which histone H3 is methylated by the Enhancer of zeste [E(z)] Polycomb group silencing protein. Here we show that transcription is hypersensitive to cohesin levels in two exceptional cases where cohesin and the E(z)-mediated histone methylation simultaneously coat the entire Enhancer of split and invected-engrailed gene complexes in cells derived from Drosophila central nervous system. These gene complexes are modestly transcribed, and produce seven of the twelve transcripts that increase the most with cohesin knockdown genome-wide. Cohesin mutations alter eye development in the same manner as increased Enhancer of split activity, suggesting that similar regulation occurs in vivo. We propose that cohesin helps restrain transcription of these gene complexes, and that deregulation of similarly cohesin-hypersensitive genes may underlie developmental deficits in Cornelia de Lange syndrome.
2348	Hemophagocytic syndrome associated with cytomegalovirus infection in a severely immunocompromised AIDS patient: case report.	19578635	Hemophagocytic syndrome is a clinical condition characterized by the infiltration of the bone marrow and reticuloendothelial system by macrophages and activated histiocytes, leading to uncontrolled phagocytosis of platelets, erythrocytes, lymphocytes and precursor cells. It is a severe inflammatory and aggressive condition, characterized by high fever, hepatosplenomegaly, lymphadenopathy and cytopenia, and it may lead to organ dysfunction. This syndrome is classified as familial or acquired; the latter is more frequent and is associated with diverse conditions, such as infections, malignancies and rheumatic diseases. We report a case of HLH associated with cytomegalovirus infection in a patient with acquired-immunodeficiency syndrome and Burkitt's lymphoma.
2349	[The early diagnosis and clinical analysis of 57 cases of acquired hemophagocytic lymphohistiocytosis].	19576122	To explore the significance of NK cell activity, interleukin-2 receptors (sCD(25)) and glycosylated ferritin in the early diagnostic of acquired hemophagocytic lymphohistiocytosis (HLH).57 patients suspected of HLH from June 2005 to May 2008 and 25 healthy subjects were enrolled in the study. The patients suspected of HLH were divided into three groups i.e. (1) a group with diagnosis confirmed at first visit; (2) a group with diagnosis confirmed at subsequent visit and (3) a group with diagnosis unconfirmed according to HLH-2004 diagnostic criteria. Healthy subjects were enrolled as control. NK cell activity was determined with a released LDH assay. The percentage of glycosylated ferritin was determined with phytohemagglutinin adsorption assay. sCD(25) was examined with ELISA double antibody sandwich assay. We compared the coincidence of each diagnostic index before and after diagnosis.The median percentage of NK cell activity was significantly lower in the first group (18.3 +/- 5.6)% and the second (16.7 +/- 6.7)% than that in the third group (33.4 +/- 6.8)% or in the controls (36.6 +/- 5.0)%. The median percentage of glycosylated ferritin was also significantly lower in the first group (15.4 +/- 2.0)% and the second group (16.9 +/- 3.4)% than that in the third group (40.4 +/- 3.0)% or in the controls (45.2 +/- 2.2)%. Meanwhile, the median level of sCD(25) was significantly higher in the first group (12 916 +/- 4328) ng/L and the second group (12 117 +/- 5465) ng/L than that in the third group (4728 +/- 1482) ng/L or in the controls (3841 +/- 993) ng/L. Furthermore, NK cell activity, sCD(25) and glycosylated ferritin were abnormal in all the patients in the early stage of HLH.NK cell activity, sCD(25) and glycosylated ferritin may be helpful markers for the early diagnosis of HLH.
2350	Infection Associated with Hemophagocytic Lymphohisticytosis triggered by nosocomial Infection.	22224191	Hemophagocytic lymphohistiocytosis (HLH) can occur as primary idiopathic syndrome or secondary to neoplastic, infection or autoimmune processes. It is characterized by the proliferation of histiocytes with phagocytosis of formed elements of blood. Clinical manifestations include signs and symptoms of immune activation and pancytopenia. This report presents a child with infection associated with HLH trigged by Acinetobacter baumannii sepsis. Multidrug-resistant Acinetobacter, an emergent nosocomial pathogen but so far in the literature, it has not been reported to cause HLH.
2351	Eastern equine encephalitis virus infection and hemophagocytic lymphohistiocytosis in a 5-month-old infant.	19483523	Eastern equine encephalitis virus infection is a rare sporadic central nervous system infection transmitted by a mosquito vector. Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease associated with the inability of an overactive immune system to effectively respond to infections. Many viruses are known to trigger primary, as well as secondary, HLH. We report a pediatric case of eastern equine encephalitis virus-associated HLH which caused severe neurologic injury and death.
2352	[Haemophagocytic syndrome: A common pathogenic mechanism of various aetiologies].	19481995	Haemophagocytic syndrome (HPS) is a rare syndrome characterised by the uncontrolled activation and proliferation of histiocytes and T cells, leading to a cytokines overproduction. There are two forms of HPS: primary and secondary.To analyse patients diagnosed with HPS at the Oncohaematology Department, using HLH-94 and 2004 protocol diagnostic criteria.Retrospective study of clinical files of patients diagnosed with HPS, analysing the following features: diagnostic criteria, variability in clinical presentation, aetiology, treatment and outcome.Twenty-two patients were diagnosed with HPS: 6 familial haemophagocytic lymphohistiocytosis (FHL), 11 HPS with evidence of infection, 3 HPS associated with malignant disease and 2 macrophage activation syndrome (MAS) in patients with Crohn's disease and Juvenile Idiopathic Arthritis. The onset of FHL was within 1 year of age in 83.3%, except for 1 patient who was adolescent (MUNC13-4 mutations).All patients (100%) had fever at diagnosis, 18 (85%) hepatosplenomegaly, 7 (31%) lymphadenopathy, 5 (21%) pallor, 3 (14%) rash and 3 (14%) neurological symptoms.all patients (100%) had cytopenias at diagnosis, 20 (90.9%) hypertriglyceridaemia, 19 (86%) hyperferritinaemia, 17 (77%) elevated serum liver enzymes, and 9 (40%) hypofibrinogenaemia. Decreased or absent NK-cell activity was detected in all patients (100%). Haemophagocytosis was found more frequently in bone marrow; however, liver or lymph node biopsies were required in two patients to demonstrate this.Of the ten patients (6 FHL, 3 Epstein-Barr virus-associated HPS and 1 MAS) treated with HLH-94 and 2004 protocols, six received a stem-cell transplant; of these, 2 with FHL had a favourable outcome. The remaining 12 patients received aetiological/supportive therapy, with complete remission in 83.3%.The diagnosis of FHL should be made before the age of 2 years. Advances in genetic studies allow the detection of early and late forms of FHL. Immunochemotherapy and stem-cell transplantation constitute the treatment of FHL and aetiological/supportive therapy of acquired haemophagocytic lymphohistiocytosis, except in severe forms.
2353	Familial hemophagocytic lymphohistiocytosis.	23100980	A 45-day-old infant presented with hepatosplenomegaly and fever since 15 days. Hemogram revealed bicytopenia and bone marrow aspirate showed hemophagocytosis. With the history of death of two siblings, the baby was diagnosed with hemophagocytic lymphohistiocytosis (HLH), likely to be of familial origin.
2354	Separation of the PROX1 gene from upstream conserved elements in a complex inversion/translocation patient with hypoplastic left heart.	19471316	Hypoplastic left heart (HLH) occurs in at least 1 in 10 000 live births but may be more common in utero. Its causes are poorly understood but a number of affected cases are associated with chromosomal abnormalities. We set out to localize the breakpoints in a patient with sporadic HLH and a de novo translocation. Initial studies showed that the apparently simple 1q41;3q27.1 translocation was actually combined with a 4-Mb inversion, also de novo, of material within 1q41. We therefore localized all four breakpoints and found that no known transcription units were disrupted. However we present a case, based on functional considerations, synteny and position of highly conserved non-coding sequence elements, and the heterozygous Prox1(+/-) mouse phenotype (ventricular hypoplasia), for the involvement of dysregulation of the PROX1 gene in the aetiology of HLH in this case. Accordingly, we show that the spatial expression pattern of PROX1 in the developing human heart is consistent with a role in cardiac development. We suggest that dysregulation of PROX1 gene expression due to separation from its conserved upstream elements is likely to have caused the heart defects observed in this patient, and that PROX1 should be considered as a potential candidate gene for other cases of HLH. The relevance of another breakpoint separating the cardiac gene ESRRG from a conserved downstream element is also discussed.
2355	Experimental study on burning rates of square/rectangular gasoline and methanol pool fires under longitudinal air flow in a wind tunnel.	19467784	Square pool fires with length of 5, 7.5, 10, 15, 20, 25 and 30 cm and rectangular pool fires with dimensions of 10 cm x 20 cm and 10 cm x 40 cm were burned in a wind tunnel, under a longitudinal air flow ranged from 0 to 3m/s with incremental change of about 0.5m/s. Methanol and gasoline were burned and compared, with results indicated that their burning rates showed different response to the longitudinal air flow. With the increase of the longitudinal air flow speed, the burning rates of methanol pool fires, except the 5 cm square one, first decreased and then increased, but those of the 5 cm methanol square one and the gasoline pool fires increased monotonously. The burning rate of smaller square pool fires increased more significantly than that of the larger ones, as well as the enlargement of their flame attachment length along the ground. The burning rate of a rectangular pool fire with longer rim parallel to the longitudinal flow increased faster, but the flame attachment length seemed to increase more gradually, with the increase of the longitudinal air flow speed than that perpendicular to.
2356	Pediatric acute lymphoblastic leukemia complicated by secondary hemophagocytic lymphohistiocytosis.	19459197	Hemophagocytic lymphohistiocytosis (HLH) may be familial or secondary to infections, malignancies, metabolic disorders. Infectious causes are mostly viral and EBV is the mostly frequently seen etiologic agent. In this case, we report a child with acute lymphoblastic leukemia (ALL), who had three episodes of secondary HLH, possibly due to two different viral etiologies, namely CMV and RSV together with her malignancy, during her induction-consolidation treatment.
2357	Id3 is a direct transcriptional target of Pax7 in quiescent satellite cells.	19458195	Pax7 is a key regulator of skeletal muscle stem cells and is required along with Pax3 to generate skeletal muscle precursors. We have identified a collection of genes induced by either Pax3 or Pax7 in C2C12 muscle cells. Two notable Pax3/7 targets are the inhibitory helix-loop-helix (HLH) proteins inhibitor of DNA binding (Id) 2 and Id3, both of which are coordinately expressed with Pax7 in quiescent satellite cells and are induced in quiescent C2C12 myogenic cells after ectopic expression of either Pax3 or Pax7. Ectopic Pax7 activates expression of a luciferase reporter driven by the Id3 promoter, and maximal induction of this reporter requires a conserved Pax7 binding site located upstream of the Id3 gene. Chromatin immunoprecipitation indicated that Pax7 is bound upstream of the Id3 promoter in quiescent satellite cells. In addition, short hairpin RNA-mediated knockdown of Pax7 expression in cultured satellite cells coordinately decreased both Id2 and Id3 expression. Together, these findings indicate that Id3 is a direct transcriptional target for Pax7 in quiescent satellite cells, and they suggest that Pax7 acts to block premature differentiation of quiescent satellite cells by inducing the expression of Id2 and Id3, which in turn may act to block either the precocious induction of myogenic basic (b)HLH proteins, the activity of myogenic bHLH proteins, or both.
2358	Sequestration of E12/E47 and suppression of p27KIP1 play a role in Id2-induced proliferation and tumorigenesis.	19451188	Id2 is a member of the helix-loop-helix (HLH) family of transcription regulators known to antagonize basic HLH transcription factors and proteins of the retinoblastoma tumor suppressor family and is implicated in the regulation of proliferation, differentiation, apoptosis and carcinogenesis. To investigate its proposed role in tumorigenesis, Id2 or deletion mutants were re-expressed in Id2(-/-) dermal fibroblasts. Ectopic expression of Id2 or mutants containing the central HLH domain increased S-phase cells, cell proliferation in low and normal serum and induced tumorigenesis when grafted or subcutaneously injected into athymic mice. Similar to their downregulation in human tumors, the expression of cyclin-dependent kinase inhibitors p27(KIP1) and p15(INK4b) was decreased by Id2; the former by downregulation of its promoter by the Id2 HLH domain-mediated sequestration of E12/E47. Re-expression of p27(KIP1) in Id2-overexpressing cells reverted the hyperproliferative and tumorigenic phenotype, implicating Id2 as an oncogene working through p27(KIP1). These results tie together the previously observed misregulation of Id2 with a novel mechanism for tumorigenesis.
2359	Administration of follitropin alfa and lutropin alfa combined in a single injection: a feasibility assessment.	19450267	Gonadotrophins are routinely administered in assisted reproductive technology (ART) treatment protocols. Recombinant human follicle-stimulating hormone (r-hFSH; follitropin alfa) and recombinant human luteinizing hormone (r-hLH; lutropin alfa) can be administered individually or in a fixed combination. The ability to vary the FSH to LH dose ratio in a single injection without compromising the bioactivity of either gonadotrophin or generating losses of active principle is important for physicians and patients alike.This study investigated whether follitropin alfa (GONAL-f ), as lyophilized powder for reconstitution or solution from the GONAL-f (filled-by-mass [FbM]) Prefilled Pen, could be used to reconstitute Pergoveris TM (follitropin alfa/lutropin alfa 150 IU/75 IU) lyophilized powder. In Ratio Groups 1 and 2, the r-hFSH:r-hLH ratio was 3:1; in Ratio Groups 3 and 4, the ratios of r-hFSH:r-hLH were 5:1 and 8:1, respectively. The protein content and bioactivity of each mixed solution were evaluated. The r-hFSH and r-hLH content was determined using reverse-phase high performance liquid chromatography. The biological activity of r-hFSH and r-hLH was assessed using the Steelman-Pohley and Van Hell in vivo bioassays in rats, respectively.Follitropin alfa/lutropin alfa 150 IU/75 IU lyophilized powder could be successfully mixed with follitropin alfa 75 IU FbM solution that was either reconstituted from lyophilized powder or injected directly from the prefilled pen to create solutions with ratios of r-hFSH and r-hLH from 3:1 to 8:1. The measured content of r-hFSH and r-hLH corresponded favourably with the target protein content in Ratio Groups 1-4. The in vivo target and measured bioactivity of r-hFSH and r-hLH were also closely matched in all Ratio Groups.Follitropin alfa lyophilized powder or solution can be accurately mixed with follitropin alfa/lutropin alfa 150 IU/75 IU lyophilized powder to enable physicians to administer ratios of r-hFSH and r-hLH in the IU range from 3:1 to 8:1. Mixing of recombinant formulations offers flexibility for patients receiving follitropin alfa and lutropin alfa for ART protocols in clinical practice.
2360	Max-E47, a designed minimalist protein that targets the E-box DNA site in vivo and in vitro.	19449889	Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nanomolar K(d) values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14, 15, 9, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site.
2361	Characteristics of hemophagocytic lymphohistiocytosis in neonates: a nationwide survey in Japan.	19446847	To assess the etiology, prognosis, and appropriate treatment of hemophagocytic lymphohistiocytosis (HLH) in neonates.We collected information on neonates in whom HLH was diagnosed between 1997 and 2007 from participating members of the Japanese Society of Pediatric Hematology.HLH was diagnosed in 20 patients within 4 weeks after birth. Of the diagnostic criteria for HLH-2004, the incidence of fever was quite low in preterm infants, and hypertriglyceridemia and neutropenia were uncommon. Familial HLH (n = 6) or severe combined immunodeficiency-associated HLH (n = 1) was diagnosed in 7 patients, and 2 of them have survived. Herpes simplex virus-associated HLH was diagnosed in 6 patients, and 2 of them have survived. The overall survival rate for the 20 patients was 40%.HLH is rare in neonates and has a poor prognosis. Early diagnosis and immediate treatment are required when considering the possibility of herpes simplex virus-associated or familial HLH.
2362	Analysis of triglyceride value in the diagnosis and treatment response of secondary hemophagocytic syndrome.	19443971	Secondary hemophagocytic syndrome (hemophagocytic lymphohistiocytosis, HLH) follows viral infection, malignant disorders, and autoimmune disease. Criteria for HLH diagnosis, which were proposed in 2004, include hypertriglyceridemia. However, some studies reported the absence of hypertriglyceridemia in patients with secondary HLH, differing from those with primary HLH.In this study, we investigated the presence or absence of hypertriglyceridemia in 28 patients who were diagnosed with secondary HLH between 1997 and 2007 retrospectively. There were no patients undergoing treatment for those with a history of hyperlipidemia.The subjects consisted of 14 patients with lymphoma-associated HLH, 11 with virus-associated HLH, 2 with autoimmune disease-associated HLH, and 1 with post transplantation HLH. In 19 patients (68%), hypertriglyceridemia was noted on diagnosis or during the disease period (mean: 242 mg/dL). Furthermore, the triglyceride (TG) level decreased with the treatment-related amelioration of HLH (mean level before and after treatment: 297 and 136 mg/dL, respectively, p=0.0001).These results suggest that the TG level is useful for diagnosing HLH and evaluating the treatment response. TG measurement is simple and inexpensive; therefore, this parameter can be determined several times to evaluate the treatment response.
2363	Secondary acute myeloid leukemia after etoposide therapy for haemophagocytic lymphohistiocytosis.	19434733	Haemophagocytic lymphohistiocytosis (HLH) is an uncommon disease with a high fatality rate. Etoposide is an important component of current HLH treatment regimes. Two patients with HLH developed etoposide-related secondary acute myeloid leukemia (sAML) following therapy for HLH. Etoposide, an epipodophyllotoxin, is a topoisomerase II inhibitor that interacts with DNA to potentiate leukaemogenesis. The risk of developing sAML is estimated to be between 1% and 5%, 2-20 years after exposure to etoposide but may also be related to cumulative drug doses, treatment schedules, host factors and co-administration of other antineoplastic agents.
2364	Hemophagocytic lymphohistiocytosis and Kawasaki disease: combined manifestation and differential diagnosis.	1943472820658642	Both hemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD) are diagnosed in patients with prolonged resistant fever by using a scoring system. Concurrent manifestation of both conditions has been reported previously. We describe an infant of 7 weeks whose condition fulfilled the criteria of HLH, but who, after clinical response to treatment, suddenly died from a myocardial infarction at 11 weeks. Post-mortem examination revealed a previously unknown coronary arteritis typical for KD. Since it is difficult to distinguish between KD and HLH, both diseases should be considered in young children with overlapping symptoms. Repeated echocardiograms may be helpful in these cases.
2365	An unusual cause of multiple organ dysfunction syndrome in the pediatric intensive care unit: hemophagocytic lymphohistiocytosis.	19433941	To report our experience in children with primary or secondary hemophagocytic lymphohistiocytosis (HLH) presented with multiple organ dysfunction syndrome (MODS) in pediatric intensive care unit (PICU).The records of patients with a diagnosis of HLH and MODS between January 2005 and January 2008 were reviewed. The patients' characteristics, treatment modalities, and outcomes were assessed.PICU of Ege University Hospital.Twelve children who were hospitalized in the PICU met the diagnostic criteria for HLH, and presented with MODS were entered into the study.The median age of the patients was 3 years (range, 2 months-15.5 years). Six patients had a history of parental consanguinity and two had an affected sibling. Five of the patients were classified as primary HLH. All of the patients had hepatosplenomegaly, elevated ferritin levels, hypofibrinogenemia, anemia, thrombocytopenia, and hemophagocytosis in bone marrow examination at presentation. The median Pediatric Logistic Organ Dysfunction score of the patients at onset was 51 (range, 12-62). Four patients had six, four had five, two had four, and the remaining two had three organ dysfunctions. Organ dysfunction, other than hematologic dysfunction which was present in all patients, was most commonly seen in hepatic (n = 11, 91.7%), respiratory (n = 11, 91.7%), and cardiovascular systems (n = 10, 83.3%). Although nine patients showed neurologic dysfunction including convulsion and coma, renal failure was detected in five patients. Eleven patients were supported with mechanical ventilation and four patients required hemodialysis. Eight patients were treated according to the HLH 2004 treatment protocol, consisting of cyclosporine A, etoposide, and dexamethasone. The remaining four patients received only intravenous immunoglobulin and supportive treatment. Seven of the patients died.HLH is a frequently lethal disease and with a clinical presentation similar to severe sepsis, MODS, disseminated intravascular coagulation, or septic shock, which are frequent diagnoses in the PICU. In the PICU, HLH should be considered in the case of prolonged fever, splenomegaly, cytopenia, and MODS. It is important for pediatricians and particularly pediatric intensivists to know the diagnostic criteria and possible clinical presentations of HLH so treatment is initiated promptly.
2366	A conserved Six-Eya cassette acts downstream of Wnt signaling to direct non-myogenic versus myogenic fates in the C. elegans postembryonic mesoderm.	19427847	The subdivision of mesodermal cells into muscle and non-muscle cells is crucial to animal development. In the C. elegans postembryonic mesoderm, this subdivision is a result of an asymmetric cell division that leads to the formation of striated body wall muscles and non-muscle coelomocytes. Here we report that the Six homeodomain protein CEH-34 and its cofactor Eyes Absent, EYA-1, function synergistically to promote the non-muscle fate in cells also competent to form muscles. We further show that the asymmetric expression of ceh-34 and eya-1 is regulated by a combination of 1) mesodermal intrinsic factors MAB-5, HLH-1 and FOZI-1, 2) differential POP-1 (TCF/LEF) transcriptional activity along the anterior-posterior axis, and 3) coelomocyte competence factor(s). These factors are conserved in both vertebrates and invertebrates, suggesting a conserved paradigm for mesoderm development in metazoans.
2367	Hematopoietic stem cell transplantation in Griscelli syndrome type 2: a single-center report on 10 patients.	19403888	Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for Griscelli syndrome type 2, an inherited immune disorder causing fatal hemophagocytic lymphohistiocytosis (HLH). Optimal therapeutic modalities are not yet well known. We retrospectively analyzed the outcome for 10 patients who underwent HSCT in a single center between 1996 and 2008. Seven patients (70%) were cured of the primary immune defect (mean follow-up, 5.2 years; range, 0.8-12.0 years), 4 of them without neurologic sequelae. In the 3 deceased patients, death occurred within 110 days of HSCT and was probably due to adverse reaction to HSCT in 2 patients and to HLH relapse in one patient. One patient received 2 transplants because of graft failure. Clinical events included veno-occlusive disease (n = 5), acute (n = 7) or chronic (n = 1) graft-versus-host disease II-III, and Epstein-Barr virus-induced lymphoproliferative disease (n = 2). Of the 7 patients with neurologic involvement before HSCT, 4 survived and 2 presented sequelae. Furthermore, 1 patient lacking neurologic involvement before HSCT developed long-term sequelae. These results demonstrate the efficacy of HSCT in curing the immune disorder but also show that neurologic HLH before HSCT is a major factor, given the neurologic sequelae after otherwise successful HSCT. Additional studies are required to improve treatment.
2368	Dlx1&2 and Mash1 transcription factors control MGE and CGE patterning and differentiation through parallel and overlapping pathways.	19386638	Here we define the expression of approximately 100 transcription factors (TFs) in progenitors and neurons of the developing mouse medial and caudal ganglionic eminences, anlage of the basal ganglia and pallial interneurons. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most gamma-aminobutyric acidergic projection neurons of the basal ganglia. This analysis identified Dlx-dependent and Dlx-independent pathways. The Dlx-independent pathway depends in part on the function of the Mash1 basic helix-loop-helix (b-HLH) TF. These analyses define core transcriptional components that differentially specify the identity and differentiation of the globus pallidus, basal telencephalon, and pallial interneurons.
2369	Linking asymmetric cell division to the terminal differentiation program of postmitotic neurons in C. elegans.	19386265	How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. We establish here a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/beta-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. Our study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/beta-catenin pathway is widely used to control the identity of neuronal lineages.
2370	Reactive haemophagocytic lymphohistiocytosis.	19381497	We present a series of five cases diagnosed and treated as reactive haemophagocytic lymphohistiocytosis (HLH) in three tertiary referral centers of Kolkata, within a time frame of 3 months. The initial presentations were very variable, the most prominent clinical feature being--acute renal failure in the first patient, convulsions in the second, encephalopathy the third, marked cervical lymphadenopathy in the fourth and polyserositis in the fifth. All had a history of prolonged fever preceding admission and hepatosplenomegaly on examination. Investigations revealed multi-organ involvement with pancytopenia; haemophagocytosis was eventually diagnosed by bone marrow examination. These cases highlight the diagnostic challenge posed by infection associated haemophagocytosis and the need for maintaining a high index of suspicion to promptly diagnose and treat this potentially life threatening condition.
2371	C. elegans HLH-2/E/Daughterless controls key regulatory cells during gonadogenesis.	19376107	The Caenorhabditis elegans distal tip cell (DTC) provides a niche for germline stem cells in both hermaphrodites and males. The hermaphrodite distal tip cell (hDTC) also provides "leader" function to control gonadal elongation and shape, while in males, leader function is allocated to the linker cell (LC). Therefore, the male distal tip cell (mDTC) serves as a niche but not as a leader. The C. elegans homolog of E/Daughterless, HLH-2, was previously implicated in hDTC specification. Here we report that HLH-2 is also critical for hDTC maintenance, hDTC niche function and hDTC expression of a lag-2/DSL ligand reporter. We also find that HLH-2 functions in males to direct linker cell specification and to promote both mDTC maintenance and the mDTC niche function. We conclude that HLH-2 functions in both sexes to promote leader cell specification and DTC niche function.
2372	The C. elegans Snail homolog CES-1 can activate gene expression in vivo and share targets with bHLH transcription factors.	19372275	Snail-type transcription factors (TFs) are found in numerous metazoan organisms and function in a plethora of cellular and developmental processes including mesoderm and neuronal development, apoptosis and cancer. So far, Snail-type TFs are exclusively known as transcriptional repressors. They repress gene expression by recruiting transcriptional co-repressors and/or by preventing DNA binding of activators from the basic helix-loop-helix (bHLH) family of TFs to CAGGTG E-box sequences. Here we report that the Caenorhabditis elegans Snail-type TF CES-1 can activate transcription in vivo. Moreover, we provide results that suggest that CES-1 can share its binding site with bHLH TFs, in different tissues, rather than only occluding bHLH DNA binding. Together, our data indicate that there are at least two types of CES-1 target genes and, therefore, that the molecular function of Snail-type TFs is more plastic than previously appreciated.
2373	Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples.	19361471	Direct determination of four pituitary peptide hormones: human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), and luteinizing hormone (hLH) has been carried out using a portable surface plasmon resonance (SPR) immunosensor.A commercial SPR biosensor was employed. The immobilization of the hormones was optimized and monoclonal antibodies were selected in order to obtain the best sensor performance. Assay parameters as running buffer and regeneration solution composition or antibody concentration were adjusted to achieve a sensitive analyte detection.The performance of the assays was assessed in buffer solution, serum and urine, showing sensitivity in the range from 1 to 6 ng/mL. The covalent attachment of the hormones ensured the stability of the SPR signal through repeated use in up to 100 consecutive assay cycles. Mean intra- and inter-day coefficients of variation were all <7%, while batch-assay variability using different sensor surfaces was <5%.Taking account both the excellent reutilization performance and the outstanding reproducibility, this SPR immunoassay method turns on a highly reliable tool for endocrine monitoring in laboratory and point-of-care (POC) settings.
2374	[Characteristics of re-suspended road dust and its significant effect on the airborne particulate pollution in Beijing].	19353848	The re-suspended road dust samples from 53 sites that could cover basically the entire Beijing in summer, and aerosol samples from three representative sites in Beijing in four seasons were collected to study concentrations and spatial variations of elements and ions in resuspended road dust and its contribution to the air-borne particulate pollution. Ca, S, Cu, Zn, Ni, Pb and Cd were main pollution elements, and Ca2+, SO4(2-), Cl-, K+, Na+ and NO3- were major ions in re-suspended road dust. Al, Ti, Sc, Co and Mg in re-suspended road dust were mainly originated from crustal source, while Cu, Zn, Ni and Pb were mainly originated from traffic emissions and coal burning, and Fe, Mn and Cd were mainly from industrial emissions and coal combustion. Ca2+ and SO4(2-) mainly came from construction activities, construction materials and secondary gas-particle conversions, Cl- and Na+ were derived from industrial wastewater disposal and chemical industrial emissions, and NO3- and K+ were from vehicle emissions, photochemical reactions of NO,, biomass and vegetable burning. The contribution of mineral aerosol from inside Beijing to the total mineral aerosols was 30% in spring of 2002, 70% in summer of 2002, 80% in autumn of 2003, 20% in winter of 2002. The pollution levels of some major pollution species, Ca, S, Cu, Zn, Ni, Pb, Fe, Mn, and Cd in resuspended road dust reached 76%, 87%, 75%, 80%, 82%, 90%, 45%, 51%, and 94%, respectively, and their contributions to the related elements in PM10 were 20%-45%, 5%-18%, 4%-50%, 2%-46%, 4%-52%, 5%-20%, 30%-60%, 20%-40% and 2%-25%. Re-suspended road dust from the traffic and construction activities is one of the major sources of the air-born particulate pollution in Beijing.
2375	Atypical varicella zoster infection associated with hemophagocytic lymphohistiocytosis.	19353624	Two adolescents, on immunosuppressive therapy for graft-versus-host disease, developed hemophagocytic lymphohistiocytosis (HLH) after varicella zoster virus (VZV) reactivation. In the absence of dermatome restricted characteristic skin lesions, VZV reactivation was not immediately recognized and treatment with acyclovir was delayed. The first patient developed optical neuritis and died 2 months after the VZV episode due to massive intracranial hemorrhage. The second patient presented with severe abdominal pain and pancreatitis, followed by atypical skin eruptions, which prompted a faster diagnosis. Both patients recovered from their HLH, the first patient being successfully treated with immunosuppressive agents and the second with VZV treatment only. These two cases demonstrate the difficulties in recognizing VZV reactivation, and in order to start adequate and timely treatment, the need to consider VZV as a possible cause of HLH in severely immunocompromised patients.
2376	Infection of T lymphocytes in Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children of non-Asian origin.	1935362119533652	Epstein-Barr virus (EBV) is one of the most frequent triggers of hemophagocytic lymphohistiocytosis (HLH). EBV-associated HLH (EBV-HLH) and ectopic infection of T cells has been particularly described in patients from Far East Asia.In a cohort of 12 children with EBV-HLH treated in Germany, the EB viral load was detected by real-time polymerase chain reaction in plasma and peripheral blood mononuclear cells (PBMC). Virological and clinical data were analyzed retrospectively.Among the 12 mainly German patients, children with underlying immunodeficiencies as well as otherwise healthy individuals were affected. The clinical course ranged from a steroid-responding to a fatal disease despite intensive treatment. Increased EBV copy numbers in plasma and/or PBMC were found in all patients. Serial measurements reflected the course of the disease. Cell-type specific viral load was determined in seven patients and revealed EBV-infection of T cells in all of them. In contrast to the reported Asian patients a significant viral load was also found in B cells.T cell infection appears to be a typical feature of EBV-associated HLH irrespective of patients ethnic background and the clinical course. Evaluation of cell-type specific infection should be considered when targeted therapy is applied.
2377	Engineering of dominant active basic helix-loop-helix proteins that are resistant to negative regulation by postnatal central nervous system antineurogenic cues.	19350686	Neural precursor cells (NPCs) are present in most regions of the adult central nervous system (CNS). Using NPCs in a therapeutical perspective, that is, to regenerate CNS tissue after injury or in neurodegenerative diseases, will require the efficient manipulation of their fate. Proneural gene overexpression in NPCs represents a promising strategy to promote neuronal differentiation. The activity of the proneural proteins is, however, context-dependent and can be inhibited/modulated by binding with other bHLH (basic helix-loop-helix) or HLH transcription factors. In this study, we show that the two proneural proteins, Ngn2 and Mash1, are differentially sensitive to negative regulation by gliogenic factors or a gliogenic substrate (i.e., postnatal spinal cord slices). Coexpressing E-proteins with proneural proteins was efficient to rescue proneural proteins neurogenic activity, suggesting a central role for E-protein sequestration in mediating postnatal CNS gliogenic inhibition. Tethering of proneural proteins with E47 further insulated Mash1 from negative environmental influences whereas this strategy was not successful with Ngn2, suggesting that mechanisms of inhibition differ in between these two proneural proteins. Our results demonstrate that a better understanding of proneural protein modulation by environmental cues is a prerequisite to develop innovative approaches that will permit the manipulation of the fate of NPCs in the adult CNS after trauma or disease.
2378	Suppression of chondrogenesis by Id helix-loop-helix proteins in murine embryonic orofacial tissue.	19349107	Inhibitors of differentiation (Id) proteins are helix-loop-helix (HLH) transcription factors lacking a DNA-binding domain. Id proteins modulate cell proliferation, apoptosis and differentiation in embryonic/fetal tissue. Perturbation of any of these processes in cells of the developing orofacial region results in orofacial anomalies. Chondrogenesis, a process integral to normal orofacial ontogenesis, is known to be modulated, in part, by Id proteins. In the present study, the mRNA and protein expression patterns of Id1, Id2, Id3 and Id4 were examined in developing murine orofacial tissue in vivo, as well as in murine embryonic maxillary mesenchymal cells in vitro. The functional role of Ids during chondrogenesis was also explored in vitro. Results reveal that cells derived from developing murine orofacial tissue (1) express Id1, Id2, Id3 and Id4 mRNAs and proteins on each of gestational days 12-14, (2) express all four Id proteins in a developmentally regulated manner, (3) undergo chondrogenesis and express genes encoding various chondrogenic marker proteins (e.g. Runx2, Type X collagen, Sox9) when cultured under micromass conditions and (4) can have their chondrogenic potential regulated via alteration of Id protein function through overexpression of a basic HLH factor. In summary, results from the current report reveal for the first time the expression of all four Id proteins in cells derived from developing murine orofacial tissue, and demonstrate a functional role for the Ids in regulating the ability of these cells to undergo chondrogenesis.
2379	Differential expression of the Enhancer of split genes in the developing Drosophila midgut.	19348652	The Notch signaling pathway plays an important role during development in animals from worms to humans and pathway components are required for the differentiation of many different cell types. In Drosophila, Su(H) dependent Notch activation up-regulates transcription of the Enhancer of split-Complex (E(spl)-C). The E(spl) genes are known to function during neurogenesis, although expression and genetics studies suggest that they also play roles in the development of other tissues. The majority of the E(spl) genes contain upstream binding sites for Su(H), proneural proteins, and E(spl) bHLH proteins resulting in overlapping expression patterns during embryonic development. However, their expression patterns are quite distinct during later embryonic stages and in larval imaginal discs. In order to characterize expression patterns of the E(spl) genes during development and determine potential mechanisms through which expression is controlled, we examined the expression levels and patterns of the E(spl) genes in the midgut during metamorphosis. Quantitative Reverse Transcriptase-PCR and X-Gal staining results show that the genes have different levels and patterns of expression in the developing midgut. Two ancestral E(spl) genes, malpha and mbeta, are highly expressed and increase significantly at puparium formation, whereas another gene, mgamma, is expressed at low levels and decreases in expression at puparium formation. We also show that mbeta is expressed in cells throughout the midgut, while mgamma is expressed in two small regions. These results provide further evidence that the E(spl) genes function during midgut development and that they are regulated by different factors.
2380	Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation.	19330020	Inhibitor of DNA-binding (Id) proteins prevent cell differentiation, promote growth and sustain tumour development. They do so by binding to E proteins and other transcription factors through the helix-loop-helix (HLH) domain, and inhibiting transcription. This makes HLH-mediated Id protein interactions an appealing therapeutic target. We have used the dominant interfering HLH dimerization mutant 13I to model the impact of Id inhibition in two human neuroblastoma cell lines: LA-N-5, similar to immature neuroblasts, and SH-EP, resembling more immature precursor cells. We have validated 13I as an Id inhibitor by showing that it selectively binds to Ids, impairs complex formation with RB, and relieves repression of E protein-activated transcription. Id inactivation by 13I enhances LA-N-5 neural features and causes SH-EP cells to acquire neuronal morphology, express neuronal proteins such as N-CAM and NF-160, proliferate more slowly, and become responsive to retinoic acid. Concomitantly, 13I augments the cell-cycle inhibitor p27(Kip1) and reduces the angiogenic factor vascular endothelial growth factor. These effects are Id specific, being counteracted by Id overexpression. Furthermore, 13I strongly impairs tumorigenic properties in agar colony formation and cell invasion assays. Targeting Id dimerization may therefore be effective for triggering differentiation and restraining neuroblastoma cell tumorigenicity.
2381	Secondary hemophagocytic lymphohistiocytosis and severe sepsis/ systemic inflammatory response syndrome/multiorgan dysfunction syndrome/macrophage activation syndrome share common intermediate phenotypes on a spectrum of inflammation.	19325510	In an effort to attain earlier diagnoses in children with hemophagocytic lymphohistiocytosis (HLH), the International Histiocyte Society has now broadened their diagnostic criteria to no longer differentiate primary (HLH) and secondary hemophagocytic lymphohistiocytosis (SHLH). Five of the following eight diagnostic criteria needed to be met: 1) fever, 2) cytopenia of two lines, 3) hypertriglyceridemia and/or hypofibrinogenemia, 4) hyperferritinemia (>500 microg/L), 5) hemophagocytosis, 6) elevated soluble interleukin-2 receptor (CD25), 7) decreased natural killer-cell activity, and 8) splenomegaly can also commonly be found in patients with sepsis, systemic inflammatory response syndrome (SIRS), multiorgan dysfunction syndrome (MODS), and macrophage activation syndrome (MAS). Nevertheless, the therapeutic options for these are radically different. Chemotherapy and bone marrow transplant have been used for treatment of HLH/SHLH, whereas antibiotics and supportive treatment are used in severe sepsis/SIRS and MODS. MAS is treated with limited immune suppression. Outcomes are also different, SHLH has a mortality rate around 50%, whereas pediatric septic shock and MODS have a mortality of 10.3% and 18%, respectively, and severe sepsis in previously healthy children has a mortality rate of 2%. MAS has a mortality rate between 8% and 22%. Because SHLH and severe sepsis/SIRS/MODS/MAS share clinical and laboratory inflammatory phenotypes, we recommend extreme caution when considering applying HLH therapies to children with sepsis/SIRS/MODS/MAS. HLH therapies are clearly warranted for children with HLH; however, a quantitative functional estimate of cytotoxic lymphocyte function may be a more precise approach to define the overlap of these conditions, better identify these processes, and develop novel therapeutic protocols that may lead to improved treatments and outcomes.
2382	Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma.	19321746	Although the identification and characterization of translocations have rapidly increased, little is known about the mechanisms of how translocations occur in vivo. We used anaplastic large cell lymphoma (ALCL) with and without the characteristic t(2;5)(p23;q35) translocation to study the mechanisms of formation of translocations and of ALCL transformation. We report deregulation of several genes located near the ALCL translocation breakpoint, regardless of whether the tumor contains the t(2;5). The affected genes include the oncogenic transcription factor Fra2 (located on 2p23), the HLH protein Id2 (2p25), and the oncogenic tyrosine kinase CSF1-receptor (5q33.1). Their up-regulation promotes cell survival and repression of T cell-specific gene expression programs that are characteristic for ALCL. The deregulated genes are in spatial proximity within the nuclear space of t(2;5)-negative ALCL cells, facilitating their translocation on induction of double-strand breaks. These data suggest that deregulation of breakpoint-proximal genes occurs before the formation of translocations, and that aberrant transcriptional activity of genomic regions is linked to their propensity to undergo chromosomal translocations. Also, our data demonstrate that deregulation of breakpoint-proximal genes has a key role in ALCL.
2383	Daxx inhibits muscle differentiation by repressing E2A-mediated transcription.	19308989	The basic helix-loop-helix (HLH) E2A transcription factors bind to DNA as homodimers or as heterodimers formed with other basic HLH factors, activate gene expression, and promote differentiation of muscle, lymphoid, neuronal, and other cell types. These E2A functions can be inhibited by the Id proteins, HLH factors that sequester E2A in non-DNA binding dimers. Here we describe the direct interaction of E2A with Daxx, a broadly expressed non-HLH protein previously associated with apoptosis and transcriptional repression. Daxx inhibits E2A function, but not via an Id-like mechanism; rather, it recruits histone deacetylase activity to E2A-dependent promoters. Increased Daxx expression during muscle differentiation inhibits E2A-dependent expression of key myogenic genes and reduces myotube formation, while decreased Daxx expression promotes myotube formation. These results identify a new mechanism for limiting E2A activity and establish a link between Daxx-mediated gene regulation and control of cellular differentiation.
2384	Pregnancy-induced hemophagocytic lymphohistiocytosis combined with autoimmune hemolytic anemia.	19299225	Hemophagocytic lymphohistiocytosis (HLH), presenting with fever, cytopenia, liver dysfunction, hepatosplenomegaly, hypertriglyceridemia, and hyperferritinemia, is associated with various etiologies, including infections, collagen vascular diseases, and malignancies. The present report describes a 28-year-old woman who developed HLH combined with autoimmune hemolytic anemia (AIHA) at 23 weeks of gestation. Without response to corticosteroid, the patient completely recovered from both HLH and AIHA after termination of the pregnancy. Pregnancy-induced immune dysregulation and cytokine overproduction in genetically susceptible women may play critical roles in HLH. The differential diagnosis of pregnant women with fever and cytopenia should include HLH. Pregnancy termination should be considered when pregnancy-induced HLH is refractory to medical treatment.
2385	A rapid flow cytometric screening test for X-linked lymphoproliferative disease due to XIAP deficiency.	19288545	Deficiency of X-linked inhibitor of apoptosis (XIAP), caused by BIRC4 gene mutations, is the second known cause of X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency that often presents with life-threatening hemophagocytic lymphohistiocytosis (HLH). Rapid diagnosis of the known genetic causes of HLH, including XIAP deficiency, facilitates the initiation of life-saving treatment and preparation for allogeneic hematopoietic cell transplantation (HCT). Until now, a rapid screening test for XIAP deficiency has not been available.To develop a flow cytometric screening test for XIAP deficiency, we first used lymphoblastic cell lines generated from controls and patients with BIRC4 mutations to identify two commercially available antibodies specific for native intracellular XIAP. Next, we used these antibodies to study control whole blood leukocyte XIAP expression. We then studied XIAP expression in leukocytes from patients with XLP due to BIRC4 mutations, maternal carriers, and patients following HCT.XIAP was expressed by the majority of all whole blood nucleated cells in normal controls. In contrast, XIAP was absent or decreased in all lymphocyte subsets, monocytes and granulocytes from four unrelated patients with XLP due to BIRC4 mutations. Bimodal distribution of XIAP expression was evident in two maternal carriers, with significant skewing toward cells expressing normal XIAP. Bimodal distribution was also observed in a patient following HCT.Flow cytometric analysis of intracellular XIAP provides a rapid screening test for XLP due to XIAP deficiency. It also allows carrier detection and can be used to monitor donor versus recipient reconstitution following HCT.
2386	Griscelli syndrome type 2: long-term follow-up after unrelated donor bone marrow transplantation.	19270433	Griscelli syndrome (GS) is a rare autosomal recessive disease characterized by silvery hair ('partial albinism'). Three forms exist; GS type 2 (GS2), the most common one, is characterized by severe primary immunodeficiency with acute episodes of hemophagocytic lymphohistiocytosis (HLH) which may be fatal in the absence of hematopoietic stem cell transplantation. A 5-year-old boy with HLH was referred to us because of silvery-gray hair present since birth. Abnormal pigment clumps were observed in the medulla of hair shafts on light microscopy. Electron microscopy of a skin biopsy revealed melanosomes in melanocytes, but not in keratinocytes. Leukocytes were devoid of intracytoplasmic granules on blood smear. Neurological signs were absent. Genotyping revealed a homozygous haplotype for polymorphic markers linked to the RAB27A locus, but no RAB27A mutation. A diagnosis of GS2 was established. The patient received bone marrow transplantation (BMT) from an unrelated donor, and after 72 months he did not show relapse of HLH. The long, uneventful follow-up supports the use of BMT from an unrelated donor if transplantation from a relative is not possible.
2387	A basic-HLH transcription factor, HLH54F, is highly expressed in the prothoracic gland in the silkworm Bombyx mori and the fruit fly Drosophila melanogaster.	19270399	We describe our findings on HLH54F, a basic helix-loop-helix transcription factor gene that was highly expressed in the prothoracic gland, an organ producing the insect steroid ecdysone. HLH54F was uncovered by the use of an expressed sequence tag database of the silkworm Bombyx mori. It was also highly expressed in the prothoracic gland of the fruit fly Drosophila melanogaster.
2388	Type II Gaucher disease manifesting as haemophagocytic lymphohistiocytosis.	19267217	Haemophagocytic lymphohistiocytosis (HLH) is a rare and rapidly progressive disease which, untreated, invariably leads to death. Gaucher disease is a rare lysosomal storage disorder. The acute neuronopathic variant; type II, is also rapidly progressive. We report an infant with Gaucher disease type II manifesting as HLH. Immunoblot revealed a deficiency of Munc 13-4, an intracellular protein responsible for docking of secretory lysosomes. This, and other possible pathogenetic mechanisms to explain the link are discussed.
2389	Caudal-like PAL-1 directly activates the bodywall muscle module regulator hlh-1 in C. elegans to initiate the embryonic muscle gene regulatory network.	19261701	Previous work in C. elegans has shown that posterior embryonic bodywall muscle lineages are regulated through a genetically defined transcriptional cascade that includes PAL-1/Caudal-mediated activation of muscle-specific transcription factors, including HLH-1/MRF and UNC-120/SRF, which together orchestrate specification and differentiation. Using chromatin immunoprecipitation (ChIP) in embryos, we now demonstrate direct binding of PAL-1 in vivo to an hlh-1 enhancer element. Through mutational analysis of the evolutionarily conserved sequences within this enhancer, we identify two cis-acting elements and their associated transacting factors (PAL-1 and HLH-1) that are crucial for the temporal-spatial expression of hlh-1 and proper myogenesis. Our data demonstrate that hlh-1 is indeed a direct target of PAL-1 in the posterior embryonic C. elegans muscle lineages, defining a novel in vivo binding site for this crucial developmental regulator. We find that the same enhancer element is also a target of HLH-1 positive auto regulation, underlying (at least in part) the sustained high levels of CeMyoD in bodywall muscle throughout development. Together, these results provide a molecular framework for the gene regulatory network activating the muscle module during embryogenesis.
2390	Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors.	19257909	Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor alpha (ERalpha)-positive MCF-7 and SKOV-3 cancer cells.MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.Overexpression of Id2 in ERalpha-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.
2391	A role for Id2 in regulating photic entrainment of the mammalian circadian system.	1921729219368876	Inhibitor of DNA binding genes (Id1-Id4) encode helix-loop-helix (HLH) transcriptional repressors associated with development and tumorigenesis [1, 2], but little is known concerning the function(s) of these genes in normal adult animals. Id2 was identified in DNA microarray screens for rhythmically expressed genes [3-5], and further analysis revealed a circadian pattern of expression of all four Id genes in multiple tissues including the suprachiasmatic nucleus. To explore an in vivo function, we generated and characterized deletion mutations of Id2 and of Id4. Id2(-/-) mice exhibit abnormally rapid entrainment and an increase in the magnitude of the phase shift of the pacemaker. A significant proportion of mice also exhibit disrupted rhythms when maintained under constant darkness. Conversely, Id4(-/-) mice did not exhibit a noticeable circadian phenotype. In vitro studies using an mPer1 and an AVP promoter reporter revealed the potential for ID1, ID2, and ID3 proteins to interact with the canonical basic HLH clock proteins BMAL1 and CLOCK. These data suggest that the Id genes may be important for entrainment and operation of the mammalian circadian system, potentially acting through BMAL1 and CLOCK targets.
2392	Hyperferritinemia as the diagnostic clue in life-threatening hemophagocytic lymphohistiocytosis.	19199227	We report on a seventeen year old girl with persistent fever of unknown origin. An initial episode of abdominal pain led to laparotomy and appendectomy, which did not reveal any pathological findings. In the course of the next 3 weeks, the girl's general condition progressively deteriorated. Despite extensive diagnostics, no explanation was found. In summary, the girl was cared for by five different departments in two hospitals before she was admitted to our Children's Hospital. We too were initially misguided by the clinical picture of an infectious disease and treated the girl unsuccessfully with antibiotics. The clue in this case was the finding of a markedly elevated level of serum ferritin. While a normal upper value of less than 400 microg/l is reported, our patient displayed levels above 60,000 microg/l. Such extreme elevations of serum ferritin have been almost exclusively reported for hemophagocytic syndromes, as hemophagocytic lymphohistiocytosis (HLH). In HLH, impaired cytolytic function of T cells and natural killer cells leads to a state of hyperinflammation. We treated our patient with immunoglobulins and corticosteroids. Simultaneously, we started immunomodulatory therapy with oral cyclosporine A. The clinical response to this treatment was remarkable. The child's general condition stabilized quickly and the fever vanished. Additionally, the recovery was accompanied by a normalization of laboratory findings. In conclusion, HLH is a potentially life-threatening disease, which has to be considered in cases with fever of unknown origin. In our case, the diagnostic clue was an extreme elevation of ferritin.
2393	Experience with hemophagocytic lymphohistiocytosis/macrophage activation syndrome at a single institution.	19194188	Hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS) is a serious and potentially life threatening histiocytic disorder in children and adults. The most commonly used protocol-based therapy includes corticosteroids, cyclosporine-A, and etoposide. However, patients are often started on corticosteroid alone with or without the addition of intravenous gamma-globulin. The role of the various therapies in HLH/MAS remains undefined.To identify patient-related factors that led to the use of full protocol therapy (HLH 1994/2004) and to determine treatment-related factors that were associated with adverse outcome including relapse and death.Patients who were diagnosed with HLH/MAS between January 1998 and December 2005 were included in this study.Thirty-eight patients had a median age of 9.1 years at diagnosis. Underlying diagnoses were: viral/other 42%; rheumatologic 37%; and malignancy 21%. Initial treatment included corticosteroids 29%; intravenous immunoglobulin (IVIG) 18%; steroids+IVIG 8%; cyclosporine 5%; etoposide 5%; HLH protocol 32%. Etoposide was eventually used in 21% (3/14) of rheumatology and 75% (18/25) viral/other patients. In all, 5/14 (36%) rheumatology and 12/16 (75%) viral/other patients required intensive care unit admission, and 1/14 (7.1%) rheumatology, and 6/16 (38%) viral/other patients died. Three children received a bone marrow transplant. Eleven of 38 (29%) patients died, despite 8 having received etoposide therapy. Three deaths were secondary to underlying malignancy and one from transplant-related complication for malignancy.Patients with HLH are at high risk for death early in their disease course. However, corticosteroids and/or IVIG may be sufficient as first-line therapy for patients with underlying rheumatologic disease who present with HLH/MAS. Further prospective studies are required to more precisely define early risk factors for poor outcomes in this often fatal disease.
2394	The Max homodimeric b-HLH-LZ significantly interferes with the specific heterodimerization between the c-Myc and Max b-HLH-LZ in absence of DNA: a quantitative analysis.	19189276	Specific heterodimerization plays a crucial role in the regulation of the biology of the cell. For example, the specific heterodimerization between the b-HLH-LZ transcription factors c-Myc and Max is a prerequisite for c-Myc transcriptional activity that leads to cell growth, proliferation and tumorigenesis. On the other hand, the Mad proteins can compete with c-Myc for Max. The Mad/Max heterodimer antagonizes the effect of the c-Myc/Max heterodimer. In this contribution, we have focused on the specific heterodimerization between the b-HLH-LZ domains of c-Myc and Max using CD and NMR. While the c-Myc and Max b-HLH-LZ domains are found to preferentially form a heterodimer; we demonstrate for the first time that a significant population of the Max homodimeric b-HLH-LZ can also form and hence interferes significantly with the specific heterodimerization. This indicates that the Max/Max homodimer can also interfere with c-Myc/Max functions, therefore adding to the complexity of the regulation of transcription by the Myc/Max/Mad network. The demonstration of the existence of the homodimeric population was made possible by the application of numerical routines that enable the simulation of composite spectroscopic signal (e.g. CD) as a function of temperature and total concentration of proteins. From a systems biology perspective, our routines may be of general interest as they offer the opportunity to treat many competing equilibriums in order to predict the probability of existence of protein complexes.
2395	Large eddy simulation of fire-induced buoyancy driven plume dispersion in an urban street canyon under perpendicular wind flow.	19153006	The dispersion of fire-induced buoyancy driven plume in and above an idealized street canyon of 18 m (width) x 18 m (height) x 40 m (length) with a wind flow perpendicular to its axis was investigated by Fire Dynamics Simulator (FDS), Large Eddy Simulation (LES). Former studies, such as that by Oka [T.R. Oke, Street design and urban canopy layer climate, Energy Build. 11 (1988) 103-113], Gayev and Savory [Y.A. Gayev, E. Savory, Influence of street obstructions on flow processes within street canyons. J. Wind Eng. Ind. Aerodyn. 82 (1999) 89-103], Xie et al. [S. Xie, Y. Zhang, L. Qi, X. Tang, Spatial distribution of traffic-related pollutant concentrations in street canyons. Atmos. Environ. 37 (2003) 3213-3224], Baker et al. [J. Baker, H. L. Walker, X. M. Cai, A study of the dispersion and transport of reactive pollutants in and above street canyons--a large eddy simulation, Atmos. Environ. 38 (2004) 6883-6892] and Baik et al. [J.-J. Baik, Y.-S. Kang, J.-J. Kim, Modeling reactive pollutant dispersion in an urban street canyon, Atmos. Environ. 41 (2007) 934-949], focus on the flow pattern and pollutant dispersion in the street canyon with no buoyancy effect. Results showed that with the increase of the wind flow velocity, the dispersion pattern of a buoyant plume fell into four regimes. When the wind flow velocity increased up to a certain critical level, the buoyancy driven upward rising plume was re-entrained back into the street canyon. This is a dangerous situation as the harmful fire smoke will accumulate to pollute the environment and thus threaten the safety of the people in the street canyon. This critical re-entrainment wind velocity, as an important parameter to be concerned, was further revealed to increase asymptotically with the heat/buoyancy release rate of the fire.
2396	Severe neurologic side effects in patients being treated for hemophagocytic lymphohistiocytosis.	19137570	Hemophagocytic Lymphohistiocytosis (HLH) is characterized by uncontrolled inflammation that is generally fatal without immune modulating chemotherapy. At Texas Children's Hospital, we have observed significant central nervous system (CNS) toxicity in several patients treated for HLH according to the Histiocyte Society protocol HLH-2004 in which cyclosporine is given early in the treatment regimen.Patients diagnosed with HLH at Texas Children's Hospital between April 2004 and October 2007 were identified and charts were reviewed. A reference group of patients treated between August 2001 and March 2004, prior to the introduction of HLH-2004, was also evaluated.Five of 17 patients in the study group developed severe neurotoxicity. Four had new onset seizures associated with significant MRI abnormalities, while the fifth died of intracerebral hemorrhage. Timing of the development of neurologic side effects ranged from day 5 to week 6 of therapy. Cyclosporine levels were outside the therapeutic range (200-300 ng/ml) prior to the onset of symptoms in two of the five patients. Systolic blood pressures for all five patients were greater than the 95th percentile for age on at least one measurement within 24 hr of the onset of neurologic symptoms. MRI scans obtained within 24 hr of seizure activity in four patients were consistent with posterior reversible encephalopathy syndrome (PRES). By comparison only one patient in the reference group (n = 15) had neurotoxicity (PRES).Patients being treated for HLH appear to be at risk for neurotoxicity, particularly PRES. Elevated blood pressure, worsening renal and liver function, increased cyclosporine levels, and CNS involvement of HLH may be triggers for the neurotoxic side effects of treatment. Patients being treated on HLH-2004 require close monitoring of their neurologic status and modifiable risk factors such as hypertension should managed aggressively. If larger studies validate our observations, it will be important to determine if up-front cyclosporine in HLH protocols confers a survival benefit that outweighs the potential risk of increased neurotoxicity.
2397	Neonatal primary hemophagocytic lymphohistiocytosis in Turkish children.	19131769	Although the data on hemophagocytic lymphohistiocytosis (HLH) has gradually increased, the neonatal-onset HLH patients have usually been reported as case reports or together with other age groups of patients. The aim of this study was to draw attention to the clinical and laboratory characteristics of neonatal HLH cases. Herein, the data of 8 primary, neonatal-onset HLH patients are reported. Mutational analyses were performed in 7 of the patients and mutations in UNC13D gene were detected in 3 of the patients, whereas 2 patients were found to have perforin gene mutation. Four of the patients were symptomatic within the initial 10 days of life. One patient with perforin mutation (1122 G>A) had a very severe clinical course and died on the seventh day of life before receiving any specific treatment. Another patient with UNC13D 2783 G>C, who became symptomatic on the sixth day of life, underwent early hematopoietic stem cell transplantation and is currently alive at 8 years of age. Two of these 4 patients had extensively high serum ferritin levels mimicking neonatal hemochromatosis. Of the 4 patients who became symptomatic after 20th day of newborn period, 1 was found to have perforin gene mutation (445 G>A) and 2 siblings were detected to have a missense mutation in UNC13D (640 C>T) gene. The latter patients with UNC13D mutations could survive 3 and 4 months, although their parents ceased therapy. The patient with perforin mutation survived 11 months.
2398	The HLH protein Extramacrochaetae is required for R7 cell and cone cell fates in the Drosophila eye.	19118542	Notch signaling is one of the most important pathways in development and homeostasis, and is altered in multiple pathologies. Study of Drosophila eye development shows that Notch signaling depends on the HLH protein Extramacrochaetae. Null mutant clones show that extramacrochaetae is required for multiple aspects of eye development that depend on Notch signaling, including morphogenetic furrow progression, differentiation of R4, R7 and cone cell types, and rotation of ommatidial clusters. Detailed analysis of R7 and cone cell specification reveals that extramacrochaetae acts cell autonomously and epistatically to Notch, and is required for normal expression of bHLH genes encoded by the E(spl)-C which are effectors of most Notch signaling. A model is proposed in which Extramacrochaetae acts in parallel to or as a feed-forward regulator of the E(spl)-Complex to promote Notch signaling in particular cellular contexts.
2399	Hemophagocytic lymphohistiocytosis-late diagnosis in an adult patient.	21847419	We present a case of hemophagocytic lymphohistiocytosis (HLH) in a previously healthy 30-year-old woman. The patient presented with features consistent with HLH: persistent fever, neurological abnormalities, lymphadenopathy, anaemia, leucopoenia and markedly elevated serum lactate dehydrogenase and ferritin levels. Diagnosis was delayed for approximately 1 month beyond presentation. Once initiated on treatment, the patient rapidly improved and was discharged from the intensive care unit and subsequently sent home. Unfortunately, she succumbed to progressive HLH 5 months after her initial presentation. This case highlights key clinical features associated with HLH to help prevent late diagnosis as delayed treatment may lead to irreversible multi-organ failure and/or death.
2400	Cloak and dagger: the case for adult onset still disease and hemophagocytic lymphohistiocytosis.	19115058	Adult onset Still's disease (AOSD) is a rare systemic inflammatory disorder of unknown etiology. Systemic onset juvenile idiopathic arthritis (SoJIA) is the preferred nomenclature of Still's disease. Strong association with so-called macrophage activation syndrome (MAS) may provide a clue to the understanding of the distinctive pathogenetic features of SoJIA. MAS is a severe, potentially life-threatening complication characterized by the excessive activation of well-differentiated macrophages. It is more appropriately named autoimmune disease associated reactive hemophagocytic lymphohistiocytosis (ReHLH), a subset of a histiocytic disorder: class II histiocytosis hemophagocytic lymphohistiocytosis (HLH). The relation of SoJIA with HLH is still under debate. We propose that MAS, HLH, SoJIA, and AOSD are indeed the same disease, in different clinical presentations that may be classified based on severity and laboratory findings, but with essentially the same physiopathogenesis. We propose that the case described by Hong & Lee (Rheumatol Int 2008) was actually an AOSD-associated MAS/RHS/ReHLH fulminant disease.
2401	Mice lacking asparaginyl endopeptidase develop disorders resembling hemophagocytic syndrome.	19106291	Asparaginyl endopeptidase (AEP or legumain) is a lysosomal cysteine protease that cleaves protein substrates on the C-terminal side of asparagine. AEP plays a pivotal role in the endosome/lysosomal degradation system and is implicated in antigen processing. The processing of the lysosomal proteases cathepsins in kidney is completely defective in AEP-deficient mice with accumulation of macromolecules in the lysosomes, which is typically seen in lysosomal disorders. Here we show that mutant mice lacking AEP develop fever, cytopenia, hepatosplenomegaly, and hemophagocytosis, which are primary pathological manifestations of hemophagocytic syndrome/hemophagocytic lymphohistiocytosis (HLH). Moreover, AEP deficiency provokes extramedullary hematopoiesis in the spleen and abnormally enlarged histiocytes with ingested red blood cells (RBCs) in bone marrow. Interestingly, RBCs from AEP-null mice are defective in plasma membrane components. Further, AEP-null mice display lower natural killer cell activity, but none of the major cytokines is substantially abnormal. These results indicate that AEP might be a previously unrecognized component in HLH pathophysiology.
2402	Combined informatic and expression screen identifies the novel DAF-16 target HLH-13.	19103192	Insulin/IGF-signaling (IIS) affects longevity, stress resistance and metabolism in worms, flies, and mammals. The Forkhead transcription factor DAF-16/FOXO is the major downstream effector of IIS and is responsible for the activation and repression of genes that mediate the diverse effects of IIS. We surveyed a set of informatically predicted conserved DAF-16/FOXO target genes and identified the novel DAF-16 direct target hlh-13. hlh-13 is the predicted homolog of the mammalian transcription factor Ptf1a, a critical determinant of pancreatic development. We found that an hlh-13 mutant exits L1 arrest and IIS-dependent dauer diapause faster than control worms, but is not involved in lifespan or resistance to a variety of stresses. Our results have identified a novel DAF-16 target gene and linked its function to known outputs of IIS. Considering the high conservation of IIS in diverse species, our results also hint at an intriguing connection of IIS and Ptf1a in mammals.
2403	[Diagnostic significance of glycosylated ferritin for patients with secondary hemophagocytic lymphohistiocytosis].	19099648	This study was aimed to explore the level of glycosylated ferritin in the patients with secondary hemophagocytic lymphohistiocytosis (HLH) and its diagnostic significance. 29 suspected HLH patients from October 2007 to October 2008 were enrolled in the study, and 25 healthy subjects were selected as control. The 29 suspected HLH patients were divided into confirmed group (22 out of 29) and unconfirmed group (7 out of 29) according to HLH-2004 diagnostic criteria. The percentage of glycosylated ferritin in peripheral blood was determined by phytohemagglutinin adsorption assay. The results showed that the median level of total serum ferritin in patients of confirmed group (2897.6+/-1837.2 microg/L) was significantly higher than that in patients of unconfirmed group (653.1+/-249.1 microg/L) (p<0.01), and was also higher than that in controls (414.6+/-212.6 microg/L) (p<0.01). The median percentage of glycosylated ferritin in patients of confirmed group was significantly lower (17.0+/-4.2%) than that in patients of unconfirmed group (40.7+/-4.5%) (p<0.01) and was lower than that in controls (53.6+/-13.3%) (p<0.01). The sensitivity (86.4% vs 77.3%) and specificity (71.4% vs 42.9%) of glycosylated ferritin for the diagnosis of HLH were higher than that of total serum ferritin. In conclusions, glycosylated ferritin may be a helpful marker for the diagnosis of HLH.
2404	Fatal hemophagocytic lymphohistiocytosis in X-linked chronic granulomatous disease associated with a perforin gene variant.	19058215	A patient with previously unrecognized X-linked chronic granulomatous disease (X-CGD) died of multi-organ failure, secondary to ongoing infection and hemophagocytic lymphohistiocytosis (HLH). Post mortem histological investigations were compatible with X-CGD, and a CYBB gene mutation was confirmed. No homozygous mutations in the genes encoding perforin (PRF1), MUNC 13-4 or syntaxin-11 (STX11) were found; however, there was a heterozygous alteration c.1471G>A in the PRF1 gene causing a p.Asp491Asn substitution. Although this substitution has not been reported to cause primary or secondary HLH, we speculate that it may have made the patient more susceptible for HLH under the circumstances of ongoing infection associated with X-CGD.
2405	Clinical aspects, immunologic assessment, and genetic analysis in Taiwanese children with hemophagocytic lymphohistiocytosis.	19057461	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by immune disturbance associated with certain genetic defects. This study aimed to define the clinical spectrum, immunology, and candidate genes in HLH in Taiwanese patients.The medical records of children who met the updated diagnostic criteria and had confirmed hemophagocytosis by bone marrow aspiration between 1992 and 2007 were retrospectively evaluated. The clinical course and prognosis were analyzed. Quantitative immunoglobulin, lymphocyte subsets, cytotoxicity to K562 cells, intracellular natural killer perforin expression, and candidate genes including SH2D1A, PFR1, Mun13-4, and STX11 genes were also investigated.Thirty-two patients (16 male) were evaluated during the 15-year period. The underlying diseases were acute lymphoblastic leukemia, systemic lupus erythematosus, natural killer malignancy, juvenile idiopathic arthritis, and Chediak-Higashi syndrome. Epstein-Barr virus (EBV)-associated HLH was present in 12 patients, 6 of whom died, while 12 of 20 non-EBV-associated patients died despite aggressive polychemotherapy. Extreme immunoglobulin values occurred in 3 fatal cases. There was decreasing percentage of CD4, CD1656, and B-memory cells and increasing CD8, activated lymphocyte, and T-memory cells among the patients. Cytotoxicity in 14 patients and intracellular perforin expression in 13 were diminished but restored to borderline normal range during the convalescent stage. None of the mutations of SH2D1A, PFR1, Mun13-4, and STX11 genes were found.In the treatment of Taiwanese HLH patients, aggressive chemotherapy in EBV-associated patients and stem cell transplantation in non-EBV-associated patients are recommended to improve survival. Individualized immune dysregulation instead of the well-known candidate genetic mutations can explain the genetic variation in our cohort.
2406	[Two young adult cases of Epstein-Barr virus associated-hemophagocytic lymphohistiocytosis with monoclonal proliferation of virus-infected cells within a short period after primary infection].	19047785	Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe complication of Epstein-Barr virus (EBV) infection. Interactions between EBV-infected T cells and activated macrophages cause several conditions such as pancytopenia, liver dysfunction and coagulopathy. We describe here two young adults with EBV-associated HLH with monoclonal proliferation of EBV-infected T cells within a short period after infectious mononucleosis as a primary infection. One patient was a 16-year-old man who developed severe pancytopenia and liver dysfunction two months after infectious mononucleosis. Bone marrow examination showed hemophagocytosis, and laboratory data demonstrated monoclonal proliferation of EBV-infected T cells. Several treatments such as immunosuppressive therapy, chemotherapy and hematopoietic stem cell transplantation were not effective, and the patient died of progressive disease. The other patient was a 19-year-old woman who developed thrombocytopenia and liver dysfunction two months after infectious mononucleosis. Findings of hemophagocytosis and monoclonal proliferation of EBV-infected T cells were similar to those in the first case. Clinical signs and symptoms were resolved completely by immunosuppressive therapy containing methyl-prednisolone and cyclosporine. Since these two cases each demonstrated a distinct clinical course, an investigation of the prognostic factors and treatment strategies for EBV-HLH is warranted.
2407	Dlx1&2 and Mash1 transcription factors control striatal patterning and differentiation through parallel and overlapping pathways.	19030180	Here we define the expression of approximately 100 transcription factors in progenitors and neurons of the developing basal ganglia. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most GABAergic projection neurons of the basal ganglia. This analysis identified Dlx-dependent and Dlx-independent pathways. The Dlx-independent pathway depends in part on the function of the Mash1 b-HLH transcription factor. These analyses define core transcriptional components that differentially specify the identity and differentiation of the striatum, nucleus accumbens, and septum.
2408	TAL1/SCL relieves the E2-2-mediated repression of VEGFR2 promoter activity.	1902914321172954	The basic helix-loop-helix (bHLH) protein TAL1/SCL is essential for embryonic-vascular development. TAL1/SCL regulates the activation of endothelial cells by binding directly or indirectly to DNA sequences in critical target genes. We recently demonstrated that E-box protein E2-2 blocks endothelial cell activation via perturbation of VEGFR2 promoter activity. Herein, we report that TAL1/SCL interacts with E2-2 and inhibits E2-2-mediated effects on reporter activity. Mutational analysis revealed that the HLH domain of TAL1/SCL, but not its basic region, is required for interaction with E2-2. Importantly, TAL1/SCL relieves the E2-2-mediated repression of VEGFR2 reporter activity in endothelial cells. Our data elaborate on the bHLH protein interactions that regulate endothelial cell activation.
2409	The use of reduced-intensity stem cell transplantation in haemophagocytic lymphohistiocytosis and Langerhans cell histiocytosis.	18978744	Allogeneic stem cell transplant is curative for haemophagocytic lymphohistiocytosis (HLH) and refractory Langerhans cell histiocytosis (LCH). However, patients frequently have significant pre-transplant morbidity and there is high TRM. Because HLH is caused by immune dysregulation, we surmised that a reduced-intensity conditioned (RIC) regimen might be sufficient for cure, while decreasing the TRM. In 2006, we reported the outcome of 12 patients treated with RIC SCT from a matched family/unrelated or haploidentical donor. Here we discuss the update of these patients, including a total of 25 patients treated with RIC SCT for HLH and three for LCH. Twenty-one of the twenty-five patients with HLH (84%) are alive and well with remission at a median of 36 months from SCT. Mortality included pneumonitis (n=3) and hepatic rupture (n=1). All three patients treated with RIC SCT for LCH remain alive and in remission at a median of 5.1 years from SCT. Seven of twenty-four survivors (one with LCH) have mixed chimerism but remain disease-free. These data are supported by other groups including 100% survival in seven patients with HLH and 78% survival of nine patients with LCH. In summary, RIC compares favourably with conventional SCT with long-term disease control in surviving patients with both HLH and LCL, despite a significant incidence of mixed chimerism.
2410	Association of monoclonal expansion of Epstein-Barr virus-negative CD158a+ NK cells secreting large amounts of gamma interferon with hemophagocytic lymphohistiocytosis.	19020108	We report the first case of hemophagocytic lymphohistiocytosis (HLH) induced by the monoclonal expansion of Epstein-Barr virus (EBV)-negative NK cells. Consanguinity of the patient's parents made it necessary to discard familial HLH in the patient and her sister with identical HLA markers and demonstrate that no cause other than the expansion of NK cells, which secrete high levels of gamma interferon, was inducing HLH in this patient.
2411	Comment on HLH in a child with anaplastic large cell lymphoma (ALCL).	1901146619011482	NaN
2412	Expression of the inhibitor of DNA-binding (ID)-1 protein as an angiogenic mediator in tumour advancement of uterine cervical cancers.	19002177	The ID protein, an inhibitor of basic helix-loop-helix (HLH) transcription factors, has been involved in multiple cellular processes. To investigate the association between tumour advancement and ID expressions of uterine cervical cancers, the levels of ID-1, ID-2 and ID-3 mRNAs were determined by real-time reverse transcription-polymerase chain reaction and the histoscore with the localisation of ID-1 was determined by immunohistochemistry and patient survival in 60 patients. ID-1 histoscores and mRNA levels both significantly (P<0.05) increased in uterine cervical cancers according to clinical stage regardless of histopathological type or lymph node metastasis. Furthermore, the 36-month survival rate of the 30 patients with high ID-1 was poor (60%), whereas that of the other 30 patients with low ID-1 was significantly higher (83%). ID-1 histoscores and mRNA levels significantly (P<0.0001) correlated with microvessel counts in uterine cervical cancers. Tumour cells show mostly diffuse to strong cytoplasmic expression of ID-1 and also very faint expression in endothelial cells. Moreover, ID-1 expression not only correlated with microvessel counts but also correlated significantly with histoscore. Therefore, ID-1 might work on tumour advancement through angiogenic activity and is considered to be a candidate for a prognostic indicator in uterine cervical cancers.
2413	A Griscelli syndrome type 2 murine model of hemophagocytic lymphohistiocytosis (HLH).	18991284	Griscelli syndrome type 2 is caused by mutations in the RAB27A gene and is a rare and potentially fatal immune disorder associated with hemophagocytic lymphohistiocytosis (HLH). Animal models could provide assistance for better understanding the mechanisms and finding new treatments. Rab27a-deficient (ashen) mice do not spontaneously develop HLH. When injected with lymphocytic choriomeningitis virus (LCMV) strain WE, Rab27a-deficient C57BL/6 mice developed wasting disease, hypothermia, splenomegaly, cytopenia (anemia, neutropenia and thrombocytopenia), hypertriglyceridemia and increased levels of IFN-gamma, TNF-alpha, GM-CSF, IL-12, CCL5 and IL-10. Activated macrophages with hemophagocytosis were found in liver sections of these mice. Compared with perforin-deficient mice, LCMV-infected Rab27a-deficient mice showed a substantially better survival rate and slightly higher viral doses were needed to trigger HLH in Rab27a-deficient mice. This study demonstrates that LCMV-infected Rab27a-deficient C57BL/6 mice develop features consistent with HLH and, therefore, represent a murine model of HLH in human Griscelli syndrome type 2.
2414	Clinical assessment of Mycoplasma pneumoniae-associated hemophagocytic lymphohistiocytosis.	18937751	Mycoplasma pneumoniae has been reported to be an etiologic pathogen of infection-associated hemophagocytic lymphohistiocytosis (HLH), but few case reports have been available to date.The clinical features of four childhood cases of M. pneumoniae-associated hemophagocytic lymphohistiocytosis (Mp-HLH) were retrospectively assessed to obtain data that might be useful for early diagnosis and effective management. The previous English-language literature pertaining to Mp-HLH was also reviewed.The patients were two boys and two girls, aged between 1 and 11 years of age. One patient was demonstrated to have concurrent infection with rubella. All the patients had typical radiographic features of M. pneumoniae pneumonia, and one patient also had encephalopathy as a complication. All the children underwent bone marrow examination because of antibiotic-refractory fever, mild hepatosplenomegaly, cytopenia, hyperferritinemia and elevated levels of urine beta2-microglobulin. Cytopenia and hepatosplenomegaly in the present patients were relatively mild as compared to those in cases of other infection-associated HLH such as Epstein-Barr virus infection-associated HLH. Treatment with corticosteroids resulted in prompt and complete resolution in two cases, and i.v. high-dose gammaglobulin therapy achieved a complete response in another child. Spontaneous resolution under treatment with antibiotics alone was observed in one case.Although Mp-HLH is a rare complication of M. pneumoniae infection, it should always be considered in patients with antibiotic-refractory M. pneumoniae infections with cytopenia. Mp-HLH might be effectively treated by corticosteroids or high-dose gammaglobulin. To clarify the diverse clinical manifestations of M. pneumoniae infections, immunological interactions between M. pneumoniae and the host immune system should be further investigated.
2415	Treatment of the X-linked lymphoproliferative, Griscelli and Chédiak-Higashi syndromes by HLH directed therapy.	18937330	Griscelli syndrome type 2 (GS2), the X-linked lymphoproliferative (XLP) and the Chédiak-Higashi (CHS) syndromes are diseases that all may develop hemophagocytic syndromes. We wanted to investigate whether the treatment protocols for hemophagocytic lymphohistiocytosis (HLH) can also be used for these syndromes.In the HLH-94/HLH-2004 treatment study registries, we evaluated all patients with GS2 (n = 5), XLP (n = 2) or CHS (n = 2) treated between 1994 and 2004.All patients responded to the therapy, and all are alive but one (suffering from CHS), with a mean follow-up of 5.6 years. All GS2, one XLP and one CHS patient underwent hematopoietic stem cell transplant. Mean follow-up post transplant was 6.0 years. Six of the seven transplanted children achieved non-active disease status at the time for SCT. Neurological sequelae were reported in all, except for the XLP patients.Our results indicate that HLH treatment can be an effective first line treatment to induce remission in patients with GS2, XLP and CHS that have developed a hemophagocytic syndrome. We suggest that these patients should be included as a separate cohort in the international HLH study.
2416	Expression and regulation of tumor necrosis factor (TNF) and TNF-receptor family members in the macaque corpus luteum during the menstrual cycle.	18932199	Members of the tumor necrosis factor (TNF)-receptor (R) family may be involved in the tissue remodeling that occurs in the primate corpus luteum (CL) during development and regression. As a first step towards addressing this issue, studies assessed TNF ligand-R expression and regulation in CL collected from monkeys during the early (ECL, Days 3-5), mid (MCL, Days 7-8), mid-late (MLCL, Days 10-11), late (LCL, Days 14-16), and very late (VLCL, menses) luteal phase of the menstrual cycle. CL were also collected after gonadotropin and/or steroid ablation and replacement (with hLH and the progestin R5020) for 3 days at mid-late luteal phase. TNF-alpha, -beta, FAS ligand (FASL), and TNF-R1 mRNA levels were two- to sixfold greater (P < 0.05) at the MLCL or LCL phase as compared to earlier (ECL, MCL). In contrast, TNF-R2 and FAS mRNA levels did not change during the luteal phase. Immunohistochemical staining for TNF-beta, TNF-R1, TNF-R2, FAS, and FASL was observed in luteal cells, whereas only TNF-beta staining was observed in endothelial cells. Several TNF-R components were influenced by LH and/or steroid ablation; notably, steroid ablation reduced (P < 0.05) luteal TNF-alpha, but not TNF-beta, mRNA levels, which was prevented by progestin treatment. In contrast, steroid ablation increased (P < 0.05) luteal cell immunostaining for FAS and FASL, which was reduced by progestin treatment. Thus, several members of the TNF R-ligand family are expressed in the primate CL in an LH- and/or progestin-dependent manner. Peak expression in the late luteal phase may signify a role for the TNF-R system in death receptor-mediated apoptosis during luteolysis.
2417	[Diagnostic significance of NK cell activity and soluble CD25 level in serum from patients with secondary hemophagocytic lymphohistiocytosis].	18928616	This study was aimed to explore the clinical significance of NK cell activity and serum soluble CD25 (sCD25) level in early diagnosis of the patients with secondary hemophagocytic lymphohistiocytosis (HLH). 38 suspected secondary HLH patients from June 2005 to June 2008 and 25 healthy subjects were enrolled in the study. The NK cell activity in peripheral blood was determined by a released LDH assay, The sCD25 level in serum was detected with ELISA double antibody sandwich assay. The 38 suspected secondary HLH patients were divided into diagnosed and excluded group according to HLH-2004 diagnostic criteria, The NK cell activity and sCD25 level were compared between the two groups. The results showed that 22 out of 38 suspected patients were diagnosed as secondary HLH, the NK cell activity in peripheral blood of these 22 patients was significantly lower than that of healthy control (p < 0.001), the sCD25 level in peripheral blood of these 22 diagnosed patients was higher than that of healthy control (p < 0.05). In conclusion, detection of NK cell activity and sCD25 level may be valuable in the early diagnosis of secondary HLH.
2418	The HLH-6 transcription factor regulates C. elegans pharyngeal gland development and function.	18927627	The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the "organ identity factor" PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.
2419	Successful treatment of secondary hemophagocytic lymphohistiocytosis in a patient with disseminated histoplasmosis.	18854090	Hemophagocytic lymphohistiocytosis (HLH) is an overwhelming inflammatory response, associated with an outpouring of cytokines and inappropriate activation of the macrophage system, causing severe morbidity and possible death. HLH has inherited and acquired forms. Secondary HLH can be related to any number of underlying conditions including infections, malignancy and autoimmune diseases. There are similarities between "cytokine storm" seen in HLH and the clinical findings of avian influenza. We report a patient with a history of sarcoidosis on chronic steroid treatment, who developed HLH secondary to an infection with Histoplasma capsulatum.
2420	Multiorgan failure due to hemophagocytic syndrome: A case report.	18834507	Hemophagocytic syndrome (HFS) is a potentially lethal disorder due to an uncontrolled immune response to a triggering agent. Our objective is to raise the importance of HFS early diagnosis by presenting a representative case.A sixteen-year-old girl with Still disease diagnosis developed a progressive multiorgan failure including acute respiratory distress (ARDS), anemia and thrombopenia, elevated liver enzymes, renal failure, coagulopathy with hypofibrinogenemia, and acute phase reactants elevation despite broad-spectrum antibiotics. A bone marrow puncture-biopsy was performed, and hemophagocytosis was found. Prolonged fever, splenomegaly, bicytopenia, hypofibrinogenemia, hyperferritinemia and hypertriglyceridemia confirmed HFS diagnosis. She received intensive care support therapy including mechanical ventilation and specific therapy according to HLH 2004 protocol, with a very good response.Our case shows complexity of HFS diagnosis, due to septic shock-like manifestations. Early diagnosis is essential to start appropriate treatment achieving a better outcome.
2421	hLH/hCG-receptor expression correlates with in vitro invasiveness in human primary endometrial cancer.	18829089	Endometrial cancer (EC) is the most frequent cancer of the female genital tract. It has been hypothesized that those ECs that occur in the postmenopausal period, might be sensitive to elevated levels of luteinizing hormone/human chorionic gonadotropin (LH/hCG). Based on previous indications, we analyzed the functional expression of LH/hCG receptors (LH/hCG-R) in primary ECs.We studied a cohort of primary ECs, in which both the LH/hCG-R mRNA and the LH/hCG-R protein were analyzed. Results were correlated with both clinical-pathological data and the effects of LH addition on cell invasion in vitro.The LH/hCG-R mRNA levels ranged from 4.67 e(-02) to 2.36 e(+03). The transcript was properly translated into a functional LH/hCG-R protein. The analysis of cell invasion in vitro in response to LH/hCG allowed us to divide the EC samples into two groups, one with a null or very low response (non-responders=NR) and the other with a significant response to LH (responders=R). The two groups had significantly different levels of LH/hCG-R mRNA expression: the NR group had a median value of 1.40 e(+)(00), while the R group of 7.42 e(+)(01) (p=0.043).In primary ECs a statistically significant correlation emerged between the levels of LH/hCG-R mRNA and the LH-induced cell invasion in vitro. These results suggest that therapies aimed at decreasing LH levels, through Gonadotropin Releasing Hormone (Gn-RH) analogues, could produce benefits in the treatment of recurrent or metastatic EC, especially in patients displaying high LH/hCG-R levels.
2422	Hemophagoctic lymphohistiocytosis--recent concept.	18822626	Hemophagocytic lymphohistiocytosis is a rare condition characterized by highly stimulated but inactive immune response. The disease may be inherited or acquired due to infections, collagen vascular diseases and malignancies. The pathological hallmark of the syndrome is aggressive proliferation of macrophages and histiocytes. Decreased NK cell activity results in increased T cell activation resulting production of large quantities of interferon gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha) and granulocyte macrophage colony stimulating factor (GM-CSF). This causes sustained macrophage activation and tissue infiltration as well as production of interleukin 1 (IL1) and interleukin 6 (IL6).The resulting inflammatory reaction causes extensive damage and associated symptoms. Patients with HLH commonly present with high fever, anemia and splenomegaly. Minimal diagnostic parameters are a complete hemogram, liver function test, serum triglycerides and ferritin, coagulation profile including fibrinogen and bone marrow aspiration. Two highly sensitive diagnostic marker are an increased plasma concentration of the alpha chain of soluble IL2 receptor (CD25) and impaired NK cell activity. Hyperinflammation can be treated with steroid, Cyclosporine prevents T lymphocytes and immunoglobulin infusion helps to control the infection. Etoposide may be life saving specially in case of HLH with Ebstein Barr Viruses infection. The Histiocyte Society in 1994 developed a common treatment protocol (HLH-94). In January 2004 a revised HLH treatment protocol was opened entitled HLH-2004, which is based on HLH-94 with minor modifications. There is a high remission rate on the HLH-94 and HLH-2004 treatment protocols.
2423	Pre- and post-natal treatment of hemophagocytic lymphohistiocytosis.	18819128	Hemophagocytic lymphohistiocytosis (HLH) is a rare autosomal recessive disorder of infancy and childhood that is invariably fatal if not treated. We report on the first patient to receive post-natal HSCT for HLH after receiving in utero chemotherapy for disease stabilization.
2424	Hemophagocytic lymphohistiocytosis in very young infants.	1881670019350640	We report three cases of hemophagocytic lymphohistiocytosis (HLH) in infants within the first 6 weeks of life. Diagnosis of HLH was made early after symptoms started. All three cases were successfully treated with dexamethasone and none relapsed, indicating that not all cases of HLH in very young infants are familial.
2425	Hemophagocytic lymphohistiocytosis in a pancreas-kidney transplant recipient: response to dexamethasone and cyclosporine.	18793556	We describe a severe case of hemophagocytic lymphohistiocytosis (HLH), secondary to a candida glabrata retroperitoneal abscess in a 41-year-old simultaneous pancreas-kidney transplantation (SPKT) recipient. Despite percutaneous abscess drainage and antifungal therapy, general status deteriorated with persistent fever, severe pancytopenia and liver dysfunction. Presence of hypertriglyceridemia, very high serum levels of ferritin and hemophagocytosis in a bone-marrow aspirate gave the diagnosis of HLH. Of note, change from tacrolimus to cyclosporine together with dexamethasone produced rapid response with status improvement. We concluded that HLH, a rare but often fatal condition characterized by excessive activation of lymphocytes and macrophages, is a diagnostic and therapeutic challenge in solid-organ transplanted patients and must be suspected in the presence of fever, blood cytopenia and liver dysfunction. Specific antiinfectious therapy together with cyclosporine and dexamethasone may be a therapeutic approach.
2426	Macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis is associated with MUNC13-4 polymorphisms.	18759271	Systemic juvenile idiopathic arthritis (JIA) is associated with macrophage activation syndrome. Macrophage activation syndrome bears a close resemblance to familial hemophagocytic lymphohistiocytosis (HLH). The development of familial HLH has been recently associated with mutations in MUNC13-4. The purpose of this study was to assess for possible sequence alterations in MUNC13-4 in patients with systemic JIA/macrophage activation syndrome.The MUNC13-4 sequence was analyzed in 18 unrelated patients with systemic JIA/macrophage activation syndrome, using 32 primer pair sets designed to amplify the 32 exons and at least 100 basepairs of the adjacent intronic regions. DNA samples obtained from 73 unrelated patients with systemic JIA and no history of macrophage activation syndrome and 229 unrelated healthy individuals were used as controls.The biallelic sequence variants in MUNC13-4 reported in familial HLH were present in 2 of the 18 patients with JIA/macrophage activation syndrome. Further analysis of the MUNC13-4 sequences revealed an identical combination of 12 single-nucleotide polymorphisms (SNPs) in 9 of the remaining 16 patients with systemic JIA/macrophage activation syndrome (56%). Additional analysis suggested that these 12 SNPs (154[-19] g>a, 261[+26] c>g, 388[+81] g>a, 388[+122] c>t, 570[-60] t>g, 888 G>C, 1389[+36] g>a, 1992[+5] g>a, 2447[+144] c>t, 2599 A>G, 2830[+37] c>g, 3198 A>G) were inherited as an extended haplotype. In several patients, in addition to the described haplotype, there were other SNPs in the second allele of MUNC13-4. Moreover, 1 patient had a complex mutation with 2 changes, 2542 A>C and 2943 G>C, in a cis configuration. The haplotype was present in only 27 (12%) of 229 healthy control subjects (chi(2) = 23.5) and in 6 (8.2%) of 73 patients with systemic JIA and no history of macrophage activation syndrome.The data suggest an association between MUNC13-4 polymorphisms and macrophage activation syndrome in patients with systemic JIA.
2427	Novel function of the class I bHLH protein Daughterless in the negative regulation of proneural gene expression in the Drosophila eye.	18758436	Two types of basic helix-loop-helix (bHLH) family transcription factor have functions in neurogenesis. Class II bHLH proteins are expressed in tissue-specific patterns, whereas class I proteins are broadly expressed as general cofactors for class II proteins. Here, we show that the Drosophila class I factor Daughterless (Da) is upregulated by Hedgehog (Hh) and Decapentaplegic (Dpp) signalling during retinal neurogenesis. Our data suggest that Da is accumulated in the cells surrounding the neuronal precursor cells to repress the proneural gene atonal (ato), thereby generating a single R8 neuron from each proneural cluster. Upregulation of Da depends on Notch signalling, and, in turn, induces the expression of the Enhancer-of-split proteins for the repression of ato. We propose that the dual functions of Da--as a proneural and as an anti-proneural factor--are crucial for initial neural patterning in the eye.
2428	Regulation of the bHLH transcription factor E2A in epithelial cells by interaction with the Na/K-ATPase beta1 subunit.	18727914	The bHLH transcription factor E2A controls proliferation and differentiation in many cell types including kidney epithelial cells. To identify regulatory binding partners of E2A in the kidney, a yeast two-hybrid assay with a human adult kidney cDNA library was performed. Results demonstrated E2A interactions with other HLH proteins including Id1-3 and Pod1 and the Na/K-ATPase beta1 subunit. The specificity of beta1 subunit binding was confirmed by co-immunoprecipitation of E2A and beta1 subunit deletion constructs in HEK cells demonstrating E2A binding to the cytoplasmic tail of the beta1 subunit. Immunofluorescence and Western analysis of HEK cells co-transfected with GFP-beta1 subunit and E2A demonstrated E2A membrane binding and increased beta1 subunit membrane localization. Increased beta1 subunit expression resulted in decreased nuclear E2A expression and protein half-life and reduced E2A induced gene expression. These results suggest that E2A and Na/K-ATPase beta1 subunit expression in epithelial cells are regulated by interactions between these proteins.
2429	Follicular development induced by recombinant luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in anovulatory women with LH and FSH deficiency: evidence of a threshold effect.	18727841	To assess the requirement for luteinizing hormone (LH) in women deficient in LH and follicle-stimulating hormone (FSH).A prospective, randomized, parallel-group, multicentre study was carried out in tertiary care and academic medical centres. Women with anovulatory amenorrhoea > or = 1 year, serum oestradiol (E(2)) < 60 pg/mL (< 220 pmol/L) and low normal serum gonadotrophins were randomized in cycle A to a fixed daily dose of recombinant human (r-h) FSH (150 IU) and r-hLH 0, 25, 75 or 225 IU. Cycles B and C were not randomized.Follicular development, ovulation and luteinization.In cycle A, follicular development was achieved by 63.6% (7/11), 100% (9/9), 72.7% (8/11) and 66.7% (6/9) of patients who received r-hFSH and r-hLH 0, 25, 75 or 225 IU/day, respectively (p = not significant). Among patients with basal serum LH of < 1.2 IU/L, a dose-response relationship of r-hLH to follicular development was observed (p = 0.039). Fourteen of 34 patients (41.2%) wishing to conceive became pregnant. Among patients with hypogonadotrophic hypogonadism (HH) treated with r-hFSH alone, a transition from LH dependence to independence was observed between basal LH values of > or = 1.2 IU/L and < or = 1.6 IU/L. The r-hLH was well tolerated and no serious adverse events occurred during treatment. The most common treatment-related events were related to the reproductive system and the gastrointestinal tract.Recombinant human LH provides a safe treatment option for women with HH. This small study also provided evidence suggestive of an LH threshold: follicular development was suboptimal when less than 75 IU/day r-hLH was administered.
2430	Suppression of E-protein activity interferes with the development of BCR-ABL-mediated myeloproliferative disease.	18725623	E-proteins are a class of helix-loop-helix (HLH) proteins, which play multiple roles throughout lymphoid development. The DNA binding activities of the E-proteins are regulated by a distinct class of antagonistic HLH proteins, named Id1-4. Here we demonstrate that Id2 deficient mice in a C57BL/6 genetic background exhibit increased cellularity in the granulocyte/myeloid progenitor compartment and show significantly higher numbers of maturing neutrophils. Within 6 months of age, Id2 deficient mice succumbed from overwhelming granulocytosis. The disease closely mimicked the distinctive features of human chronic myeloid leukemia: leukocytosis with maturing neutrophils, splenomegaly, hepatomegaly, and myeloid infiltration into peripheral tissues, including spleen, liver, and lungs. Strikingly, forced Id2 expression in murine bone marrow cells substantially delayed the onset of myeloproliferative disease (MPD). Collectively, these studies show that suppression of E-protein activity interferes with the development of BCR-ABL-mediated MPD.
2431	A "FLP-Out" system for controlled gene expression in Caenorhabditis elegans.	18723890	We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest.
2432	Hairy2-Id3 interactions play an essential role in Xenopus neural crest progenitor specification.	18721802	Loss of function studies have shown that the Xenopus helix-loop-helix transcription factor Hairy2 is essential for neural crest formation and maintains cells in a mitotic undifferentiated state. However, its position in the genetic cascade regulating neural crest formation and its relationship with other neural crest regulators remain largely unknown. Here we find that Hairy2 is regulated by BMP, FGF and Wnt and that it is only required downstream of BMP and FGF for neural crest formation. We show that Hairy2 overexpression represses neural crest and upregulates neural border genes at early stages while it expands a subset of them in later embryos. We show that Hairy2 downregulates Id3, another essential HLH neural crest regulator, through attenuation of BMP signaling. Knockdown and rescue experiments indicate that Id3 protein, which physically interacts with Hairy2, negatively regulates Hairy2 activity. However, Id3 is required to allow Hairy2 to promote neural crest formation. Together, our results provide evidence that Hairy2 acts downstream of FGF and BMP signals at the neural border to maintain cells in an undifferentiated state, and that Hairy2-Id3 interactions play an essential role in neural crest progenitor specification.
2433	The CSL transcription factor LAG-1 directly represses hlh-6 expression in C. elegans.	18706403	The Caenorhabditis elegans gene hlh-6 is expressed specifically in pharyngeal glands, one of five distinct pharyngeal cell types. Expression of hlh-6 is controlled by a discrete set of cis-regulatory elements, including a negative element called HRL1. Here we demonstrate that HRL1 is a functional binding site for LAG-1, the CSL transcriptional effector of Notch in C. elegans, and that regulation of hlh-6 by LAG-1 is direct. Regulation of hlh-6 by LAG-1 is strictly negative: removal of HRL1 or LAG-1 regulation results in ectopic expression of hlh-6, but does not affect expression in pharyngeal glands. Furthermore, direct regulation of hlh-6 expression does not appear to involve Notch signaling, contrary to the canonical mechanism by which CSL factors regulate target genes. We also identify an additional cis-regulatory element in the hlh-6 promoter that, together with previously identified elements, is sufficient to overcome repression by LAG-1 and activate hlh-6 expression in pharyngeal glands.
2434	Cytologic diagnosis of hemophagocytic lymphohistiocytosis in ascitic fluid: a case report.	18702370	Hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome, is a rare, fatal hematopoietic disease. Its cytologic features may be subtle because the abnormal histiocytes may not be recognized if one is not aware of this entity. We report a case of HLH involving the ascitic fluid.A 73-year-old man developed weakness, lethargy, decreased appetite and progressive shortness of breath after a cholecystectomy. Physical examination revealed hypotension, tachycardia and chest dullness with decreased breath sounds bilaterally. Radiologic examination revealed bilateral pleural effusions. The patient accumulated fluid in the peritoneal cavity, lungs, retroperitoneum and mediastinum. Bone marrow biopsy showed abundant histiocytes infiltrating the marrow cavity, and many of these histiocytes contained cellular debris. A diagnosis of HLH was therefore made. The abdominal paracentesis specimen contained many similar histiocytes exhibiting erythrophagocytosis and lymphophagocytosis. These abnormal histiocytes were positive for CD68 and negative for AE1/AE3, confirming the diagnosis of HLH. The patient died soon after from disseminated aspergillosis.HLH is cytologically characterized by the presence of abnormal histiocytes with ingested cellular debris. In serous effusions they should not be confused with mesothelial cells. Immunohistochemical studies may help confirm the diagnosis.
2435	Hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis: a journey of a thousand miles begins with a single (big) step.	18679369	Hemophagocytic lymphohistiocytosis (HLH) is a rare, highly fatal disorder of uncontrolled inflammation, usually affecting infants. Significant progress in the treatment of this disorder has been achieved during the last decade, and outcomes for larger series of patients have been reported in recent years. Although medical therapy has advanced, hematopoietic cell transplantation remains the only curative therapy for patients with the familial form of this disorder. Unfortunately, these patients have demonstrated relatively poor post-transplant outcomes for a nonmalignant disorder, with approximately 30% mortality in the first 100 days. Early deaths were attributable to infection, GVHD, and unusually high rates of primary nonengraftment, venoocclusive disease and pneumonitis. In addition, a significant number of deaths were due to HLH reactivation, a unique complication seen in this patient group. In contrast, late complications were relatively infrequent and essentially all patients with durable engraftment remained in remission indefinitely. In this review, we will discuss recent progress in the transplant management of patients with HLH and potential future strategies, including the use of reduced intensity conditioning regimens.
2436	Id-1 induces proteasome-dependent degradation of the HBX protein.	18674781	Id-1 is a member of the HLH protein family that regulates a wide range of cellular processes such as cell proliferation, apoptosis, senescence and overexpression of Id-1 was recently suggested to play roles in the development and progression of different cancers. Previously, Id-1 was shown to physically interact with the viral protein E1A. Meanwhile, Id-1 expression was found to be regulated by several of the virus-encoded proteins, suggesting that Id-1 may be a common cellular target of the viral proteins. Here, we report that Id-1 interacts with the Hepatitis-B virus (HBV)-encoded protein HBX and regulates its stability in hepatocellular carcinoma (HCC) cells. We found that in HCC cells, ectopic Id-1 expression significantly decreased the half-life of the HBX protein, indicating that HBX is destabilized by Id-1. Meanwhile, the Id-1-induced HBX degradation was found to be inhibited by treatment with proteasome inhibitor, suggesting that this process is mediated through the proteasome pathway. Interestingly, while Id-1 did not induce HBX-ubiquitination, we found that removal of all the lysine residues of the HBX protein protects it from the effect of Id-1, indicating that ubiquitination is still required for the Id-1-mediated HBX degradation. Meanwhile, we found that Id-1 binds to the proteasome subunit C8 and facilitates its interaction with the HBX protein and disruption of this interaction completely abolishes the negative effect of Id-1 on HBX protein stability. Taken together, our results demonstrated a novel function of Id-1 in regulating HBX protein stability through interaction with the proteasome.
2437	The helix-loop-helix motif at the N terminus of HalI is essential for its immunity function against halocin C8.	18658263	Halocin C8 (HalC8) is a stable microhalocin exhibiting strong antimicrobial activity against a wide range of haloarchaea. HalI, a 207-amino-acid peptide derived from the N terminus of the HalC8 preproprotein, is the immunity protein of HalC8. In this study, the molecular mechanism of the immunity function of HalI was investigated. Both pull-down and surface plasmon resonance assays revealed that HalI directly interacted with HalC8, and a mixture of purified HalI and HalC8 readily formed a heterocomplex, which was verified by gel filtration. Interestingly, HalC8 tended to form a self-associated complex, and one immunity protein likely sequestered multiple halocins. Significantly, the helix-loop-helix (HLH) motif containing a 4-amino-acid repeat (RELA) at the N terminus of HalI played a key role in its immunity activity. Disruption of the HLH motif or mutagenesis of the key residues of the RELA repeat resulted in loss of both the immunity function and the ability of HalI to bind to HalC8. These results demonstrated that HalI sequestered the activity of HalC8 through specific and direct binding.
2438	Intricate gene regulatory networks of helix-loop-helix (HLH) proteins support regulation of bone-tissue related genes during osteoblast differentiation.	18655182	Helix-loop-helix (HLH) transcription factors are key regulators of neurogenesis, myogenesis and osteogenesis. Here the relative contributions of multiple classes of HLH factors to the expression of bone related genes during osteoblast maturation were compared. We examined the expression of a panel of HLH proteins (e.g., Twist1/2, USF1/2, c-Myc, Id1 approximately 4, E12/47, Stra13) and one Zn finger protein (Snail which recognizes a subset of E-boxes), during osteoblast differentiation and their functional contributions to bone phenotypic gene regulation. While expression of Twist1, Stra13, E12/47 and Snail transcripts remains relatively constant, expression of Twist2 as well as the inhibitory factors Id1, Id2, Id3, and Id4 decreases and USF1 is up-regulated during osteoblastic differentiation of MC3T3 cells. Forced expression of selected HLH transcription factors shows that Myc, Snail and USF factors increase expression of the bone markers osteocalcin (OC) and/or alkaline phosphatase (AP), while E12/47, Twist and Id factors decrease their expression. None of these factors affect Runx2 gene expression. Interestingly, Snail enhances expression of osteoblast markers, while Twist1 and Twist2 factors are cross-regulated and inhibit bone specific gene expression and other HLH proteins (e.g., Id) indirectly. Thus, our data suggest that the integrated activities of negative and positive E-box related regulatory factors control osteoblast differentiation.
2439	Assessment of the biopotency of follitropin alfa and lutropin alfa combined in one injection: a comparative trial in Sprague-Dawley rats.	18647398	The current study was designed to determine if follitropin alfa (recombinant human follicle-stimulating hormone; r-hFSH) and lutropin alfa (recombinant human luteinizing hormone; r-hLH) biopotencies were unchanged by reconstituting in sterile water for injection and mixing prior to injection.The biopotencies of r-hFSH and r-hLH were determined following injection of female Sprague-Dawley rats with a mixture of follitropin alfa revised formulation female (RFF) and lutropin alfa (1:1, r-hFSH:r-hLH). Biopotencies of follitropin alfa RFF and lutropin alfa were measured using ovarian weight and ascorbic acid depletion assays, respectively, and compared with a reference standard. Stock mixtures of follitropin alfa RFF and lutropin alfa (1:1) were prepared within 1 h prior to each respective assay's injection and stored at 6 +/- 2 degrees C. Separate low dose (follitropin alfa RFF 1.5 IU/rat, lutropin alfa 2 IU/rat) and high dose (follitropin alfa RFF 3 IU/rat, lutropin alfa 8 IU/rat) treatments were prepared from stock mixtures or individual solutions by diluting with 0.22% bovine serum albumin saline solution and injected within 1 h of preparation. The main outcome measures were ovarian weight and ovarian ascorbic acid depletion.FSH bioactivities were similar (p > 0.10) between the individual follitropin alfa RFF test solution (84.2 IU) and follitropin alfa RFF/lutropin alfa (87.6 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2 degrees C. LH bioactivities were similar (p > 0.10) between lutropin alfa (94.7 IU) test solution and lutropin alfa/follitropin alfa RFF (85.3 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2 degrees C for not more than 1 h prior to injection.Mixing follitropin alfa RFF and lutropin alfa did not alter the bioactivity of either FSH or LH.
2440	The role of BMT in childhood histiocytoses.	18545250	Childhood histiocytoses comprise two main diseases, Langerhans cell histiocytosis (LCH) and hemophagocytic lymphohistiocytosis (HLH). LCH is a rare disorder with obscure pathogenesis. Data on clonality suggested neoplastic origin, yet were not convincing. Dysregulation of cytokines and of DC trafficking and cross-talk are documented. Clinical manifestations and course are highly variable, ranging from self-healing solitary bone lesion to disseminated, multi-organ involvement with 20% fatality rate despite standard chemotherapy. HSCT has been applied in less than 50 cases, outside any trial, with good disease control but elevated early toxicity. The familial form of HLH (FHL) has been recognized as congenital immune deficiency, with mutations of PRF1, Munc13-4, syntaxin11 genes resulting in defective cellular cytotoxicity machinery. Chemo-immunotherapy allows temporary disease control, but HSCT holds as the only procedure with potential for cure. Rapid identification of genetic defects allows differential diagnosis from transient, virus-associated HLH, thus indicating early HSCT. The role of HSCT in childhood histiocytoses is thus very important. Better understanding of the pathogenesis, in particular of genetic and immune function defects, will help to tailor indications and, possibly less toxic, conditioning regimens, reducing treatment-related mortality, and thus disclosing the way to final cure.
2441	Switching from the unfolded to the folded state of the helix-loop-helix domain of the Id proteins based on the O-acyl isopeptide method.	18636401	The inhibitors of DNA binding and cell differentiation Id1-4 are helix-loop-helix (HLH) proteins that negatively regulate DNA transcription by forming inactive dimers with ubiquitous and tissue-specific bHLH proteins, including E47 and MyoD, respectively. Their highly conserved HLH domains are essential for heterodimerization, but can also self-associate to highly stable, alpha-helix-rich structures at low micromolar peptide concentrations. Here, we show that the introduction of an O-acyl isodipeptide unit involving the putative N-cap serine residue of the C-terminal helix completely abrogates the propensity of the Id HLH analogue for any secondary and tertiary structure, resulting in a random coil, as shown by CD measurements in nonbuffered aqueous solutions. However, the HLH fold reappears as soon as an O-->N intramolecular acyl migration, which occurs spontaneously under physiological conditions, restores the native N-cap serine residue. These results show that changes addressing the N-terminus of the C-terminal helix can dramatically influence the HLH structure, and suggest that local interactions at the junction between the loop and the C-terminal helix might be crucial during the HLH folding process. Furthermore, the present study contributes to the evaluation of the O-acyl isodipeptide unit as a powerful tool to introduce a conformational switch into peptides.
2442	Allogeneic hematopoietic stem cell transplantation for Epstein-Barr virus-associated T/natural killer-cell lymphoproliferative disease in Japan.	18626884	Epstein-Barr virus (EBV)-associated T/NK-cell lymphoproliferative disease (LPD) has been linked to several different disorders. Its prognosis is generally poor and a treatment strategy has yet to be established. There are reports, however, that hematopoietic stem cell transplantation (HSCT) can cure this disease. To clarify the current situation regarding allogeneic hematopoietic stem cell transplantation (allo-HSCT) for EBV-associated T/NK-LPD, a nationwide survey was performed in Japan. Data for 74 patients were collected. There were 42 cases of chronic active EBV infection (CAEBV), 10 cases of EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), and 22 cases of EBV-associated lymphoma/leukemia (EBV-lymphoma/leukemia). Of those with CAEBV, 54% had the EBV-infected T-cell type and 59% with EBV-lymphoma/leukemia had the EBV-infected NK-cell type. Most patients with EBV-HLH and EBV-lymphoma/leukemia received allo-HSCT within 1 year after onset compared to only 14% of patients with CAEBV. The event-free survival (EFS) rate following allo-HSCT was 0.561 +/- 0.086 for CAEBV, 0.614 +/- 0.186 for EBV-HLH, and 0.309 +/- 0.107 for EBV-lymphoma/leukemia. The EFS of allo-HSCT with conventional conditioning was 0.488 +/- 0.074 and with reduced-intensity conditioning was 0.563 +/- 0.124. Thus, in a substantial number of cases, EBV-associated T/NK-LPD can be cured by either allogeneic conventional stem cell transplantation or reduced-intensity stem cell transplantation.
2443	Increased serum monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, and interleukin-8 concentrations in hemophagocytic lymphohistiocytosis.	18623207	Hemophagocytic lymphohistiocytosis (HLH) is characterized by hypercytokinemia caused by macrophage and T cell activation. We analyzed the serum concentrations of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1beta, and interleukin (IL)-8 to investigate the roles of these chemokines in the pathophysiology of HLH.Seven patients clinically diagnosed with HLH were examined. Serum cytokines and chemokines were measured. The differences in the serum concentrations between the patients with HLH and the controls were investigated.In patients with an active phase of HLH, the serum MCP-1, MIP-1beta, and IL-8 levels all were significantly higher than in healthy controls. The chemokine elevations decreased rapidly after initiation of chemotherapy. During increases in disease activity, elevation of MCP-1 and MIP-1beta preceded elevation of the serum ferritin level, which is a clinical indicator of HLH disease activity.These results suggest that MCP-1, MIP-1beta, and IL-8 play important roles in the pathophysiology of HLH. In addition, the serum concentrations of these chemokines may be sensitive markers for assessing disease activity in patients with HLH.
2444	Successful use of the anti-CD25 antibody daclizumab in an adult patient with hemophagocytic lymphohistiocytosis.	18615554	Hemophagocytic lymphohistiocytosis (HLH) is a rare and severe inflammatory disorder marked by abnormal cytotoxic T and natural killer cell activity, resulting in impaired clearance of pathogen, excessive cytokine production, and continued immune system activation. Soluble IL-2 receptor (sIL-2R or sCD25) is typically elevated in HLH and can serve as a marker of disease activity, although its role in the pathophysiology of the disease is unclear. Here we present a case of an adult patient with steroid-dependent HLH who was treated successfully with daclizumab, a monoclonal anti-CD25 antibody, allowing successful withdrawal of steroid therapy without an increase in symptoms.
2445	High elevated ferritin levels and the diagnosis of HLH/Sepsis/SIRS/MODS/MAS.	1861550818085676	NaN
2446	HLH-3 is a C. elegans Achaete/Scute protein required for differentiation of the hermaphrodite-specific motor neurons.	18586090	Basic helix-loop-helix (bHLH) proteins of the Achaete/Scute (Ac/Sc) family are required for neurogenesis in both Drosophila and vertebrates. These transcription factors are commonly referred to as 'proneural' factors, as they promote neural fate in many contexts. Although Ac/Sc proteins have been studied in Hydra, jellyfish, many insects, and several vertebrates, the role of these proteins in Caenorhabditis elegans neurogenesis is relatively uncharacterized. The C. elegans genome consists of three Ac/Sc genes, previously identified as hlh-3, hlh-6, and hlh-14. Here, we characterize the role of hlh-3 in nervous system development. Although hlh-3 appears to be expressed in all neural precursors, we find that hlh-3 null mutants have a mostly functional nervous system. However, these mutants are egg-laying defective, resulting from a block in differentiation of the HSN motor neurons. Detectable HSNs have misdirected axon projection, which appears to result from a lack of netrin signaling within the HSNs. Thus, our findings suggest a novel link between Ac/Sc bHLH proteins and the expression of genes required for proper interpretation of axon guidance cues. Lastly, based on sequence identity, expression pattern, and a role in neural differentiation, hlh-3 is most likely an ortholog of Drosophila asense.
2447	Treatment outcomes with CHOP chemotherapy in adult patients with hemophagocytic lymphohistiocytosis.	18583880	The objective of the current study was to investigate the treatment outcomes for the use of cyclophosphamide, adriamycin, vincristine, and prednisolone (CHOP) chemotherapy in adult patients with hemophagocytic lymphohistiocytosis (HLH). Seventeen HLH patients older than 18 yr of age were treated with CHOP chemotherapy. A response evaluation was conducted for every two cycles of chemotherapy. With CHOP chemotherapy, complete response was achieved for 7/17 patients (41.2%), a partial response for 3/17 patients (17.6%), and the overall response rate was 58.8%. The median response duration (RD) was not reached and the 2-yr RD rate was 68.6%, with a median follow-up of 100 weeks. Median overall survival (OS) was 18 weeks (95% CI, 6-30 weeks) and the 2-yr OS rate was 43.9%. Reported grade 3 or 4 non-hematological toxicities were increased serum liver enzyme levels and stomatitis. Grade 3 or 4 hematological toxicities were leukopenia (50.8%), anemia (20%), and thrombocytopenia (33.9%). Neutropenic fever was observed in 21.6% of patients (14/65 cycles), and most of the cases were resolved with supportive care including treatment with broad-spectrum antibiotics. CHOP chemotherapy seems to be effective in adult HLH patients and the toxicities are manageable.
2448	[Hemophagocytic lymphohistiocytosis as an initial manifestation in an HIV-infected patient].	18572813	A 48-year old man was admitted with idiopathic fever, and subsequently diagnosed as having hemophagocytic lymphohistiocytosis (HLH). Though an extensive series of laboratory examinations failed to demonstrate an apparent etiology, empirical use of steroids achieved remission. About two years later, the patient developed Pneumocystis Jiroveci pneumonia and was diagnosed as HIV-positive. Based on this case, HIV-screening tests would be performed whenever we encounter HLH in Japan, where the number of HIV-positive patients is increasing.
2449	Differential transfer of dietary flavour compounds into human breast milk.	18571209	Transfer of dietary flavour compounds into human milk is believed to constitute the infant's early flavour experiences. This study reports on the time-dependent transfer of flavour compounds from the mother's diet to her breast milk using a within-subject design. Eighteen lactating mothers completed three test days on which they provided a baseline milk sample prior to ingestion of capsules containing 100 mg d-carvone, l-menthol, 3-methylbutyl acetate and trans-anethole. Milk samples were collected 2, 4, 6 and 8 h post-ingestion and analysed by a dynamic headspace method and gas chromatography-mass spectroscopy. The recovery quantities were adjusted for variations in milk fat content. Concentration-time profiles for d-carvone and trans-anethole revealed a maximum around 2 h post-ingestion, whereas the profile for l-menthol showed a plateau pattern. The ester 3-methylbutyl acetate could not be detected in the milk, but a single determination showed traces (<0.4 ppb) in a 1 h milk collection. Flavour compounds appeared to be transmitted differentially from the mother's diet to her milk. The results imply that human milk provides a reservoir for time-dependent chemosensory experiences to the infant; however, volatiles from the diet are transferred selectively and in relatively low amounts.
2450	A direct immunoassay assessment of streptavidin- and N-hydroxysuccinimide-modified biochips in validation of serological TNFalpha responses in hemophagocytic lymphohistiocytosis.	18566478	The authors report 2 biochip platforms on gold manufactured by either nanoscale biotinylated self-assembled architectures to streptavidin surface or proteins containing free NH(2) groups to N-hydroxysuccinimide (NHS)-activated surfaces and investigated the potential application of tumor necrosis factor-alpha (TNFalpha) serodiagnosis of hemophagocytic lymphohistiocytosis (HLH). Interactions of TNFalpha antigen and TNFalpha antibody on the biochips were optimized using an indirect immunofluorescence method. Variation coefficients were 1.87% to 4.56% on the streptavidin biochip and 5.03% to 8.64% on the NHS biochip. The correlation coefficients (r) in TNFalpha and TNFalpha antibody assays in HLH patients between the 2 biochip formats were 0.9623 and 0.9386 and the concordance frequencies were 92.2% and 96.1%, respectively. To detect plasma TNFalpha-receptor complexes (TNFR1 and R2) in HLH, a biochip assay strategy was developed. Plasma levels of TNFalpha, TNFalpha antibody, and TNFalpha-receptor complexes (TNFR1 and R2) were detected in plasmas from 42 HLH cases using streptavidin biochips. Frequencies of the biomarkers in the plasmas were 40.5% (17/42) for TNFalpha, 30.9% (13/42) for TNFalpha antibody, 28.6% (12/42) for TNFalpha-receptor 1 complex, and 26.1% (11/42) for TNFalpha-receptor 2 complex, respectively. The streptavidin biochip format was more sensitive than the NHS surface and was demonstrated to be a valuable tool to identify individual biomarker molecules and molecular complexes in sera and cell lysates and to track therapeutic progress of patients.
2451	The role of the initial bone marrow aspirate in the diagnosis of hemophagocytic lymphohistiocytosis.	18523990	The identification of hemophagocytosis (HPC) in tissue or bone marrow (BM) represents only one of 5/8 criteria needed for the diagnosis of hemophagocytic lymphohistiocytosis (HLH). Yet, confirmation of HPC in bone marrow aspirates (BMA) is often relied upon to make therapeutic decisions. There is no standardized reporting criteria for the definition of "positive" BMA, and likely differs between institutions. The purpose of this study was to quantify the number of HPC in the initial BMA in patients diagnosed with HLH at our institution.Patient charts were retrospectively reviewed. Numbers of HPC were counted per 500 nucleated cells in initial BMA.Fifty-eight percent of patients had at least one HPC per 500 nucleated cells. Median number of HPC per 500 cells was 1 (0-12). Median time from initial BMA to HLH diagnosis was 0 days (-3 to 11), suggesting that HLH diagnosis was made regardless of the results of this initial BMA.The number of HPC at initial BMA is often low and variable, confirming that a BMA lacking HPC does not rule out the diagnosis of HLH, and a negative initial BMA should not delay therapy. We recommend that the BMA report should document negative as well as any positive findings of HPC.
2452	Cytokines as a common components of two different disorders: metabolic syndrome and hemophagocytic lymphohystiositosis.	18523932	Increased cytokines secretion occurs in several different disorders. Hemophagocytic lymphohystiositosis (HLH) and metabolic syndrome (MS) are consist two of them. Hemophagocytic lymphohystiositosis results from uncontrolled macrophage activation and huge amounts of cytokine secretion. The metabolic syndrome is a multicomponent condition characterized by insulin resistance, dyslipidemia, abdominal obesity, hypertension, and increased level of proinflammatory cytokines. It was presented a 6.8 years old girl, diagnosed as HLH. Because she was morbid obese, endocrinological investigation had been done and metabolic syndrome, thyroid hormone dysfunction, and hypercortisolemia with disturbances of diurnal rhythm were detected. During follow-up of patient, metabolic syndrome components disappear gradually while haemophagocytosis was recovered. Endocrine system can be affect during HLH attack, and MS can be developed. Cytokines seems to act central role of pathological changes for both diseases.
2453	Postrenal transplant hemophagocytic lymphohistiocytosis and thrombotic microangiopathy associated with parvovirus b19 infection.	18522549	Persistent anemia is a known consequence of Parvovirus B19 (B19) infection following renal transplantation. However, to date, no description of B19-related hemophagocytic lymphohistiocytosis (HLH) exists in renal transplant recipients. We report a 24-year-old male kidney recipient, who presented with fever, severe anemia and allograft dysfunction two years following transplantation. Hyperferritinemia, hypertriglyceridemia, elevated serum lactate dehydrogenase, pancytopenia and fragmented red blood cells on the peripheral blood were also noted. Bone marrow examination revealed giant pronormoblasts and frequent histiocytes with intracellular hematopoietic elements, consistent with HLH. Renal allograft biopsy revealed closure of the lumen of glomerular capillaries and thickening of the capillary walls compatible with thrombotic microangiopathy. The presence of anti-B19 IgM antibody and viral DNA in the patient's serum (detected by real-time PCR) confirmed an acute B19 infection. Following high-dose intravenous immunoglobulin therapy, the anemia gradually resolved and renal function improved. As far as we know, this is the first report of B19-associated HLH and thrombotic microangiopathy in a renal transplant recipient.
2454	mls-2 and vab-3 Control glia development, hlh-17/Olig expression and glia-dependent neurite extension in C. elegans.	18508862	Glia are essential components of nervous systems. However, genetic programs promoting glia development and regulating glia-neuron interactions have not been extensively explored. Here we describe transcriptional programs required for development and function of the C. elegans cephalic sheath (CEPsh) glia. We demonstrate ventral- and dorsal-restricted roles for the mls-2/Nkx/Hmx and vab-3/Pax6/Pax7 genes, respectively, in CEPsh glia differentiation and expression of the genes hlh-17/Olig and ptr-10/Patched-related. Using mls-2 and vab-3 mutants, as well as CEPsh glia-ablated animals, we show that CEPsh glia are important for sensory dendrite extension, axon guidance/branching within the nerve ring, and nerve ring assembly. We demonstrate that UNC-6/Netrin, expressed in ventral CEPsh glia, mediates glia-dependent axon guidance. Our results suggest possible similarities between CEPsh glia development and oligodendrocyte development in vertebrates, and demonstrate that C. elegans provides a unique environment for studying glial functions in vivo.
2455	Transcription factor FIGLA is mutated in patients with premature ovarian failure.	18499083	Premature Ovarian Failure (POF) is a genetically heterogenous disorder that leads to hypergonadotropic ovarian failure and infertility. We screened 100 Chinese women with POF for mutations in the oocyte-specific gene FIGLA and identified three variants in four women: missense mutation c.11C --> A (p.A4E) was found in two women; deletion c. 15-36 del (p.G6fsX66), resulting in a frameshift that leads to haploinsufficiency, was found in one woman; and deletion c.419-421 delACA (p.140 delN) was found in one. Functional analyses by the yeast two-hybrid assay demonstrated that the p.140 delN mutation disrupted FIGLA binding to the TCF3 helix-loop-helix (HLH) domain. Our findings show that a subset of Chinese women with sporadic, premature ovarian failure harbor mutations in FIGLA.
2456	Hemophagocytic lymphohistiocytosis preceded by Kikuchi disease in children.	18496683	Kikuchi disease (KD) is a type of benign, self-limiting lymphadenitis, but it has also been associated with hemophagocytic lymphohistiocytosis (HLH). To date, only a few reports have suggested an association between HLH and KD.To report the imaging findings and clinical characteristics of KD accompanied by HLH in children.Five children with a prolonged fever and cervical lymphadenopathy were diagnosed as having HLH accompanied by KD. The authors retrospectively analyzed the clinical characteristics and the imaging findings in these children. The histology of excision biopsy samples of cervical lymph nodes in all children confirmed the diagnosis of KD. HLH was confirmed by bone marrow biopsy and laboratory criteria provided by the Histiocyte Society.The greatest dimension of the enlarged nodes ranged from 0.5 cm to 2.5 cm and the nodes were most frequently located at level V. CT scans visualized perinodal infiltrates in most of the affected cervical nodes (four of five children) and extracervical nodes (three of three children). On enhanced CT scans, nonenhancing necrosis within the affected cervical nodes was noted in three children.KD might be related to HLH in children. Systemic evaluations and follow-up of children with KD might help to identify HLH related to KD.
2457	The metazoan history of the COE transcription factors. Selection of a variant HLH motif by mandatory inclusion of a duplicated exon in vertebrates.	18454855	The increasing number of available genomic sequences makes it now possible to study the evolutionary history of specific genes or gene families. Transcription factors (TFs) involved in regulation of gene-specific expression are key players in the evolution of metazoan development. The low complexity COE (Collier/Olfactory-1/Early B-Cell Factor) family of transcription factors constitutes a well-suited paradigm for studying evolution of TF structure and function, including the specific question of protein modularity. Here, we compare the structure of coe genes within the metazoan kingdom and report on the mechanism behind a vertebrate-specific exon duplication.COE proteins display a modular organisation, with three highly conserved domains : a COE-specific DNA-binding domain (DBD), an Immunoglobulin/Plexin/transcription (IPT) domain and an atypical Helix-Loop-Helix (HLH) motif. Comparison of the splice structure of coe genes between cnidariae and bilateriae shows that the ancestral COE DBD was built from 7 separate exons, with no evidence for exon shuffling with other metazoan gene families. It also confirms the presence of an ancestral H1LH2 motif present in all COE proteins which partly overlaps the repeated H2d-H2a motif first identified in rodent EBF. Electrophoretic Mobility Shift Assays show that formation of COE dimers is mediated by this ancestral motif. The H2d-H2a alpha-helical repetition appears to be a vertebrate characteristic that originated from a tandem exon duplication having taken place prior to the splitting between gnathostomes and cyclostomes. We put-forward a two-step model for the inclusion of this exon in the vertebrate transcripts.Three main features in the history of the coe gene family can be inferred from these analyses: (i) each conserved domain of the ancestral coe gene was built from multiple exons and the same scattered structure has been maintained throughout metazoan evolution. (ii) There exists a single coe gene copy per metazoan genome except in vertebrates. The H2a-H2d duplication that is specific to vertebrate proteins provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage. (iii) This duplication provides an interesting example of counter-selection of alternative splicing.
2458	Unrelated donor hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis.	18454181	We report outcomes after unrelated donor hematopoietic cell transplantation (HCT) for 91 patients with hemophagocytic lymphohistiocytosis (HLH) transplanted in the US in 1989-2005. Fifty-one percent were <1 year at HCT and 29% had Lansky performance scores<90%. Most (80%) were conditioned with BU, CY, and etoposide (VP16) with or without anti-thymocyte globulin. Bone marrow was the predominant graft source. Neutrophil recovery was 91% at day-42. The probabilities of grades 2-4 acute GVHD at day-100 and chronic GVHD at 5 years were 41 and 23%, respectively. The overall mortality rate was higher in patients who did not receive BU/CY/VP16-conditioning regimen (RR 1.95, P=0.035). The 5-year probability of overall survival was 53% in patients who received BU/CY/VP16 compared to 24% in those who received other regimens. In the subset of patients with known disease-specific characteristics, only one of five patients with active disease at HCT is alive. For those in clinical remission at HCT (n=46), the 5-year probability of overall survival was 49%. Early mortality rates after HCT were high, 35% at day-100. These data demonstrate that a BU/CY/VP16-conditioning regimen provides cure in approximately 50% of patients and future studies should explore strategies to lower early mortality.
2459	Acute human immunodeficiency virus syndrome presenting with hemophagocytic lymphohistiocytosis.	18452272	Hemophagocytic lymphohistiocytosis (HLH) has been described in patients with advanced stages of human immunodeficiency virus (HIV) infection, but rarely occurs during the seroconversion stage of acute HIV infection. We report a case of acute HIV syndrome that presented with virus-associated HLH. The patient recovered spontaneously without any immunomodulating therapy. This case suggests that acute HIV infection should be included in the differential diagnosis of HLH and indicates that HLH associated with acute HIV infection can have a favorable outcome.
2460	A hydraulic-photosynthetic model based on extended HLH and its application to Coast redwood (Sequoia sempervirens).	18440559	Although recent investigations [Ryan, M.G., Yoder, B.J., 1997. Hydraulic limits to tree height and tree growth. Bioscience 47, 235-242; Koch, G.W., Sillett, S.C.,Jennings, G.M.,Davis, S.D., 2004. The limits to tree height. Nature 428, 851-854; Niklas, K.J., Spatz, H., 2004. Growth and hydraulic (not mechanical) constraints govern the scaling of tree height and mass. Proc. Natl Acad. Sci. 101, 15661-15663; Ryan, M.G., Phillips, N., Bond, B.J., 2006. Hydraulic limitation hypothesis revisited. Plant Cell Environ. 29, 367-381; Niklas, K.J., 2007. Maximum plant height and the biophysical factors that limit it. Tree Physiol. 27, 433-440; Burgess, S.S.O., Dawson, T.E., 2007. Predicting the limits to tree height using statistical regressions of leaf traits. New Phytol. 174, 626-636] suggested that the hydraulic limitation hypothesis (HLH) is the most plausible theory to explain the biophysical limits to maximum tree height and the decline in tree growth rate with age, the analysis is largely qualitative or based on statistical regression. Here we present an integrated biophysical model based on the principle that trees develop physiological compensations (e.g. the declined leaf water potential and the tapering of conduits with heights [West, G.B., Brown, J.H., Enquist, B.J., 1999. A general model for the structure and allometry of plant vascular systems. Nature 400, 664-667]) to resist the increasing water stress with height, the classical HLH and the biochemical limitations on photosynthesis [von Caemmerer, S., 2000. Biochemical Models of Leaf Photosynthesis. CSIRO Publishing, Australia]. The model has been applied to the tallest trees in the world (viz. Coast redwood (Sequoia sempervirens)). Xylem water potential, leaf carbon isotope composition, leaf mass to area ratio at different heights derived from the model show good agreements with the experimental measurements of Koch et al. [2004. The limits to tree height. Nature 428, 851-854]. The model also well explains the universal trend of declining growth rate with age.
2461	Regulation of inhibitors of differentiation family proteins by thyroid-stimulating hormone in FRTL-5 thyroid cells.	18437010	Members of the inhibitors of differentiation (Id) family of helix-loop-helix (HLH) proteins are known to play important roles in the proliferation and differentiation of many cell types. Thyroid-stimulating hormone (TSH) regulates proliferation and differentiation by activating TSH receptor (TSHR) in thyrocytes. In this study, we found that Id2, one of the Id family proteins, is a major target for regulation by TSH in FRTL-5 thyroid cells. TSH rapidly increases the Id2 mRNA level in FRTL-5 thyroid cells but the Id2 protein showed biphasic regulatory patterns, being transiently reduced and subsequently induced by TSH treatment. Transient reduction of Id2 protein was noted within 2 hr of TSH treatment and was mediated by proteasomal degradation. Moreover, reduced Id2 expression correlated with the activity of the phosphatidylinositol 3 kinase pathway, which is activated by TSH. Although TSH increases the activity of the Id2 promoter, TSH-induced activation of this promoter was independent of c-Myc. Id2 did not alter TTF-1- and Pax-8-mediated effects on the regulation of the Tg promoter. Thus, in summary, we found that TSH regulates Id2 expression, but that Id2 does not alter the expression of thyroid-specific genes, such as Tg, in FRTL-5 thyroid cells.
2462	The spectrum of Epstein-Barr virus-associated lymphoproliferative disease in Korea: incidence of disease entities by age groups.	18436998	This study is to identify the spectrum of Epstein-Barr virus (EBV)-positive lymphoproliferative diseases (LPD) and relationships between these diseases in Korea. The EBV status and clinicopathology of 764 patients, including acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV (CAEBV) infections, B-LPD arising in chronic latent EBV infection, T & natural killer (NK) cell non-Hodgkin's lymphomas (NHL), B-NHLs, and Hodgkin's lymphomas (HD), were analyzed. T or NK cell NHLs were the most common forms of EBV-positive NHLs (107/167, 64%); among these, nasal-type NK/T cell lymphomas were the most common (89/107, 83%). According to the age, Burkitt's lymphoma was the most common in early childhood; in teenagers, chronic (active) EBV infection-associated LPD was the most common type. The incidence of NK/T cell lymphoma began to increase from the twenties and formed the major type of EBV-associated tumor throughout life. Diffuse large B cell lymphoma formed the major type in the sixties and seventies. In conclusion, primary infections in early childhood are complicated by the development of CAEBV infections that are main predisposing factors for EBV-associated T or NK cell malignancies in young adults. In old patients, decreased immunity associated with old age and environmental cofactors may provoke the development of peripheral T cell lymphoma, unspecified, and diffuse large B cell lymphoma.
2463	Primary splenic lymphoma associated with hemophagocytic lymphohistiocytosis complicated with splenic rupture.	18436505	Primary splenic lymphoma (PSL) is a rare disease with ambiguous definition, comprising less than 1% of non-Hodgkin's lymphoma. Even rarer is PSL combined with hemophagocytic lymphohistiocytosis (HLH), which has presentations of fever, cytopenia, hepatosplenomegaly, hyperferritinemia, and phagocytosis of hematopoietic cells in the reticuloendothelial system. We report the case of a 77-year-old man who presented with HLH initially. Refusing diagnostic splenectomy, he received chemotherapy. Spontaneous splenic rupture occurred after chemotherapy. In the following emergency operation, PSL was diagnosed. He received another 5 courses of chemotherapy with the R-CNOP regimen (rituximab, cyclophosphamide, mitoxantrone, vincristine, prednisolone). Now he has no residual or relapsed disease. Diagnostic splenectomy for adult HLH patients without definite etiologies may play an important role.
2464	A boy with fever, lymphadenopathy, hepatosplenomegaly, and lymphocytosis.	18430321	Proliferation of the lymphoid system should arouse suspicion of a potentially serious illness. We present a 4.5-year-old boy who developed fever, vomiting, diarrhea, lymphadenopathy, hepatosplenomegaly, lymphocytosis, anemia, thrombocytopenia, and increased liver enzymes. Lymph node and bone marrow biopsies showed lymphoproliferation, Epstein-Barr virus (EBV) infection, and hemophagocytosis leading to the diagnosis of hemophagocytic lymphohistiocytosis (HLH). Chemotherapy was initiated for HLH with dexamethasone, etoposide, and cyclosporine. Because of a high level of EBV viremia, rituximab was added a few days later and resulted in a remarkable drop in the EBV in the circulation but not in the cerebrospinal fluid. However, the patient succumbed to encephalitis, pneumonia, and cardiopulmonary failure. Autopsy revealed the presence of EBV in the brain, indicating the ineffectiveness of rituximab therapy in treating central nervous system infection with EBV.
2465	Understanding organ dysfunction in hemophagocytic lymphohistiocytosis.	18427781	This review aims to help critical care clinicians maintain a high level of suspicion regarding the diagnosis of Hemophagocytic Histiolymphocytosis (HLH). It describes the clinical and laboratory features of HLH, outlines its pathophysiology and reviews the most frequent etiologies related to HLH. Prognostic factors and therapeutic options are also reported.Review of the literature.The diagnosis of HLH relies on the association of clinical abnormalities and hemophagocytosis in bone marrow, spleen, or lymph node specimens. Liver, pulmonary, renal, cardiac and skin involvement may occur at various degrees possibly leading to multiple organ failure. Three main etiologies can be found, namely infections, lymphoproliferative diseases, or connective tissue diseases. Immune deficiency is often retrieved. Mortality can be as high as 50%. Although clinically mimicking severe sepsis, HLH has a distinct pathophysiology on which specific therapy is based. Early diagnosis and treatment is mandatory to increase the chances of survival.The comprehensive management of severe HLH requires the involvement of a multidisciplinary team in order to determine the best therapeutic strategy and to identify the underlying cause.
2466	Familial hemophagocytic lymphohistiocytosis in two brothers with X-linked agammaglobulinemia.	18421721	Hemophagocytic lymphohistiocytosis (HLH) is often familial and is associated with high mortality. Primary (familial) HLH is known to occur in children with mutations in perforin, Munc13-4, or syntaxin 11. We describe a case series of two brothers who developed HLH in the setting of X-linked agammaglobulinemia (XLA, Bruton's disease) and adenovirus infection. Further studies revealed absence of Bruton's tyrosine kinase (BTK) protein expression and a novel BTK mutation.
2467	The helix-loop-helix protein Id1 requires cyclin D1 to promote the proliferation of mammary epithelial cell acini.	18413773	Overexpression of the helix-loop-helix (HLH) protein Id1 has been associated with metastasis in breast cancer, but its role in models of early breast tumorigenesis is not well characterized. We show that the down-regulation of endogenous Id1 via proteosomal degradation and relocalization from the nucleus to the cytoplasm is an early event in the formation of mammary epithelial acini. Overexpression of Id1 in both human MCF-10A and primary mouse mammary epithelial cells disrupted normal acinar development by increasing acinar volume. This occurred in an HLH domain-dependent fashion via an increase in S phase. Id1 overexpression also increased apoptosis leading to accelerated luminal clearance, and this was reversed by coexpression of the proto-oncogene Bcl2, leading to large, disorganized structures with filled lumina. Id1 overexpression was unable to increase the volume of cyclin D1(-/-) acini, indicating that Id1 is dependent on cyclin D1 for its proliferative effects. In summary, Id1 may contribute to early breast cancer by promoting excessive proliferation through cyclin D1.
2468	Visceral leishmaniasis associated hemophagocytic lymphohistiocytosis--case report and systematic review.	18405976	The clinical features of leishmaniasis overlap with that of hemophagocytic lymphohistiocytosis (HLH) and the diagnosis of visceral leishmaniasis (VL) related HLH can be challenging.To review information available on disease course, treatment, adjunctive therapy used and the outcomes of VL related HLH.We describe an illustrative case and review all reported cases of VL associated HLH in the English literature till March 2007.VL associated HLH is rare, with 56 cases reported in the English literature. Clinical features lack discriminating value to recognize VL as the inciting etiology. Bone marrow aspiration (BMA) establishes the diagnosis in 78% of cases but is often negative at onset of the syndrome due to the pauci-microbial nature of the disease and patchy involvement. Repeated marrow aspiration, liver biopsy, blood cultures and serology may be required to establish the diagnosis. Liposomal amphotericin is the drug of choice. IVIG may be considered when there is an inadequate response to anti-leishmanial therapy in severe and refractory disease.VL related HLH is often under-recognized because of overlapping clinical features and negative marrow evaluation at onset, leading to high mortality rates.
2469	Recognition of the helix-loop-helix domain of the Id proteins by an artificial luminescent metal complex receptor.	18383104	Synthetic agents specifically interacting with a protein interface are important not only for the better understanding of protein dimer or complex formation but also for medical applications. Here we describe the recognition of the helix-loop-helix (HLH) dimerization domain of the Id proteins by an artificial luminescent receptor containing two binding sites for a Lewis acid and a Lewis base, respectively. The Id proteins are inhibitors of bHLH transcription factors and play key roles during development of cancer. We show that a receptor/Id-HLH-domain complex was formed cooperatively (K(0.5) approximately 2 microM under physiological conditions) and with moderate specificity, as compared to the related MyoD and Max HLH domains. Accordingly, a preferred receptor binding motif, CYSR(K), was identified within the Id HLH domains. These results are promising and may be exploited to design highly selective synthetic receptors for the Id HLH domain.
2470	Methylmercury activates enhancer-of-split and bearded complex genes independent of the notch receptor.	18367466	Methylmercury (MeHg) is a persistent environmental toxin that has targeted effects on fetal neural development. Although a number of cytotoxic mechanisms of MeHg have been characterized in cultured cells, its mode of action in the developing nervous system in vivo is less clear. Studies of MeHg-affected rodent and human brains show disrupted cortical and cerebellar architecture suggestive of mechanisms that augment cell signaling pathways potentially affecting cell migration and proliferation. We previously identified the Notch receptor pathway, a highly conserved signaling mechanism fundamental for neural development, as a target for MeHg-induced signaling in Drosophila neural cell lines. Here we have expanded our use of the Drosophila model to resolve a broader spectrum of transcriptional changes resulting from MeHg exposure in vivo and in vitro. Several Notch target genes within the Enhancer-of-split (E(spl)C) and Bearded (BrdC) complexes are upregulated with MeHg exposure in the embryo and in cultured neural cells. However, the profile of MeHg-induced E(spl)C and BrdC gene expression differs significantly from that seen with activation of the Notch receptor. Targeted knockdown of Notch and of the downstream coactivator Suppressor of Hairless (Su(H)), shows no effect on MeHg-induced transcription, indicating a novel Notch-independent mechanism of action for MeHg. MeHg transcriptional activation is partially mimicked by iodoacetamide but not by N-ethylmaleimide, two thiol-specific electrophiles, revealing a degree of specificity of cellular thiol targets in MeHg-induced transcriptional events.
2471	Integration of CREB and bHLH transcriptional signaling pathways through direct heterodimerization of the proteins: role in muscle and testis development.	18361414	The cAMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors is hormone responsive and critical for nearly all mammalian cell types. The basic helix-loop-helix (bHLH) family of transcription factors is important during the development and differentiation of a wide variety of cell types. Independent studies of the role of the bHLH protein scleraxis in testicular Sertoli cells and paraxis in muscle development using yeast-2-hybrid screens provided the novel observation that bHLH proteins can directly interact with ATF/CREB family members. Analysis of the interactions demonstrated the helix-loop-helix domain of bHLH proteins directly interacts with the leucine zipper (ZIP) region of CREB2/ATF4 to form heterodimers. The direct bHLH-CREB2 binding interactions were supported using co-immunoprecipitation of recombinant proteins. Structural analysis of bHLH and ATF4 heterodimer using previous crystal structures demonstrated the heterodimer likely involves the HLH and Zip domains and has the potential capacity to bind DNA. Transfection assays demonstrated CREB2/ATF4 over-expression blocked stimulatory actions of scleraxis or paraxis. CREB1 inhibited MyoD induced myogenic conversion of C3H10T1/2 cells. CREB2/ATF4 and scleraxis are expressed throughout embryonic and postnatal testis development, with scleraxis specifically expressed in Sertoli cells. ATF4 and scleraxis null mutant mice both had similar adult testis phenotypes of reduced spermatogenic capacity. In summary, bHLH and CREB family members were found to directly heterodimerize and inhibit the actions of bHLH dimers on Sertoli cells and myogenic precursor cells. The observations suggest a mechanism for direct cross-talk between cAMP induced and bHLH controlled cellular differentiation.
2472	Bioequivalence of recombinant human FSH and recombinant human LH in a fixed 2:1 combination: two phase I, randomised, crossover studies.	18348746	To assess bioequivalence of recombinant human follicle stimulating hormone (r-hFSH, follitropin alfa) and recombinant human luteinising hormone (r-hLH, lutropin alfa) in a fixed 2:1 combination (Pergoveris) compared with injection of each of the hormones separately.Two, two-way crossover, phase I studies in healthy female volunteers after gonadotrophin-releasing hormone agonist down-regulation. Volunteers were randomised to the order in which they received subcutaneous injections. In the r-hFSH study, volunteers received one injection of r-hFSH (300 IU) and one of r-hFSH (300 IU)/r-hLH (150 IU) > or = 7 days apart; in the r-hLH study they received r-hLH (450 IU) and r-hFSH (900 IU)/r-hLH (450 IU) > 21 days apart.The serum concentration-time profiles of FSH in the r-hFSH study and LH in the r-hLH study from zero to the last measurable concentration (AUC(0-last)) and the peak FSH/LH serum concentrations (C(max)) were assessed by non-compartmental analysis. The pre-defined range for bioequivalence was 0.8-1.25 for 90% confidence intervals (CI) of the ratio (fixed combination/single gonadotrophin) of the mean for each pharmacokinetic parameter.Bioequivalence criteria were met for the r-hFSH study (n = 34) for C(max) (ratio of means 1.0024, 90% confidence interval (CI) 0.9611-1.0454) and AUC(0-last) (ratio of means 1.0167, 90% CI 0.9933-1.0407), and for the r-hLH study (n = 63) for C(max) (ratio of means 0.9687, 90% CI 0.9194-1.0207) and AUC(0-last) (ratio of means 0.9753, 90% CI 0.8990-1.0581). In the r-hFSH study, 20 adverse events (AEs) were reported after injection of r-hFSH and 20 after r-hLH/r-hFSH. In the r-hLH study, 179 AEs were reported after injection of r-hLH and 193 after the fixed-dose combination. Across both studies, headache was the most commonly reported AE. No serious AEs occurred.These studies demonstrated bioequivalence between r-hFSH and r-hLH administered alone or in fixed 2:1 combination. The 2:1 combination of follitropin alfa and lutropin alfa allows administration of both recombinant gonadotrophins in a single injection.
2473	Genetic loci contributing to hemophagocytic lymphohistiocytosis do not confer susceptibility to systemic-onset juvenile idiopathic arthritis.	18311812	To investigate whether single-nucleotide polymorphisms (SNPs) within the genes PRF1, GZMB, UNC13D, and Rab27a, which are involved in natural killer cell dysfunction and known to contribute to the risk of hemophagocytic lymphohistiocytosis (HLH), confer an increased risk of susceptibility to systemic-onset juvenile idiopathic arthritis (JIA).Four SNPs across the PRF1 gene locus, 5 for GZMB, 7 for UNC13D, and 11 for Rab27a were investigated using MassArray genotyping in 133 UK Caucasian patients with systemic-onset JIA and 384 ethnically matched unrelated control subjects. Additional control genotypes were accessed from the data generated by the Wellcome Trust Case Control Consortium.No significant association was found between any SNP within the 4 selected loci and systemic-onset JIA, by either single-point or haplotype analysis.The results of this study demonstrate that genes involved in HLH do not confer a significant risk of association with systemic-onset JIA.
2474	Macrophage activation syndrome in rheumatic disease: what is the role of the antigen presenting cell?	18295734	Macrophage Activation Syndrome (MAS), alternatively referred to as secondary hemophagocytic lymphohistiocytosis (HLH), is a complication of many rheumatic diseases, most commonly Systemic Juvenile Idiopathic Arthritis (SJIA). MAS consists of a fulminant picture of pan-cytopenia, hectic high fevers, hepatosplenomegaly, lymphadenopathy, rash, and central nervous systemic inflammation. It can arise from genetic defects in the cytotoxic effector response of CD8+ T-cells, resulting in an inability to terminate antigen presentation, which in turn leads to uncontrolled immune activation. However, in the case of most rheumatic diseases, no such defect in cytotoxic killing is present. Little is known about what the contributions from the antigen presenting cells are in the pathogenesis of MAS. In fact, macrophages may be playing a regulatory, anti-inflammatory in MAS. We review the proposed pathogenesis of MAS/HLH, what role macrophages may play in the disease, and the relationship of MAS to its most common associated rheumatic disease, SJIA.
2475	Ascl1 is required for oligodendrocyte development in the spinal cord.	18287202	Development of oligodendrocytes, myelin-forming glia in the central nervous system (CNS), proceeds on a protracted schedule. Specification of oligodendrocyte progenitors (OLPs) begins early in development, whereas their terminal differentiation occurs at late embryonic and postnatal periods. How these distinct steps are controlled remains unclear. Our previous study demonstrated an important role of the helix-loop-helix (HLH) transcription factor Ascl1 in early generation of OLPs in the developing spinal cord. Here, we show that Ascl1 is also involved in terminal differentiation of oligodendrocytes late in development. Ascl1-/- mutant mice showed a deficiency in differentiation of myelin-expressing oligodendrocytes at birth. In vitro culture studies demonstrate that the induction and maintenance of co-expression of Olig2 and Nkx2-2 in OLPs, and thyroid hormone-responsive induction of myelin proteins are impaired in Ascl1-/- mutants. Gain-of-function studies further showed that Ascl1 collaborates with Olig2 and Nkx2-2 in promoting differentiation of OLPs into oligodendrocytes in vitro. Overexpression of Ascl1, Olig2 and Nkx2-2 alone stimulated the specification of OLPs, but the combinatorial action of Ascl1 and Olig2 or Nkx2-2 was required for further promoting their differentiation into oligodendrocytes. Thus, Ascl1 regulates multiple aspects of oligodendrocyte development in the spinal cord.
2476	Confinement of fire-induced smoke and carbon monoxide transportation by air curtain in channels.	18258356	Experimental and numerical studies were performed in this paper to study the possibility of utilizing air curtain for confinement of fire-induced smoke and carbon monoxide transportation along channels. Bench scale experiments were preliminarily performed in a 3.6 m long model channel. Complementary computational fluid dynamics (CFD) simulation was carried out by Fire Dynamics Simulator (FDS) for an 88 m long full scale channel, in order to see the longitudinal carbon monoxide concentration distribution along the real channel with air curtain discharged. Results showed that both the smoke and CO gases released by the fire were well confined to almost remain in the near fire region of the channel at one side of the air curtain. The gas temperature and CO concentration in the protection zone at the other side reduced significantly by an exponential trend with the increase of discharge velocity of the air curtain. These indicated that the air curtain can be an effective measure for confining the transportation of smoke and carbon monoxide species in long channel fires.
2477	Myelofibrosis in a patient with familial hemophagocytic lymphohistiocytosis.	18253962	We report a unique case of primary hemophagocytic lymphohistiocytosis (HLH) detected in an infant who had bone marrow biopsies demonstrating myelofibrosis, a finding not previously reported in primary HLH.
2478	Hemophagocytic lymphohistiocytosis as severe adverse event of antineoplastic treatment in children.	18245652	Hemophagocytic lymphohistiocytosis (HLH) during childhood cancer treatment is a rare adverse event posing major diagnostic and therapeutic challenges. Between 1995 and 2006, 6 children developed HLH while on conventional chemotherapy (n=4) or after allogeneic stem cell transplantation (n=2). Treatment of HLH included dexamethasone and etoposide, 2 children additionally received infliximab or daclizumab. Three children survived, whereas 3 children died 2, 5, and 47 days after diagnosis of HLH. HLH is a severe adverse event of childhood cancer therapy. Early diagnosis and immediate initiation of adequate treatment are mandatory to overcome this severe condition.
2479	Mutations of the hemophagocytic lymphohistiocytosis-associated gene UNC13D in a patient with systemic juvenile idiopathic arthritis.	18240215	The clinical syndromes of hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are both characterized by dysregulated inflammation with prolonged fever, hepatosplenomegaly, coagulopathy, hematologic cytopenias, and evidence of hemophagocytosis in the bone marrow or liver. While HLH is either inherited or acquired, children with severe rheumatic diseases, most notably systemic juvenile idiopathic arthritis, are at risk for MAS. The phenotypic similarity between HLH and MAS raises the possibility that they share common pathogenetic mechanisms. Familial forms of HLH have been attributed to mutations in the genes encoding perforin (PRF1) and Munc13-4 (UNC13D), among others, and are characterized by defective cytotoxic lymphocyte function. While some patients with systemic JIA have decreased levels of perforin protein expression and natural killer (NK) cell function, mutations of HLH-associated genes in patients with systemic JIA have not been reported. We report the case of an 8-year-old girl with systemic JIA without MAS who was found to have compound heterozygous mutations of UNC13D and reduced NK cell cytotoxic function. This case broadens the range of clinical phenotypes attributable to UNC13D mutations and offers new insights into the etiology and pathogenesis of systemic JIA.
2480	Structure-based classification of 45 FK506-binding proteins.	18214965	The FK506-binding proteins (FKBPs) are a unique group of chaperones found in a wide variety of organisms. They perform a number of cellular functions including protein folding, regulation of cytokines, transport of steroid receptor complexes, nucleic acid binding, histone assembly, and modulation of apoptosis. These functions are mediated by specific domains that adopt distinct tertiary conformations. Using the Threading/ASSEmbly/Refinement (TASSER) approach, tertiary structures were predicted for a total of 45 FKBPs in 23 species. These models were compared with previously characterized FKBP solution structures and the predicted structures were employed to identify groups of homologous proteins. The resulting classification may be utilized to infer functional roles of newly discovered FKBPs. The three-dimensional conformations revealed that this family may have undergone several modifications throughout evolution, including loss of N- and C-terminal regions, duplication of FKBP domains as well as insertions of entire functional motifs. Docking simulations suggest that additional sequence segments outside FKBP domains may modulate the binding affinity of FKBPs to immunosuppressive drugs. The docking models also indicate the presence of a helix-loop-helix (HLH) region within a subset of FKBPs, which may be responsible for the interaction between this group of proteins and nucleic acids.
2481	Successful treatment of neonatal herpes simplex-type 1 infection complicated by hemophagocytic lymphohistiocytosis and acute liver failure.	18212481	Neonatal disseminated herpes simplex virus (HSV) infection with acute liver failure (ALF) and neonatal hemophagocytic lymphohistiocytosis (HLH) are severe diseases. We recently experienced a male infant with HLH and ALF induced by HSV type 1 (HSV-1). The infant, born at 39 weeks of gestation by normal delivery, developed a fever on day 4. On day 9, laboratory investigations showed progressive liver dysfunction and coagulopathy, and the serum ferritin was excessively elevated. Furthermore, the blood levels of interleukin (IL)-6, IL-10, and interferon-gamma were also elevated. HSV-1 DNA was detected in the serum and cerebrospinal fluid by the real-time PCR method. A diagnosis of HLH was established based upon the following criteria: fever, splenomegaly, cytopenia (two cell lines), serum ferritin (> 500 mug/l) and hypofibrinogenemia (< 150 mg/dl). High-dose acyclovir therapy, steroid pulse therapy using methylprednisolone, high-dose gamma globulin therapy and a blood transfusion were given. The patient recovered without neurological deficit. Neonatal disseminated HSV infections may be complicated by the development of HLH and hypercyokinemia. If HLH is suspected, not only high-dose acyclovir therapy but also anti-cytokine therapy should be considered.
2482	Retinal microangiopathy and rapidly fatal cerebral edema in a patient with adult-onset Still's disease and concurrent macrophage activation syndrome.	18183612	Hemophagocytic lymphohistiocytosis (HLH) is a complex inflammatory disease with multiple diagnostic and therapeutic pitfalls. The congenital form, referred to as familial hemophagocytic lymphohistiocytosis (FHL), is often associated with cerebromeningeal involvement, whereas neurological complications are not characteristic of the adult form of secondary HLH (sHLH). Here we report the case of a 20-year-old woman with adult-onset Still's disease (AOSD), retinal microangiopathy and concurrent macrophage activation syndrome (MAS), in the context of sHLH. Following treatment with etanercept, ibuprofen, methylprednisolone, and phenylbutazone for 3 weeks, MAS deteriorated and fatal cerebral edema occurred within only 24 h. The clinical signs and neuropathological findings are discussed with special emphasis on possible relationships between the aggravation of MAS and therapeutic interventions for AOSD. In conclusion, even the slightest sign of mental decline in a patient with AOSD must be considered central nervous system MAS which can be rapidly fatal.
2483	Extensive left ventricular to coronary artery connections in hypoplastic left heart syndrome.	18177385	We demonstrate the echocardiographic features of a rare case of very extensive left ventricular to coronary arterial connections (VCC) to both the right and left coronary arteries in a fetus and subsequent neonate with hypoplastic left heart syndrome (HLH). The right coronary artery, after receiving multiple VCC supplied the only antegrade ascending aortic flow. The left main coronary artery was very hypoplastic and the left anterior descending coronary artery was dilated and tortuous with multiple large VCC. The left circumflex coronary artery arose exclusively from its own VCC. To our knowledge this is the first such echocardiographic demonstration of HLH with extensive bilateral VCC and coronary artery stenosis and interruption.
2484	Mesp-family genes are required for segmental patterning and segmental border formation.	21038773	Elaborate somite patterning is based on the dynamic gene regulation within the presomitic mesoderm (PSM) derived from the primitive streak and tailbud in the later stage embryo. Notch signaling and the regulators are major players involved in the all events required for the temporally and spatially coordinated somite formation. PSM can be subdivided at least two domains based on the regulation and maybe the function of genes expressed. In the posterior PSM, a basic-HLH protein Hes7 plays a central role to generate traveling wave of gene expression by negatively regulating the transcription of the target genes, which may lead defining soimte spacing and future segmental unit. In the anterior PSM, cells start to prepare segmental pattering by acquiring rostral or caudal identity of somite primordia and defining segmental border. In this process, Mesp2, another basic HLH protein plays a critical role. Genetic evidence is provided how Mesp2 regulates Notch signaling to establish segmental identity in the anterior PSM.
2485	Elucidation of the structural determinants responsible for the specific formation of heterodimeric Mxd1/Max b-HLH-LZ and its binding to E-box sequences.	18155722	The proteins of the Mxd family (formally known as Mad) are antagonists of the oncoprotein c-Myc. They compete with c-Myc for their obligate partner Max to prevent the c-Myc/Max heterodimer from binding to E-box sequences in the target gene promoters. In cancer cells, where Myc is overexpressed, the expression of Mxd proteins is usually insufficient or abrogated. However, the reintroduction of Mxd1 expression in these cells prevents growth and proliferation. While the antagonism of c-Myc functions by Mxd proteins is of potential relevance for the development of cancer treatment strategies, the structural determinants responsible for the specific heterodimerization between the Mxd and the Max b-helix-loop-helix-leucine zippers are not fully understood. Moreover, whether the heterodimer is assembled on DNA or in the nucleoplasm prior to DNA binding is under debate. In this article, we demonstrate that Mxd1 D112a and Max N78a and H81d, which are located in the leucine zippers of the proteins, can dictate the specificity of heterodimerization and whether or not the Mxd1/Max/DNA complex forms. Our results also indicate that additional specific determinants exist in the helix-loop-helix domains of Max and Mxd1. Finally, we provide evidence that heterodimerization must precede DNA binding in vivo.
2486	[A case of therapy-related acute monocytic leukemia following low-dose of etoposide treatment for hemophagocytic lymphohistiocytosis].	18094583	We report a case of therapy-related acute myeloid leukemia after low-dosed topoisomerase II inhibitor (etoposide) treatment for hemophagocytic lymphohistiocytosis (HLH). A 62-yr-old female patient had previously been treated with a HLH-94 protocol containing a low-dose of etoposide (total dose of 300 mg/m2). Thirty-one months later, the patient was admitted to the hematology department with general weakness and upper respiratory infection symptoms. Peripheral blood smear and bone marrow study revealed acute monocytic leukemia. There was no evidence of myelodysplastic syndrome, and a cytogenetic study showed no chromosomal abnormalities.
2487	Systemic type Epstein-Barr virus-related lymphoproliferative diseases in children and young adults: challenges for pediatric hemato-oncologists and infectious disease specialists.	18092246	Involvement of Epstein-Barr virus (EBV) has long been known in the development of various tumor-forming proliferating diseases, such as nasopharyngeal carcinoma in adults. However, in children and young adults more attention should be focused on systemic, severe type EBV-related diseases, such as fatal infectious mononucleosis, hemophagocytic syndrome, or chronic active EBV infection (CAEBV). These disorders show the typical clinical features of hemophagocytic lymphohistiocytosis (HLH). Although viral infectious diseases are mostly taken care of by infectious disease specialists, pediatric hemato-oncologists need to intervene in the treatment of this kind of disease because of their clonal and neoplastic disease characteristics and of their hematologically problematic, rapid, and life-threatening clinical courses.
2488	Highly elevated ferritin levels and the diagnosis of hemophagocytic lymphohistiocytosis.	18085676182601231830032318615508	Hemophagocytic lymphohistiocytosis (HLH) is a potentially lethal condition characterized by a pathologic inflammation. The diagnostic criteria for HLH include fever, splenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, abnormal natural killer cell (NK cell) functional assay, elevated soluble IL-2Ralpha level, and elevated ferritin level (>500 microg/L). Institution of timely therapy in these critically ill patients may be delayed by difficulties establishing the diagnosis. NK cell functional assay and soluble IL-2Ralpha level may require send-out to a specialized lab. However, ferritin level is available on a same-day basis at most institutions. In this study, we examined the utility of quantitative ferritin levels in diagnosing HLH.All patients with ferritin values >500 microg/L obtained at Texas Children's Hospital between January 10, 2003 and January 10, 2005 were identified. Patient charts were reviewed for ferritin levels and hospital course.During the study interval, 330 patients had ferritin levels >500 microg/L. Ten of the 330 patients were diagnosed with HLH. A ferritin level over 10,000 microg/L was 90% sensitive and 96% specific for HLH. Another diagnostic category with significantly elevated ferritin level was illness of unknown cause (n = 10), and only two of these patients were fully evaluated for HLH.Ferritin levels above 10,000 microg/L appear to be specific and sensitive for HLH. In patients without a significant medical history and a new onset of febrile illness with highly elevated ferritin levels, the diagnosis of HLH should be evaluated.
2489	Effects of recombinant LH treatment on folliculogenesis and responsiveness to FSH stimulation.	18084048	The role of LH in sensitizing antral follicles to FSH is unclear. LH is required for normal hormone production and normal oocyte and embryo development, but follicular responses to LH may depend upon the stage of development. Potential roles at the early follicular phase were explored in a clinical setting by employing a sequential approach to stimulation by recombinant human (r-h) LH followed by r-hFSH in women who were profoundly down-regulated by depo GnRH agonist.We employed a multi-centre, prospective, randomized approach. Women (n = 146) were treated in a long course high-dose GnRH agonist (Decapeptyl, 4.2 mg s.c.) protocol and were randomized to receive r-hLH (Luveris, 300 IU/day) for a fixed 7 days, or no r-hLH treatment. This was followed by a standard r-hFSH stimulation regime (Gonal-F, 150 IU/day). Ultrasound and hormone assessments of responses were measured at the start of r-hLH treatment, on FSH stimulation Days 0 and 8 and at the time of HCG administration.The LH treatment was associated with increased small antral follicles prior to FSH stimulation (P = 0.007), and an increased yield of normally fertilized (2 PN) embryos (P = 0.03). There was no influence of the r-hLH pretreatment upon hormone profiles or ultrasound assessments during the FSH phase. Anti-mullerian hormone increased in both groups during the week prior to FSH stimulation (P = 0.002).This sequential approach to the use of r-hLH in standard IVF showed a possible modest clinical benefit. The results support other recent work exploring up-regulated androgen drive upon follicular metabolism indicating that clinical benefit may be obtainable after further practical explorations of the concept.
2490	Direct control of the proneural gene atonal by retinal determination factors during Drosophila eye development.	18083159	The determination of neuronal identity in Drosophila cells depends on the accurate expression of proneural genes. The proneural gene atonal (ato) encodes a basic-HLH protein required for photoreceptor and chordotonal organ formation. The initial expression of ato in imaginal discs is regulated by sequences that lie 3' to its open reading frame. In this report, we show that the initial ato transcription in different imaginal discs is regulated by distinct 3' cis-regulatory sequences. The eye-specific ato 3' cis-regulatory sequence consists of two distinct elements we term 2.8 PB and 3.6 BP that regulate ato transcription during different stages of eye development. The 2.8 PB enhancer contains a highly conserved consensus binding site for the retinal determination (RD) factor Sine oculis (So). Mutation of this So binding site abolishes 2.8 PB enhancer activity. Furthermore the RD factors So and Eyes absent (Eya) are required for 2.8 PB enhancer activity and can induce ectopic 2.8 PB reporter expression. In contrast, ectopic Dpp signaling is not sufficient to induce ato 3' enhancer activation but can induce increased levels of RD factor Dachshund (Dac) and synergize with So and Eya to increase ato 3' enhancer activity. These results demonstrate a direct mechanism by which the RD factors regulate ato expression and suggest an important role of Dpp in the activation of ato 3' enhancer is to regulate the levels of RD factors.
2491	Frequency and spectrum of central nervous system involvement in 193 children with haemophagocytic lymphohistiocytosis.	18076710	Haemophagocytic lymphohistiocytosis (HLH) may cause meningoencephalitis and significant neurological sequelae. We examined the relationship between neurological symptoms and cerebrospinal fluid (CSF) at diagnosis, and long-term outcome, in all children enroled in the HLH-94-study prior to July 1, 2003, for whom information on CSF at diagnosis was available (n = 193). Patients were divided into four groups: (i) normal CSF (cells/protein) and no neurological symptoms (n = 71); (ii) normal CSF but neurological symptoms (n = 21); (iii) abnormal CSF but no symptoms (n = 50); and (iv) abnormal CSF with neurological symptoms (n = 51). At diagnosis, neurological symptoms were reported in 72/193 (37%) (seizures = 23); abnormal CSF in 101/193 (52%), and either or both in 122/193 (63%). Altogether 16/107 (15%) survivors had neurological sequelae at follow-up (median 5.3 years). Multivariate hazard ratios (HR) for mortality were 0.98 [95% confidence interval (CI) = 0.42-2.31], 1.52 (0.82-2.82) and 2.05 (1.13-3.72) for groups 2-4, compared with group 1. Moreover, sequelae were more frequent in group 4 (7/21, 33%) compared to groups 1-3 (9/86, 10%) (P = 0.015). Patients with abnormal CSF at diagnosis had significantly increased mortality [HR = 1.78 (95% CI = 1.08-2.92), P = 0.023]. Thus, a substantial proportion of HLH survivors suffer neurological sequelae, and children with abnormal CSF have increased risk of mortality and neurological sequelae. Prompt treatment of HLH at onset or relapse may reduce these complications.
2492	Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology.	18074397	This paper describes immunization studies with CLIPS-constrained peptides covering only the major part (beta3-loop) of a structurally complex antigenic site on human Follicle Stimulating Hormone beta-subunit (FSH-beta). In cases where linear and SS-constrained peptides fail, the CLIPS-constrained peptides generate polyclonal antibodies with high neutralizing activity for hFSH. The sera were shown to be specific for hFSH over human Luteinizing Hormone (hLH) and human Chorionic Gonadotropin (hCG). ELISA-competition studies and circular dichroism (CD)-measurements illustrate clearly that activity of the peptides in antibody binding and generation relates directly to precise and appropriate fixation of the peptide conformation. Design of the CLIPS-peptides was entirely based on epitope mapping studies with two neutralizing anti-hFSH mAbs. Both mAbs were shown to bind to a conformational epitope located at the top of the beta1-beta3-loop covering the amino acid sequences Y58-P77 (beta3-loop). The results described in this paper show that CLIPS-constrained peptides covering the Y58-P77 sequence provide the minimally required structural entity necessary to generate reproducibly sera with high hFSH-neutralizing activity.
2493	Programmed cell death.	18061982	Programmed cell death is an integral component of C. elegans development. Genetic studies in C. elegans have led to the identification of more than two dozen genes that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular and biochemical studies have revealed the underlying conserved mechanisms that control these three phases of programmed cell death. In particular, an interplay of transcriptional regulatory cascades and networks involving CES-1, CES-2, HLH-1/HLH-2, TRA-1, and other transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4 and CED-3 results in the activation of the key cell death protease CED-3. The activation of CED-3 initiates the cell disassembly process and nuclear DNA fragmentation, which is mediated by the release of apoptogenic mitochondrial factors (CPS-6 and WAH-1) and which involves multiple endo- and exo-nucleases such as NUC-1 and seven CRN nucleases. The recognition and removal of the dying cell is mediated by two partially redundant signaling pathways involving CED-1, CED-6 and CED-7 in one pathway and CED-2, CED-5, CED-10, CED-12 and PSR-1 in the other pathway. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in multicellular organisms.
2494	Transcriptional regulation.	18050428	The regulation of transcription in C. elegans shares many similarities to transcription in other organisms. The details of how specific transcription factors bind to target promoters and act as either activators or repressors are still being examined in many cases, but an increasing number of factors and their binding sites are being characterized. This chapter reviews the general concepts that have emerged with regards to promoter function in C. elegans. Included are the methods that have been successfully employed as well as limitations encountered to date. Specific cis-acting promoter elements from myo-2, hlh-1 and lin-26 are discussed as examples of complex promoters regulated by multiple sequence elements. In addition, examples of organ-, tissue-, and cell type-specific mechanisms for generating spatial specificity in gene expression are discussed.
2495	Cerebrospinal fluid involvement in a case of visceral leishmaniasis associated with hemophagocytic lymphohistiocytosis.	21748112	Hemophagocytic Lymphohistiocytosis (HLH) implies a benign generalized histiocytic proliferate with erythrophagocytosis and it includes familial hemophagocytic lymphohistiocytosis and secondary hemophgocytosis. Spinal fluid changes like mild to moderate pleocytosis (most of the cells are lymphocytes and macrophages) and sometimes hemophagocytosis are seen in primary HLH but are not reported in secondary HLH. Here we report a case of a previously healthy 10 months old male infant who was diagnosed as familial HLH with evidence of CSF hemophagocytosis. He was started on the HLH 2004 treatment protocol with no improvement. A second bone marrow aspiration revealed leshmania donovani antibodies and he was started on anti-leishmania treatment with dramatic response.To the best of our knowledge, this is the first case of secondary HLH with evidence of hemophagocytosis in cerebrospinal fluid.
2496	Hemophagocytic lymphohistiocytosis in a human immunodeficiency virus-positive homosexual high school student.	18045310	The spread of human immunodeficiency virus (HIV) infection has exploded over the past two decades and such infections in young people are no longer uncommon. However, the major infection route of pediatric patients remains vertical transmission, and sexual, especially homosexual transmission, is highly unusual. We herein describe the case of a 17-year-old boy who developed hemophagocytic lymphohistiocytosis (HLH). Although HLH was remitted soon with dexamethasone therapy, an HIV infection caused by homosexual transmission was detected.
2497	Phylogenetic footprinting analysis in the upstream regulatory regions of the Drosophila enhancer of split genes.	18039873	During Drosophila development Suppressor of Hairless [Su(H)]-dependent Notch activation upregulates transcription of the Enhancer of split-Complex [E(spl)-C] genes. Drosophila melanogaster E(spl) genes share common transcription regulators including binding sites for Su(H), proneural, and E(spl) basic-helix-loop-helix (bHLH) proteins. However, the expression patterns of E(spl) genes during development suggest that additional factors are involved. To better understand regulators responsible for these expression patterns, recently available sequence and annotation data for multiple Drosophila genomes were used to compare the E(spl) upstream regulatory regions from more than nine Drosophila species. The mgamma and mbeta regulatory regions are the most conserved of the bHLH genes. Fine analysis of Su(H) sites showed that high-affinity Su(H) paired sites and the Su(H) paired site plus proneural site (SPS + A) architecture are completely conserved in a subset of Drosophila E(spl) genes. The SPS + A module is also present in the upstream regulatory regions of the more ancient mosquito and honeybee E(spl) bHLH genes. Additional transcription factor binding sites were identified upstream of the E(spl) genes and compared between species of Drosophila. Conserved sites provide new understandings about E(spl) regulation during development. Conserved novel sequences found upstream of multiple E(spl) genes may play a role in the expression of these genes.
2498	Association of transforming growth factor-beta1 gene polymorphism in the development of Epstein-Barr virus-related hematologic diseases.	18024394	Epstein-Barr virus (EBV) is etiologically associated with various hematologic disorders, including primary acute infectious mononucleosis (IM), hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV) and malignant lymphomas. Although cytokines play a central role in EBV-related immune responses, the exact mechanisms causing different clinical responses remain unclear. In this study, the pattern of cytokine gene polymorphisms was comparatively analyzed in EBV-related diseases.Eighty-nine patients with EBV-related disease were analyzed; 30 with IM, 28 with EBV-HLH and 31 with CAEBV. Eighty-one EBV-seropositive healthy adults were also used as controls. Associations with polymorphisms of various cytokines, including interleukin (IL)-1 alpha and IL-1 beta were evaluated. The gene polymorphisms were typed by polymerase chain reaction with sequence-specific primers.A significant difference of polymorphisms was found for transforming growth factor (TGF)-beta1; the frequency of TGF-beta1 codon 10 C allele was significantly higher in patients with EBV-related diseases than in controls (p<0.001). The difference was significant in patients with IM or HLH (p<0.001), but not in those with CAEBV (p=0.127), compared with controls. As regards other cytokines, the frequency of the IL-1 alpha -889 C allele was significantly lower in patients with IM than in controls (p<0.05).Our results suggests that TGF-beta1 codon 10 C allele plays a role in the development of EBV-related diseases and that the IL-1 alpha -889 C allele may be involved in response failure and sequential progression into the development of HLH.
2499	Monitoring response to treatment in melanoma patients: potential of a serum glycomic marker.	17960619	A mechanistic marker correlating with tumor progression and clinical response is useful for assessing therapeutic response and determining the course of therapy. Since serum-total-ganglioside (sTG) and antiganglioside-IgM antibody levels reflected tumor progression, the feasibility of utilizing sTG for assessing the response to immunotherapy of metastatic-melanoma was tested. Patients (n = 34) were immunized with dendritic cells cocultured with irradiated, IFN gamma-treated autologous tumor cells admixed with GM-CSF. Levels of sTG and antiganglioside-IgM antibody titers were measured in sera of vaccine recipients at 0, 4 and 24 weeks of treatment. Based on sTG-level, whether lower (L) or higher (H) than the mean + 1 SD of normal and healthy volunteers on weeks 0, 4 and 24, patients were categorized into cohorts-I (LLL, n = 16), II (HHL/HLL, n = 4), III (LLH/LHH/LHL, n = 7) and IV (HHH/HLH, n = 7). The cohorts were regrouped as sTG- downregulators (sTG-DR; n = 20) and upregulators (sTG-UR; n = 14). These two cohorts differed significantly in their overall (p < 0.012) and progression-free (p = 0.0001) survival post-treatment. 43% sTG-UR died within 39 months, with a median survival of 39 months, whereas 61% of the sTG-DR survived for 48 months. Both endogenous and vaccine-induced antiganglioside-IgM antibodies appeared to regulate sTG levels. Nonresponders had increased sTG with no or low IgM antibody response. The sTG level is regulated within 24 weeks post-treatment and therefore, may serve as an ideal biomarker for assessing therapeutic responses in patients. Clinical correlations of sTG indicate that sTG-downregulating therapy may be an effective treatment strategy for melanoma.
2500	Soluble TWEAK is markedly elevated in hemophagocytic lymphohistiocytosis.	17918252	Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a newly identified monocyte derived cytokine, which has weak apoptosis inducing function against sensitive tumor cell lines in vitro. Also, TWEAK has been reported to have proangiogenic and proinflammatory activities in vivo. However, its functions in pathological situation remain to be elucidated. Here, we analyzed soluble TWEAK in serum of 24 patients with hemophagocytic lymphohistiocytosis (HLH) in combination with interferon-gamma (IFN-gamma) and killer-specific secretory protein of 37 kDa (Ksp37). Soluble TWEAK was not detected in serum of healthy individuals. Soluble TWEAK was markedly elevated in all six primary HLH patients and 12 of 18 secondary HLH patients. Serum IFN-gamma, which is an only known mediator to stimulate TWEAK production in monocyte in vitro, was not elevated despite elevated serum TWEAK in three of six primary HLH patients, although IFN-gamma was markedly elevated in other cases. Ksp37 was only slightly increased in HLH patients. These results indicate that TWEAK may be involved in pathogenesis of HLH and is useful as a clinical marker.
2501	Thermodynamics of b-HLH-LZ protein binding to DNA: the energetic importance of protein-DNA contacts in site-specific E-box recognition by the complete gene product of the Max p21 transcription factor.	17915948	The Myc/Mad/Max network of dimeric basic region-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factors bind to enhancer box sequences (E-box) in the promotors of a large set of genes that control cell metabolism, proliferation, and differentiation. Max (Myc-associated factor X) is the obligate heterodimerization partner of Myc and Mad proteins. On the other hand, Max is the only member of the family capable of forming a stable homodimer. As part of the transcriptional regulation mechanism, Myc/Max and Mad/Max heterodimers and Max homodimers are thought to compete for binding to the E-box target sequences. E-box recognition is structurally supported by the b-HLH-LZ structural motif, which also promotes dimerization. However, the actual dimerization and heterodimerization constants of the complete gene products and their affinities for E-box sequences are not known. Also, the detailed thermodynamic characterization of DNA binding by these transcription factors has not been done yet. Such knowledge is necessary for complete understanding of the transcriptional regulation carried out by the Myc/Mad/Max network. Here, we report the first in-depth thermodynamic characterization of the stability and specific DNA binding of a full length gene product of the Myc/Mad/Max family, namely, Max protein isoform p21 (Max p21). Using calorimetric methods (DSC and ITC) we have determined the dimerization constant of Max p21 in the low micromolar range, and the Max p21/E-box complex dissociation constant in the low nanomolar range at 37 degrees C. The association is driven by a large exothermic effect, which is partly compensated by entropic factors. The energetic contribution to binding affinity of seven highly conserved residues that contact the DNA was probed by X-to-Ala mutagenesis. The results demonstrate that high binding affinity critically relies on the side chain of Arg 26. Furthermore, the mutational analysis points to the important role of the persistent helical turn that comprises this residue at the junction of the basic region and helix H1. Altogether, the study supports the idea that Max p21 can bind E-box sequences in vivo and likely participates directly in the regulation of transcription as homodimer.
2502	Severe bacteria-associated hemophagocytic lymphohistiocytosis in an extremely premature infant.	17888050	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition with high mortality. We report an extremely premature girl, born in the 24th gestational week (BW 732 g), that during her second month developed a severe HLH subsequent to a Serratia marcescens septicemia, with hepatosplenomegaly, cytopenias, hyperbilirubinemia (mostly conjugated, total bilirubin 916 mumol/L), hypertriglyceridemia, hypofibrinogenemia, hyperferritinemia (21266 mug/L), and elevated sIL-2 receptor levels. Genetic analysis revealed no PRF1, STX11 or UNC13D gene mutations. Treatment was provided according to the HLH-2004 protocol with etoposide, dexamethasone, and immunoglobulin, but no cyclosporin because of immature kidneys. She recovered fully from the HLH but developed a severe retinopathy as well as green teeth secondary to the hyperbilirubinemia. We conclude that secondary, bacteria-associated HLH can develop in premature infants, and that HLH can be treated with cytotoxic therapy also in premature infants. It is important to be aware of HLH in premature infants, since it is treatable.
2503	The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha.	17873906	Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFalpha protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFalpha) and aha-1 (HIFbeta), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2alpha-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2alpha, but not HIF-1alpha. These results identify TWIST1 as a direct target gene of HIF-2alpha, which may provide insight into the acquired metastatic capacity of hypoxic tumors.
2504	Genotype-phenotype study of familial haemophagocytic lymphohistiocytosis due to perforin mutations.	17873118	PRF1 gene mutations are associated with familial haemophagocytic lymphohistiocytosis type 2 (FHL2). Genotype-phenotype analysis, previously hampered by limited numbers of patients, was for the first time performed by data pooling from five large centres worldwide.Members of the Histiocyte Society were asked to report cases of FHL2 on specific forms. Data were pooled in a common database and analysed.The 124 patients had 63 different mutations (including 15 novel mutations): 11 nonsense, 10 frameshift, 38 missense and 4 in-frame deletions. Some mutations were found more commonly: 1122 G-->A (W374X), associated with Turkish origin, in 32 patients; 50delT (L17fsX22) associated with African/African American origin, in 21 patients; and 1090-91delCT (L364fsX), in 7 Japanese patients. Flow cytometry showed that perforin expression was absent in 40, reduced in 6 and normal in 4 patients. Patients presented at a median age of 3 months (quartiles: 2, 3 and 13 months), always with fever, splenomegaly and thrombocytopenia. NK activity was absent in 36 (51%), <or=2% in 18 (26%), 3-<or=5% in 10 (14%), >5% in 4 (6%), "reduced" in 2 (3%) (not reported, n = 54). Nonsense mutations were significantly associated with younger age at onset (p<0.001) and absent natural killer activity (p = 0.008).PRF1 mutations are spread over the functional domains. Specific mutations are strongly associated with Turkish, African American and Japanese ethnic groups. Later onset and residual cytotoxic function are observed in patients with at least one missense mutation.
2505	A spectrum of neuroradiological findings in children with haemophagocytic lymphohistiocytosis.	17846757	Haemophagocytic lymphohistiocytosis (HLH) is a rare multisystem disorder. CNS involvement is a frequent and poor prognostic component. Better neuroradiological surveillance may be beneficial for patient management and outcome.To describe various neuroradiological findings in nine patients with HLH with an emphasis on correlation with disease activity and treatment response.Between 1996 and 2007, nine children with HLH with CNS involvement were identified in a single centre. Neuroradiological findings from CT, MRI, and proton MR spectroscopy, and the clinical records of the nine children were retrospectively reviewed. The frequency, distribution, characteristics of abnormal neuroradiological findings and changes during follow-up were correlated with clinical findings.Initial abnormal findings included multiple nodular or ring-enhancing parenchymal lesions, a laminated pattern of nodular parenchymal lesions on T2-weighted images, leptomeningeal enhancement, confluent parenchymal lesions, mild ventriculomegaly, and diffuse brain oedema. On follow-up imaging studies, haemorrhagic transformation and atrophy were seen where brain parenchymal lesions had been previously. These abnormal neuroradiological findings showed good correlation with clinical findings. Proton MR spectroscopy also demonstrated typical changes of metabolites during the course of the disease.A spectrum of neuroradiological findings in children with HLH is well matched with the clinical course of the disease and, therefore, a comprehensive analysis of the findings is useful to monitor disease activity and treatment responses.
2506	Low resolution structural models of the basic helix-loop-helix leucine zipper domain of upstream stimulatory factor 1 and its complexes with DNA from small angle X-ray scattering data.	17827227	The upstream stimulatory factor 1 (USF1) belongs to the basic helix-loop-helix leucine zipper (b/HLH/Z) transcription factor family, recognizing the CACGTG DNA motive as a dimer and playing an important role in the regulation of transcription in a variety of cellular and viral promoters. In this study we investigate the USF1 b/HLH/Z domain and its complexes with DNA by small angle x-ray scattering. We present low resolution structural models of monomeric b/HLH/Z USF1 in the absence of DNA and USF1 dimeric (b/HLH/Z)(2)-DNA and tetrameric (b/HLH/Z)(4)-DNA(2) complexes. The data reveal a concentration-dependent USF1 dimer (b/HLH/Z)(2)-DNA-tetramer (b/HLH/Z)(4)-DNA(2) equilibrium. The ability of b/HLH/Z USF1 to form a tetrameric assembly on two distant DNA binding sites as a consequence of increased protein concentration suggest a USF1 concentration-dependant mechanism of transcription activation involving DNA loop formation.
2507	E6/E7 of HPV type 16 promotes cell invasion and metastasis of human breast cancer cells.	17721085	Human papillomaviruses (HPVs) could be important risk factors for breast carcinogenesis and metastasis, as roughly 50% of breast cancers are positive for high-risk HPVs. To determine the role of high-risk HPVs in human breast carcinogenesis and metastasis, we examined the effect of E6/E7 of HPV type 16 in two non-invasive breast cancer cell lines, MCF7 and BT20. We report that E6/E7 of HPV type 16 induces cell invasive and metastatic abilities of MCF7 and BT20 in vitro and in vivo, respectively, in comparison with the wild type cells. This is accompanied by an upregulation of Id-1, a family member of helix-loop-helix (HLH) transcription factors, in MCF7 and BT20 cell lines which express E6/E7. Earlier studies have reported that Id-1 regulates cell invasion and metastasis of human breast cancer cells. To gauge the role of Id-1 in cell invasion and metastasis induced by E6/E7 of HPV type 16, we investigated the effect of E6/E7 in mouse normal embryonic fibroblast (NEF) and knockout Id-1 (Id-1(-/-)) cells. We establish that E6/E7 induces cell invasive ability in NEF but not Id-1(-/-) cells; moreover, we were able to inhibit the invasion ability of MCF7-E6/E7 and BT20-E6/E7 using Id-1 antisense retroviruses. Furthermore, we report that E6/E7 oncoproteins up-regulate Id-1 promoter activity in MCF7 and BT20 cells. We also found that HPV type 16 is present in all invasive and metastatic breast cancer and less frequently in in-situ breast cancer as opposed to normal mammary tissue. In parallel, we demonstrate that Id-1 overexpression is correlated with the presence of HPV type 16 in human invasive and metastatic breast cancer. These data suggest that high-risk HPV infections can induce cell invasion and metastasis in breast cancer through Id-1 regulation.
2508	Direct visualization of the binding of c-Myc/Max heterodimeric b-HLH-LZ to E-box sequences on the hTERT promoter.	17705400	Myc and Max belong to the b-HLH-LZ family of transcription factors. Heterodimerization between Myc and Max or homodimerization of Max allows these proteins to bind their cognate DNA sequence known as the E-box (CACGTG). Recent evidence has suggested that the c-Myc/Max heterodimeric b-HLH-LZ could interact to form a head-to-tail dimer of dimers and induce complex topologies such as loops in promoters containing more than one E-box sequence. In an attempt to shed light on this hypothesis, the interaction between the heterodimeric b-HLH-LZ of c-Myc/Max and a fragment of the hTERT promoter containing two E-box sequences was studied by atomic force microscopy. Specific binding events were observed at both E-box sites with equal probabilities. In accordance with previous results obtained by EMSA, we observed that the specific binding of the c-Myc/Max b-HLH-LZ bends the promoter. However no looping could be observed in a wide range of concentration encompassing the Ka (association constant) of the putative tetramer and the Ka for the specific binding of the heterodimer. In contrast, experiments performed with a mandatory c-Myc/Max b-HLH-LZ tetramer incubated with the hTERT promoter fragment allowed for the visualization of loops and cross-linked DNA strands originating from specific binding. Altogether, our results indicate that the c-Myc/Max b-HLH-LZ dimer binds specifically and equally to both E-box sites of the hTERT promoter and induces a significant bending of the promoter and that the suggested oligomerization of the c-Myc/Max heterodimeric b-HLH-LZ, if existing, is most likely too weak to induce the formation of a loop in a promoter.
2509	Establishment of a tissue-specific RNAi system in C. elegans.	17681718	In C. elegans, mosaic analysis is a powerful genetic tool for determining in which tissue or specific cells a gene of interest is required. For traditional mosaic analysis, a loss-of-function mutant and a genomic fragment that can rescue the mutant phenotype are required. Here we establish an easy and rapid mosaic system using RNAi (RNA mediated interference), using a rde-1 mutant that is resistant to RNAi. Tissue-specific expression of the wild type rde-1 cDNA in rde-1 mutants limits RNAi sensitivity to a specific tissue. We established hypodermal-and muscle-specific RNAi systems by expressing rde-1 cDNA under the control of the lin-26 and hlh-1 promoters, respectively. We confirmed tissue-specific RNAi using two assays: (1) tissue-specific knockdown of GFP expression, and (2) phenocopy of mutations in essential genes that were previously known to function in a tissue-specific manner. We also applied this system to an essential gene, ajm-1, expressed in hypodermis and gut, and show that lethality in ajm-1 mutants is due to loss of expression in hypodermal cells. Although we demonstrate tissue-specific RNAi in hypodermis and muscle, this method could be easily applied to other tissues.
2510	Nationwide survey of hemophagocytic lymphohistiocytosis in Japan.	17675268	Hemophagocytic lymphohistiocytosis (HLH), a disorder of the mononuclear phagocyte system, can be classified into two distinct forms: primary HLH (FHL) and secondary HLH. To clarify the epidemiology and clinical outcome for each HLH subtype, we conducted a nationwide survey of HLH in Japan. Since 799 patients were diagnosed in 292 institutions of Japan between 2001 and 2005, the annual incidence of HLH was estimated as 1 in 800,000 per year. Among them, 567 cases were actually analyzed in this study. The most frequent subtype was Epstein-Barr virus (EBV)-associated HLH, followed by other infection- or lymphoma-associated HLH. Age distribution showed a peak of autoimmune disease- and infection-associated HLH in children, while FHL and lymphoma-associated HLH occurred almost exclusively in infants and the elderly, respectively. The 5-year overall survival rate exceeded 80% for patients with EBV- or other infection-associated HLH, was intermediate for those with FHL or B-cell lymphoma-associated HLH, and poor for those with T/NK cell lymphoma-associated HLH (<15%). Although this nationwide survey establishes the heterogeneous characteristics of HLH, the results should be useful in planning prospective studies to identify the most effective therapy for each HLH subtype.
2511	Reduced intensity allogeneic umbilical cord blood transplantation in children and adolescent recipients with malignant and non-malignant diseases.	17660841	There is a significant amount of morbidity and mortality following myeloablative umbilical cord blood transplantation (UCBT). Reduced intensity (RI) conditioning offers an alternative to myeloablative conditioning before UCBT. We investigated RI-UCBT in 21 children and adolescents with malignant (n=14), and non-malignant diseases (n=7). RI conditioning consisted of fludarabine (150-180 mg/m2) with either busulfan (< or = 8 mg/kg)+rabbit antithymocyte globulin (R-ATG; n=16) or cyclophosphamide+R-ATG+/-etoposide (n=5). Human leukocyte antigen match: 4/6 (n=13), 5/6 (n=5) and 6/6 (n=3). The median total nucleated cell and CD34+ cell dose per kilogram were 3.58 x 10(7) and 2.54 x 10(5), respectively. The median time for neutrophil and platelet engraftment was 17.5 and 52 days, respectively. There were six primary graft failures (chronic myelogenous leukemia (CML), beta-thalassemia, hemophagocytic lymphohistiocytosis (HLH) and myelodysplastic syndrome (MDS)). The probability of developing grade II to grade IV acute graft-versus-host disease (GVHD) and chronic GVHD was 28.6 and 16.7%, respectively. Incidence of transplant-related mortality (TRM) was 14%. The 5 years overall survival (OS) in all patients was 59.8%. The 5 years OS for patients with average versus poor-risk malignancy was 77.8 versus 22.2% (P=0.03). RI-UCBT may result in graft failure in specific high-risk chemo-naïve patients (CML, beta-thalassemia, HLH and MDS), but in more heavily pretreated pediatric and adolescent recipients results in rapid engraftment and may be associated with decreased severe GVHD and TRM.
2512	Childhood macrophage activation syndrome differs from infection-associated hemophagocytosis syndrome in etiology and outcome in Taiwan.	17639169	Hemophagocytic lymphohistiocytosis (HLH) is a syndrome composed of macrophage activation syndrome (MAS), infection-associated hemophagocytosis syndrome (IAHS), malignancy-associated HLH and genetic HLH. Differentiation of MAS from IAHS and other HLH is important for early appropriate treatment.A retrospective analysis was used to differentiate childhood MAS from IAHS and other HLH in Chang Gung Memorial Hospital (CGMH), Kaohsiung. All relevant clinical features, laboratory data, treatments and outcomes were analysed.Seventeen patients with childhood HLH were found at CGMH, Kaohsiung in the past decade, and could be classified into 3 categories: IAHS (9 patients), MAS (5 patients), and HLH of unknown etiology (3 patients). The diagnosis of MAS first appeared in this hospital in 2001. Patients with IAHS tended to be younger than those with MAS. Boys were more frequently found in the IAHS group whereas girls (with systemic lupus erythematosus or juvenile idiopathic arthritis) were more frequently found in the MAS group. The majority of mortality cases were noted in the IAHS group (44%, 4/9). All patients with MAS survived with early cyclosporine A treatment.Childhood MAS is different from IAHS in terms of age, gender, etiology and mortality. Early administration of cyclosporine A for MAS results in a lower mortality. Further prospective studies are required to confirm these findings.
2513	Pathogenesis and mechanism of disease progression from hemophagocytic lymphohistiocytosis to Epstein-Barr virus-associated T-cell lymphoma: nuclear factor-kappa B pathway as a potential therapeutic target.	17627615	Epstein-Barr virus (EBV) can infect T lymphocytes and manifests as hemophagocytic lymphohistiocytosis (HLH), a distinct entity of hemophagocytic syndrome (HPS) characterized by fever, hepatosplenomegaly, cytopenia, hypercytokinemia, and systemic macrophage activation with hemophagocytosis. In a substantial percentage of HLH patients, the disease may relapse or progress to T-cell lymphoma in months to years. In the present review, the authors summarize the previous studies on the pathogenesis of HLH and the potential mechanism for the progression of disease from HLH to T-cell lymphoma. The infection of T cells by EBV could activate T cells to secrete proinflammatory cytokines, particularly tumor necrosis factor-alpha (TNF-alpha), which subsequently activate macrophages. EBV latent membrane protein-1 (LMP-1) is the viral product responsible for the activation of the TNF receptor (TNFR) associated factors/nuclear factor-kappaB (NF-kappaB)/ERK pathway to enhance cytokine secretion mediated through the suppression of the SAP/SH2D1A gene. The activation of NF-kappaB will confer resistance to TNF-alpha-induced apoptosis on EBV-infected T cells through the down-regulation of TNFR-1. Consistent with in vitro observations, EBV-associated T or natural killer/T-cell lymphoma showed constitutive activation of NF-kappaB, explaining its drug resistance, hypercytokinemia, and poor prognosis. Therefore, similar to other inflammation-associated cancers, HLH provides a unique model to study the mechanism of disease progression from a benign virus-infected disorder (HLH) to T-cell lymphoma. Inhibition of the NF-kappaB signal pathway should provide a potential target for the treatment of HLH and EBV-associated T-cell lymphoma.
2514	Hemophagocytic lymphohistiocytosis associated with visceral leishmaniasis.	17626064	Visceral leishmaniasis (VL), is a systemic disease caused by the dissemination of protozoan parasite Leishmania throughout the reticuloendothelial system. It may mimic or lead to several types of hematological disorders including hemophagocytosis. Infection associated hemophagocytic syndrome implicating Leishmania is very rare and often difficult to diagnose. Here, we describe a child with hemophagocytic lymphohistiocytosis (HLH) associated with VL.
2515	Evolution of the Groucho/Tle gene family: gene organization and duplication events.	17624551	The Groucho/Tle family of corepressor proteins has important roles in development and in adult tissue in both Protostomes and Deuterostomes. In Drosophila, a single member of this family has been identified. Unlike in Protostomes, most Deutrostomes contain more than two full-length Tle genes. In this study, I analyse the genomic organization and phylogenetic relationship between the long and short forms of the Groucho/Tle family members in Chordata. The genomic location and sequence similarities suggest that Aes/Grg5 and Tle6/Grg6 arose from duplication of the Tle2 gene; each evolved independently and acquired new functions as negative regulators of the other Tle proteins. Based on these data, a model for Groucho/Tle gene evolution is proposed.
2516	Id1, but not Id3, directs long-term repopulating hematopoietic stem-cell maintenance.	17622570	E-proteins are widely expressed basic helix-loop-helix (HLH) transcription factors that regulate differentiation in many cell lineages, including lymphoid, muscle, and neuronal cells. E-protein function is controlled by HLH inhibitors such as Id and SCL/TAL1 proteins, which recently have been suggested to play a role in hematopoietic stem cell (HSC) differentiation. However, the precise stages when these proteins are expressed and their specific functions are not entirely clear. Using a knock-in mouse model where the sequence for the enhanced green fluorescent protein (GFP) was inserted downstream of the Id1 promoter, we were able to track Id1 expression on an individual cell basis and detected Id1 expression in long-term repopulating HSCs (LT-HSCs). Functional assays showed that the Id1/GFP(+)Lin(-)Sca1(+)c-kit(Hi) population was highly enriched for LT-HSCs. Consistent with this expression pattern, Id1 deficiency led to a 2-fold reduction in the number of LT-HSCs defined as Lin(-)Sca1(+)c-kit(Hi)CD48(-)CD150(+). Primary bone marrow transplantation studies revealed that Id1 is dispensable for short-term engraftment. In contrast, both Id1(-/-) whole bone marrow and Lin(-)Sca1(+)c-kit(Hi)Thy1.1(Lo)-enriched HSCs, but not Id3(-/-) marrow, displayed impaired engraftment relative to wild-type controls in secondary transplantation assays. These findings suggest a unique role for Id1 in LT-HSC maintenance and hematopoietic development.
2517	Perforin gene mutations in adult-onset hemophagocytic lymphohistiocytosis.	17606450	Perforin gene (PRF1) mutations cause the primary form of hemophagocytic lymphohistiocytosis (HLH). We report a genetic defect of PRF1 in a 62-year-old Japanese man with recurrent episodes of HLH. Sequencing of PRF1 from both peripheral blood mononuclear cells and nail clippings showed compound heterozygous mutation, including deletion of two base pairs at codons 1090 and 1091 (1090-1091delCT) and guanine-to-adenine conversion at nucleotide position 916 (916GAEA). Although primary HLH has been detected in infants and children, genetic mutation of PRF1 or other genes should be considered a differential diagnosis of HLH even in the elderly.
2518	Hemophagocytic syndromes.	17590250	Hemophagocytic syndromes (hemophagocytic lymphohistiocytosis, HLH) represent a severe hyperinflammatory condition with the cardinal symptoms prolonged fever, cytopenias, hepatosplenomegaly, and hemophagocytosis by activated, morphologically benign macrophages. Biochemical markers include elevated ferritin and triglycerides, and low fibrinogen. Whereas in children several inherited immune deficiencies may lead to this syndrome, most adults with HLH have no known underlying immune defect. Nevertheless, impaired function of natural killer (NK) cells and cytotoxic T-cells (CTL) is characteristic for both genetic and acquired forms of HLH. Frequent triggers are infectious agents, mostly viruses of the herpes group. Malignant lymphomas, especially in adults, may be associated with HLH. A special form of HLH in rheumatic diseases is called macrophage-activation syndrome. Initially HLH may masquerade as a normal infection since all symptoms, even though less pronounced, may also be found in immune competent patients. Patients with HLH, however, cannot control the hyperinflammatory response which, if untreated, is fatal in genetic cases and in a high percentage of acquired cases. Awareness of the clinical symptoms and of the diagnostic criteria of HLH is important to start life-saving therapy with immunosuppressive/immunomodulatory agents in time.
2519	bHLH transcription factors regulate organ morphogenesis via activation of an ADAMTS protease in C. elegans.	17588558	The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases plays important roles in animal development and pathogenesis. However, transcriptional regulation of ADAMTS proteins during development remains largely unexplored. Here we show that basic helix-loop-helix (bHLH) transcription factors regulate the expression of an ADAMTS protease that is required for gonad development in Caenorhabditis elegans. Mutations in the gene mig-24 cause shortened and swollen gonad arms due to a defect in gonadal leader cell migration, although leader cell specification appears to occur normally. The MIG-24 protein is a bHLH transcription factor of the Achaete-Scute family and is specifically expressed in gonadal leader cells. MIG-24 can physically interact with HLH-2, an E/Daughterless family bHLH transcription factor and bind the promoter region of gon-1, which encodes an ADAMTS protease required for gonadal leader cell migration. Mutations or RNA interference of mig-24 and hlh-2 severely impaired gon-1 expression and forced expression of GON-1 in leader cells in mig-24 mutants partially rescued the gonadal elongation defect. We propose that, unlike most previously characterized Achaete-Scute transcription factors that are involved in cell fate specification, MIG-24 acts with HLH-2 in specified cells to control cell migration by activating the expression of the GON-1 ADAMTS protease.
2520	Delta-Notch--and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors.	17586813	Hes and Hey genes are the mammalian counterparts of the Hairy and Enhancer-of-split type of genes in Drosophila and they represent the primary targets of the Delta-Notch signaling pathway. Hairy-related factors control multiple steps of embryonic development and misregulation is associated with various defects. Hes and Hey genes (also called Hesr, Chf, Hrt, Herp or gridlock) encode transcriptional regulators of the basic helix-loop-helix class that mainly act as repressors. The molecular details of how Hes and Hey proteins control transcription are still poorly understood, however. Proposed modes of action include direct binding to N- or E-box DNA sequences of target promoters as well as indirect binding through other sequence-specific transcription factors or sequestration of transcriptional activators. Repression may rely on recruitment of corepressors and induction of histone modifications, or even interference with the general transcriptional machinery. All of these models require extensive protein-protein interactions. Here we review data published on protein-protein and protein-DNA interactions of Hairy-related factors and discuss their implications for transcriptional regulation. In addition, we summarize recent progress on the identification of potential target genes and the analysis of mouse models.
2521	Importance of inhibitor of DNA binding/differentiation 2 in hepatic stellate cell differentiation and proliferation.	17559421	In liver fibrosis, activated hepatic stellate cells (HSC) are transformed into myofibroblasts. Helix-loop-helix (HLH) transcriptional factors such as MyoD regulate the differentiation of myocytes, and the inhibitor of DNA binding/differentiation (Id) family comprises dominant negative HLH transcriptional regulators that inhibit differentiation and promote cell proliferation. In the present study, we investigated how the Id family proteins regulate HSC.In primary rat HSC, inhibitor of DNA binding/differentiation (Id)2 and alpha-smooth muscle actin (alpha-SMA) mRNA expression increased 4 days after isolation. Next we established Id2 expressing HSC (HSC-T6-Id2-green fluorescent protein (GFP)) using HSC-T6 cells with retrovirus that expressed GFP-tagged Id2.HSC-T6-Id2-GFP increased cell proliferation with cyclin D1 expression. In contrast, alpha-SMA expression wassuppressed. Real-time reverse transcription-polymerase chain reaction analysis showed Id2 induction significantly suppressed alpha-SMA, collagen-1, matrix metalloproteinase (MMP)-2, and MMP-9 mRNA (P < 0.05) but had no effect on tissue inhibitor of metalloproteinase or transforming growth factor-beta1 levels.These findings suggest Id2, an HLH transcriptional regulator, plays an important regulatory role in the proliferation and differentiation of HSC.
2522	Critical wind velocity for arresting upwind gas and smoke dispersion induced by near-wall fire in a road tunnel.	17544576	In case of a tunnel fire, toxic gas and smoke particles released are the most fatal contaminations. It is important to supply fresh air from the upwind side to provide a clean and safe environment upstream from the fire source for people evacuation. Thus, the critical longitudinal wind velocity for arresting fire induced upwind gas and smoke dispersion is a key criteria for tunnel safety design. Former studies and thus, the models built for estimating the critical wind velocity are all arbitrarily assuming that the fire takes place at the centre of the tunnel. However, in many real cases in road tunnels, the fire originates near the sidewall. The critical velocity of a near-wall fire should be different with that of a free-standing central fire due to their different plume entrainment process. Theoretical analysis and CFD simulation were performed in this paper to estimate the critical velocity for the fire near the sidewall. Results showed that when fire originates near the sidewall, it needs larger critical velocity to arrest the upwind gas and smoke dispersion than when fire at the centre. The ratio of critical velocity of a near-wall fire to that of a central fire was ideally estimated to be 1.26 by theoretical analysis. Results by CFD modelling showed that the ratio decreased with the increase of the fire size till near to unity. The ratio by CFD modelling was about 1.18 for a 500kW small fire, being near to and a bit lower than the theoretically estimated value of 1.26. However, the former models, including those of Thomas (1958, 1968), Dangizer and Kenndey (1982), Oka and Atkinson (1995), Wu and Barker (2000) and Kunsch (1999, 2002), underestimated the critical velocity needed for a fire near the tunnel sidewall.
2523	Immunophenotypic analysis of Epstein-Barr virus (EBV)-infected CD8(+) T cells in a patient with EBV-associated hemophagocytic lymphohistiocytosis.	17532761	Hemophagocytic lymphohistiocytosis (HLH) is a severe and often fatal condition characterized by uncontrolled activation of T cells and macrophages. In Epstein-Barr virus (EBV)-associated HLH (EBV-HLH), the pathogenic roles of ectopic EBV infection in the T-cell population and of clonal proliferation of EBV-infected T cells has been described. However, the immunophenotype of EBV-infected T cells has not been fully characterized. Here we describe a case of EBV-HLH presenting with a massive clonal proliferation of CD8(+) T cells with TCR VB14. Analysis of in situ hybridization for EBV-encoded small RNA1 showed that only CD8(+) T cells harbored EBV in this patient. The EBV-infected TCR VB14(+) CD8(+) T cells exhibited unique immunophenotypic features including lacked CD5 expression and a markedly bright expression of HLA-DR. After initiation of treatment with prednisolone, etoposide, and cyclosporin A, the percentage of infected cells declined progressively in parallel with other serum markers such as ferritin. These findings suggest that lacking expression of CD5 on CD8(+) T cells with specific TCR VB may serve as a useful marker of dysregulated T-cell activation and proliferation in EBV-HLH.
2524	Hybrid transcatheter-surgical palliation: basis for univentricular or biventricular repair: the Giessen experience.	17487538	The outcome of patients with hypoplastic left heart (HLH) is determined by many factors, particularly by the first-step palliative procedure in newborns undergoing the Norwood procedure, its Sano modification, or, rarely, through challenging biventricular repairs. Duct stenting combined with bilateral pulmonary artery banding (PAB) is a new method employed as an alternative first-step approach in a number of centers worldwide. We describe this interventional-surgical "hybrid approach" as an additional strategy for the treatment of newborns with HLH syndrome and HLH complex. Between 1998 and April, 2006, 58 newborns underwent ductal stenting and bilateral PAB. These patients underwent surgical bilateral PAB initially, followed by percutaneous duct stenting; the only exception to this were patients in whom duct stenting was performed as a rescue procedure. Various balloon-expandable and self-expandable stents with different widths and lengths were used during the 8-year period of this study. Balloon dilatation of the atrial septum was performed when indicated. This included 5 patients in whom the atrial septum was stented. Aortic arch reconstruction (AAR) combined with a bidirectional cavopulmonary connection (BCPC) was performed at a median age of 4.8 months (range, 2.6-7.5), and total cavopulmonary connection (TCPC) was performed at a median age of 3.1 years (range, 2.5-4). Nine patients were listed for heart transplantation (HTX) and transplanted with AAR when a donor heart was available. Depending on growth of left ventricular structures, biventricular repair (BVR) was performed at a median age of 7.1 months (range, 3.5-10). Overall, 8 of 58 patients (13.8%) treated by the transcatheter-surgical hybrid approach died during the study period. The mortality rate for duct stenting was 1.7% (l/58), and it was 1.7% for bilateral PAB as well. Twenty-seven patients received an AAR/BCPC; 2 of them died (7.4%). Additionally, 1 of 2 patients with AAR/BCPC died while on the waiting list for HTX, resulting in a total mortality rate of 11% with an actuarial survival rate of 89%. One patient is still awaiting AAR + BCPC. Three patients died while on the waiting list for HTX despite successful bilateral PAB and duct stenting. The 30-day mortality rate for TCPC (n = 11), HTX (n = 8), and 18 patients with BVR was 0. The actuarial survival rate for patients with BVR is 93%. Postnatal transcatheter-surgical hybrid palliation expands the surgical options for newborns with HLH. Using hybrid palliation, Norwood stage I operation can be avoided in the neonatal period, the waiting period for children scheduled for cardiac transplantation can be extended, and observation for left ventricular growth suitable for biventricular repair as well.
2525	Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements.	17487425	Dec2, a member of the basic helix-loop-helix (bHLH) superfamily, has been shown to function as a transcriptional repressor and is implicated in cell proliferation and differentiation. In addition, Dec2 transcripts exhibit a striking circadian oscillation in the suprachiasmatic nucleus. To identify the molecular mechanisms by which Dec2 regulates gene expression, we carried out structure-function analyses. Gel retardation and luciferase assays showed that Dec2, as well as its related protein Dec1, preferentially binds to class B E-box elements (CACGTG) as a homodimer and represses the transcription of target genes in a histone deacetylase (HDAC)-dependent manner. Functional studies with the GAL4-DNA binding domain fusion proteins identified the domain responsible for the repression activity of Dec2 in its C-terminal region, which is also necessary to recruit HDAC1. In addition, the basic and HLH domains of Dec2 were required for DNA binding and homodimerization, respectively. In contrast, Dec proteins repressed a MyoD-activated promoter activity of muscle creatine kinase gene through class A E-box in an HDAC1-independent manner. Dec2 formed a heterodimer with MyoD through the basic and HLH domains. Consistent with this, both the basic and HLH domains were required for the ability of Dec2 to inhibit the transcriptional activity of MyoD. These findings indicate that Dec2 employs multiple mechanisms, including DNA-binding and protein-protein interactions, to achieve E-box-dependent transcriptional repressions.
2526	Molecular cloning and characterization of the Xenopus hypoxia-inducible factor 1alpha (xHIF1alpha).	17471499	We report the molecular cloning and the characterization of the Xenopus homolog of mammalian hypoxia-inducible factor 1alpha (HIF1alpha), a member of the bHLH/PAS transcription factor family. Searches in Xenopus genome sequences and phylogenetic analysis reveal the existence of HIF1alpha and HIF2alpha paralogs in the Xenopus laevis species. Sequence data analyses indicate that the organization of protein domains in Xenopus HIF1alpha (xHIF1alpha) is strongly conserved. We also show that xHIF1alpha heterodimerizes with the Xenopus Arnt1 protein (xArnt1) with the proteic complex being mediated by the HLH and PAS domains. Subcellular analysis in a Xenopus XTC cell line using chimeric GFP constructs show that over-expression of xHIF1alpha and xArnt1 allows us to detect the xHIF1alpha/xArnt1 complex in the nucleus, but only in the presence of both partners. Further analyses in XTC cell line show that over-producing xHIF1alpha and xArnt1 mediates trans-activation of the hypoxia response element (HRE) reporter. The trans-activation level can be increased in hypoxia conditions. Interestingly such trans-activation properties can be also observed when human Arnt1 is used together with the xHIF1alpha.
2527	Control of bleeding associated with hemophagocytic syndrome in children: an audit of the clinical use of recombinant activated factor VII.	17454777	This paper presents 2 cases of hemophagocytic lymphohistiocytosis (HLH) in whom recombinant factor VIIa (rFVIIa) was used for the management of hemorrhage. Both patients were diagnosed as HLH and were bleeding from the gut, which could not be controlled. Patients received rFVIIa at total doses of between 90 and 180 microg/kg body weight. Hemostatic affect was achieved in both of the patients but lasted only a short time. The response was achieved after 1 h of administration of rFVIIa, lasting for 24 h. The use of rFVIIa was well tolerated. These 2 patients suggest that rFVIIa is a beneficial agent in the management of hemorrhage in patients with HLH, although for a permanent homeostasis the control of primary disease is essential.
2528	Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis.	17420270	Mouse cytomegalovirus (MCMV) susceptibility often results from defects of natural killer (NK) cell function. Here we describe Jinx, an N-ethyl-N-nitrosourea-induced MCMV susceptibility mutation that permits unchecked proliferation of the virus, causing death. In Jinx homozygotes, activated NK cells and cytotoxic T lymphocytes (CTLs) fail to degranulate, although they retain the ability to produce cytokines, and cytokine levels are markedly elevated in the blood of infected mutant mice. Jinx was mapped to mouse chromosome 11 on a total of 246 meioses and confined to a 4.60-million basepair critical region encompassing 122 annotated genes. The phenotype was ascribed to the creation of a novel donor splice site in Unc13d, the mouse orthologue of human MUNC13-4, in which mutations cause type 3 familial hemophagocytic lymphohistiocytosis (FHL3), a fatal disease marked by massive hepatosplenomegaly, anemia, and thrombocytopenia. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus, exhibiting hyperactivation of CTLs and antigen-presenting cells, and inadequate restriction of viral proliferation. In contrast, neither Listeria monocytogenes nor MCMV induces the syndrome. In mice, the HLH phenotype is conditional, which suggests the existence of a specific infectious trigger of FHL3 in humans.
2529	Dip1 inhibits growth and gene transcription in MCF-7 breast cancer cells.	17407970	In previous studies we identified a novel gene Dipl, also designated CCNDBP1, which encodes a 42kDa helix-loop-helix (HLH) nuclear protein. Although this protein was originally identified by its ability to bind to cyclin D1 its precise biochemical functions are not known. In the present study we carried out mechanistic studies on Dip1 focusing on the human breast cancer cell line MCF-7. We found that overexpression of Dip1 in MCF-7 cells inhibited colony formation and cell proliferation. Reporter assays in MCF-7 cells indicated that Dip1 strongly inhibited the transcriptional activities of the cyclin D1, c-fos, NF-kappaB, SRE and p21cP1 promoters. Furthermore studies with truncated and mutant forms of the cyclin D1 promoter suggest that Dip1 does not act on specific transcriptional elements. Assays with mutant and truncated forms of Dip1 indicated that both the LXXLL motif and the HLH domain play important, but not exclusive, roles in these inhibitory effects. Dip1 co-immunoprecipitated with the histone deacetylase (HDAC) proteins HDAC1 and HDAC3. Nevertheless Dip1 markedly inhibited the stimulation of cyclin D1 promoter activity obtained with trichostatin A [1], an inhibitor of HDAC. Taken together these findings suggest that Dip1 functions as a general repressor of transcription. Although the precise mechanism by which Dip1 inhibits gene transcription and the growth of MCF-7 cells remain to be determined, the present results suggest that Dip1 is a candidate tumor suppressor gene.
2530	Segmental border is defined by the key transcription factor Mesp2, by means of the suppression of Notch activity.	17394251	Elaborate somite patterning is based upon dynamic gene regulation within the presomitic mesoderm (PSM), which is derived from the primitive streak and tail bud in the later stage mouse embryo. The Notch signaling pathway and its regulators are major components of most of the events required for temporally and spatially coordinated somite formation. The PSM can be subdivided into at least two domains, based upon transcriptional regulation and gene function. In the posterior PSM, the basic helix-loop-helix (bHLH) protein Hes7 plays a central role in generating a traveling wave of gene expression by negatively regulating the transcription of its target genes. This in turn may define the somite spacing and future segmental units. In the anterior PSM, cells begin to form segmental patterning by acquiring rostral or caudal identities of somite primordia and by defining the segmental border, which must be coupled with the segmentation clock. The link between the clock and segmental border formation is of fundamental importance during somitogenesis. During this process, Mesp2, another basic HLH protein, plays a critical role in the anterior PSM. In this review, I further clarify the dynamic processes leading to segmental border formation in the developing mouse embryo.
2531	The C. elegans Twist target gene, arg-1, is regulated by distinct E box promoter elements.	17369030	Proper metazoan mesoderm development requires the function of a basic helix-loop-helix (bHLH) transcription factor, Twist. Twist-containing dimers regulate the expression of target genes by binding to E box promoter elements containing the site CANNTG. In Caenorhabditis elegans, CeTwist functions in a subset of mesodermal cells. Our study focuses on how CeTwist controls the expression of its target gene, arg-1. We find that a 385bp promoter region of arg-1, which contains three different E box elements, is sufficient for maintaining the full CeTwist-dependent expression pattern. Interestingly, the expression of arg-1 in different tissues is regulated distinctly, and each of the three E boxes plays a unique role in the regulation. The first and the third E boxes (E1 and E3) are required for expression in a distinct subset of the mesodermal tissues where arg-1 is normally expressed, and the second E box (E2) is required for expression in the full set of those tissues. The essential role of E2 in arg-1 regulation is correlated with the finding that E2 binds with greater affinity than E1 or E3 to CeTwist dimers. A potential role for additional transcription factors in mesodermal gene regulation is suggested by the discovery of a novel site that is also required for arg-1 expression in a subset of the tissues but is not bound in vitro by CeTwist. On the basis of these results, we propose a model of CeTwist gene regulation in which expression is controlled by tissue-specific binding of distinct sets of E boxes.
2532	A haemophagocytic lymphohistiocytosis (HLH)-like picture following breastmilk transmitted cytomegalovirus infection in a preterm infant.	17366039	Due to septic illness, neutro- and thrombocytopenia, blocked myelopoiesis, CMV-positive breast milk and CMV-pp65 antigen in bone marrow mononuclear cells, CMV-related HLH was presumed in a breast fed neonate (gestational age 24 weeks). Treatment was successful with foscarnet and methylprednisolone. HLH may be a complication of post-natal CMV-infection acquired from breast milk.
2533	Two-dimensional iron(II) spin crossover complex constructed of bifurcated NH...O- hydrogen bonds and pi-pi interactions: [FeII(HLH,Me)2](ClO4)2.1.5MeCN (HLH,Me = imidazol-4-yl-methylidene-8-amino-2-methylquinoline).	17361997	A 2D iron(II) spin crossover complex, [FeII(HLH,Me)2](ClO4)2.1.5MeCN (1), was synthesized, where HLH,Me = imidazol-4-yl-methylidene-8-amino-2-methylquinoline. 1 showed a gradual spin transition between the HS (S = 2) and LS (S = 0) states from 180 to 325 K within the first warming run from 5 to 350 K, in which 1.5MeCN is removed, and there was an abrupt spin transition at T1/2 downward arrow = 174 K in the first cooling run from 350 to 5 K. Following the first cycle, the compound showed an abrupt spin transition at T1/2 upward arrow = 185 K and T1/2 downward arrow = 174 K with 11 K wide hysteresis in the second cycle. The crystal structures of 1 were determined at 296 (an intermediate between the HS and LS states) and 150 K (LS state). The structure consists of a 2D extended structure constructed of both the bifurcated NH...O- hydrogen bonds between two ClO4- ions and two neighboring imidazole NH groups of the [FeII(HLH,Me)2]2+ cations and the pi-pi interactions between the two quinolyl rings of the two adjacent cations. Thermogravimetric analysis showed that solvent molecules are gradually eliminated even at room temperature and completely removed at 369 K. Desolvated complex 1' showed an abrupt spin transition at T1/2 upward arrow = 180 K and T1/2 downward arrow = 174 K with 6 K wide hysteresis.
2534	Diagnostic difficulties of the hemophagocytic lymphohistiocytosis (HLH) associated with the Epstein-Barr virus.	17356405	NaN
2535	Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord.	17344230	During development, the three major neural cell lineages, neurons, oligodendrocytes and astrocytes, differentiate in specific temporal orders at topologically defined positions. How the timing and position of their generation are coordinately regulated remains poorly understood. Here, we provide evidence that the transcription factors Pax6, Olig2 and Nkx2.2 (Nkx2-2), which define the positional identity of multipotent progenitors early in development, also play crucial roles in controlling the timing of neurogenesis and gliogenesis in the developing ventral spinal cord. We show that each of these factors has a unique ability to either enhance or inhibit the activities of the proneural helix-loop-helix (HLH) factors Ngn1 (Neurog1), Ngn2 (Neurog2), Ngn3 (Neurog3) and Mash1 (Ascl1), and the inhibitory HLH factors Id1 and Hes1, thereby regulating both the timing of differentiation of multipotent progenitors and their fate. Consistent with this, dynamic changes in their co-expression pattern in vivo are closely correlated to stage- and domain-specific generation of three neural cell lineages. We also show that genetic manipulations of their temporal expression patterns in mice alter the timing of differentiation of neurons and glia. We propose a molecular code model whereby the combinatorial actions of two classes of transcription factors coordinately regulate the domain-specific temporal sequence of neurogenesis and gliogenesis in the developing spinal cord.
2536	What's your diagnosis? Hemophagocyytic lymphohistiocytosis (HLH) or hemophagocytic syndrome.	17338009	NaN
2537	Intense myelofibrosis in a child: unusual result of EBV-associated hemophagocytic lymphohistiocytosis.	27263483	A previously healthy 12-year-old girl was admitted to the intensive care unit with severe pulmonary bleeding. Her history revealed that she had suffered from high fever, fatigue, sore throat, myalgia and generalized rash for two weeks. Physical examination revealed hepatosplenomegaly. Laboratory investigation showed pancytopenia associated with unusual high levels of serum ferritin, triglyceride and lactate dehydrogenase (LDH) and low fibrinogen levels. Apparent hemophagocytosis was seen in bone marrow aspiration. Bone marrow biopsy revealed myelofibrosis, and confirmed hemophagocytosis. IgM for Epstein-Barr virus (EBV) viral capsid antigen was found to be positive. She received chemotherapy for 10 days according to hemophagocytic lymphohistiocytosis (HLH)-2004 treatment protocol, since the symptoms persisted despite supportive therapy and intravenous immunoglobulin (IVIG) administration. However, the clinical status and laboratory findings did not respond to treatment and she died from severe pulmonary bleeding associated with prolonged ventilator support and sepsis. Intense myelofibrosis, which is reported rarely, particularly in patients with EBV-related HLH, contributed to this fatal prognosis. Daha öncesinde sağlıklı olan 12 yaşındaki kız hasta Pediatrik Yoğun Bakım Ünitesine ağır pulmoner kanama ile yatırıldı. Öyküsünde 2 haftadır devam eden yüksek ateş, halsizlik, boğaz ağrısı, kas ağrıları ve yaygın döküntüsü olduğu tanımlanmaktaydı. Fizik bakıda karaciğer ve dalak büyüklüğü izlendi. Laboratuvar tetkiklerinde yüksek ferritin, trigliserid ve LDH değerleri ile birlikte düşük fibrinojen düzeylerine eşlik eden pansitopeni saptandı. Kemik iliği aspirasyonunda belirrgin hemofagositoz izlendi. Kemik iliği biyopsisi ile hemofagositoz doğrulandı ve yoğun miyelofibrosis saptandı. Serolojik olarak EBV Viral kapsid antijene karşı IgM antikorların pozitifliği gösterildi. Hasta destek tedaviye ve IVIG uygulamalarına yanıt vermeyince 10 gün süreyle HLH 2004 tedavi protokolüne uygun olarak kemoterapi aldı. Hasta uzun süreli mekanik ventilasyon izlemi sonrasında sepsis ve pulmoner kanama tablosunda kaybedildi. Özellikle EBV ilişkili HLH’de nadiren bildirilen ileri derecede kemik iliği myelofibrosisi bu hastadaki ölümcül gidişe katkıda bulunmuştur.
2538	Large-scale identification of c-MYC-associated proteins using a combined TAP/MudPIT approach.	17314511	The c-MYC oncogene encodes a transcription factor, which is sufficient and necessary for the induction of cellular proliferation. However, the c-MYC protein is a relatively weak transactivator suggesting that it may have other functions. To identify protein interactors which may reveal new functions or represent regulators of c-MYC we systematically identified proteins associated with c-MYC in vivo using a proteomic approach. We combined tandem affinity purification (TAP) with the mass spectral multidimensional protein identification technology (MudPIT). Thereby, 221 c-MYC-associated proteins were identified. Among them were 17 previously known c-MYC-interactors. Selected new c-MYC-associated proteins (DBC-1, FBX29, KU70, MCM7, Mi2-beta/CHD4, RNA Pol II, RFC2, RFC3, SV40 Large T Antigen, TCP1alpha, U5-116kD, ZNF281) were confirmed independently. For association with MCM7, SV40 Large T Antigen and DBC-1 the functionally important MYC-box II region was required, whereas FBX29 and Mi2-beta interacted via MYC-box II and the BR-HLH-LZ motif. In addition, regulators of c-MYC activity were identified: ectopic expression of FBX29, an E3 ubiquitin ligase, decreased c-MYC protein levels and inhibited c-MYC transactivation, whereas knock-down of FBX29 elevated the concentration of c-MYC. Furthermore, sucrose gradient analysis demonstrated that c-MYC is present in numerous complexes with varying size and composition, which may accommodate the large number of new c-MYC-associated proteins identified here and mediate the diverse functions of c-MYC. Our results suggest that c-MYC, besides acting as a mitogenic transcription factor, regulates cellular proliferation by direct association with protein complexes involved in multiple synthetic processes required for cell division, as for example DNA-replication/repair and RNA-processing. Furthermore, this first comprehensive description of the c-MYC-associated sub-proteome will facilitate further studies aimed to elucidate the biology of c-MYC.
2539	Specification of neuronal identities by feedforward combinatorial coding.	1729817620076660	Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. Here, we use the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Amongst the progeny of neuroblast 5-6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. We show the HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5-6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, we find that the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues.
2540	CNS involvement in hemophagocytic lymphohistiocytosis: CT and MR findings.	17277568	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder that is characterized by proliferation of benign histiocytes, and this commonly involves the liver, spleen, lymph nodes, bone marrow and central nervous system (CNS). We report here on the CT and MR imaging findings in a case of CNS HLH that showed multiple ring enhancing masses mimicking abscess or another mass on the CT and MR imaging.
2541	Molecular characterization of the Caenorhabditis elegans REF-1 family member, hlh-29/hlh-28.	17258327	Members of the Caenorhabditis elegans REF-1 family of bHLH proteins are atypical in that each protein contains two bHLH domains. In this study we describe a functional and molecular characterization of the REF-1 family members, hlh-29/hlh-28. 5'-RACE results confirm the presence of two bHLH domain coding regions in a single transcript and quantitative PCR (qPCR) shows that hlh-29/hlh-28 mRNA is detected in wild-type animals throughout development. A promoter fusion of hlh-29 to the green fluorescent protein shows post-embryonic reporter activity in cells of the vulva, the somatic gonad, the intestine and in neuronal cells of the head and tail. Loss of hlh-29/hlh-28 function via RNA interference (RNAi) results in multiple phenotypes including late embryonic lethality, yolk protein accumulation, everted vulva, bordering behavior, and alter chemosensory responses.
2542	2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure of normal human dermal fibroblasts results in AhR-dependent and -independent changes in gene expression.	17257637	Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of lesions in mammals including severe skin lesions. The majority of TCDD's biological effects are mediated through activation of the aryl hydrocarbon receptor (AhR). We have chosen to examine the effect of TCDD and the AhR pathway on dermal fibroblasts because this cell type plays an integral role in skin homeostasis through the production of cytokines and other factors that regulate epidermal proliferation and differentiation. Our data show that normal human dermal fibroblasts (NHDFs) are responsive to TCDD, as demonstrated by induction of cytochrome p450 1B1 (CYP1B1) expression. Further, our data demonstrate that TCDD treatment of NHDFs results in significant (75-90%) decrease in expression of Id-1 and Id-3, proteins that are involved in regulation of cell proliferation and differentiation. The Id (Inhibitor of DNA binding) proteins are transcriptional inhibitors that function by forming inactive heterodimers with other HLH proteins. TCDD-repression of Id-1 and -3 is independent of de novo protein synthesis; co-treatment with cycloheximide has no effect on TCDD inhibition of Id-1 and Id-3. Co-treatment with the AhR antagonist alpha-naphthoflavone also does not block inhibition of Id-1 and Id-3 by TCDD, suggesting that TCDD inhibition of Id-1 and Id-3 is, at least in part, mediated independently of the AhR pathway. Our data also show that TCDD inhibits expression of the cell cycle regulatory gene p16(ink4a), which is often linked to Id expression. TCDD-induced reduction of p16(ink4a) expression is also independent of protein synthesis and the AhR pathway.
2543	Molecular cloning and expression of grass carp MyoD in yeast Pichia pastoris.	17244478	MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84(th) amino acids) and HLH domain (98-142(th) amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZalphaA and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.
2544	Wnt/beta-catenin signaling is required for development of the exocrine pancreas.	17222338	Beta-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of beta-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that beta-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of beta-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of beta-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of beta-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed beta-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.Pdx1-cre floxed beta-catenin animals were viable but demonstrated small body size and shortened median survival. The pancreata from knockout mice were hypoplastic and histologically demonstrated a striking paucity of exocrine pancreas, acinar to duct metaplasia, but generally intact pancreatic islets containing all lineages of endocrine cells. In animals with extensive acinar hypoplasia, putative hepatocyte transdifferention was occasionally observed. Obvious and uniform pancreatic hypoplasia was observed by embryonic day E16.5. Transcriptional profiling of Pdx1-cre floxed beta-catenin embryonic pancreata at E14.5, before there was a morphological phenotype, revealed significant decreases in the beta-catenin target gene N-myc, and the basic HLH transcription factor PTF1, and an increase of several pancreatic zymogens compared to control animals. By E16.5, there was a dramatic loss of exocrine markers and an increase in Hoxb4, which is normally expressed anterior to the pancreas.We conclude that beta-catenin expression is required for development of the exocrine pancreas, but is not required for development of the endocrine compartment. In contrast, beta-catenin/Wnt signaling appears to be critical for proliferation of PTF1+ nascent acinar cells and may also function, in part, to maintain an undifferentiated state in exocrine/acinar cell precursors. Finally, beta-catenin may be required to maintain positional identity of the pancreatic endoderm along the anterior-posterior axis. This data is consistent with the findings of frequent beta-catenin mutations in carcinomas of acinar cell lineage seen in humans.
2545	Max is acetylated by p300 at several nuclear localization residues.	17217336	Max is a ubiquitous transcription factor with a bHLHZip [basic HLH (helix-loop-helix) leucine zipper] DNA-binding/dimerization domain and the central component of the Myc/Max/Mad transcription factor network that controls cell growth, proliferation, differentiation and apoptotic cell death in metazoans. Max is the obligatory DNA-binding and dimerization partner for all the bHLHZip regulators of the Myc/Max/Mad network, including the Myc family of oncoproteins and the Mad family of Myc antagonists, which recognize E-box DNA elements in the regulatory regions of target genes. Max lacks a transcription regulatory domain and is the only member of the network that efficiently homodimerizes. Binding of Max homodimers to E-box elements suppresses the transcription regulatory functions of its network partners and of other non-network E-box-binding regulators. In contrast with its highly regulated partners, Max is a constitutively expressed and phosphorylated protein. Phosphorylation is, however, the only Max post-translational modification identified so far. In the present study, we have analysed Max posttranslational modifications by MS. We have found that Max is acetylated at several lysine residues (Lys-57, Lys-144 and Lys-145) in mammalian cells. Max acetylation is stimulated by inhibitors of histone deacetylases and by overexpression of the p300 co-activator/HAT (histone acetyltransferase). The p300 HAT also directly acetylates Max in vitro at these three residues. Interestingly, the three Max residues acetylated in vivo and in vitro by p300 are important for Max nuclear localization and Max-mediated suppression of Myc transactivation. These results uncover novel post-translational modifications of Max and suggest the potential regulation of specific Max complexes by p300 and reversible acetylation.
2546	Two cases of ampulla (takotsubo-shaped) cardiomyopathy associated with hemophagocytic lymphohistiocytosis.	17215570	There have been many reports of patients with ampulla cardiomyopathy described as takotsubo-shaped cardiomyopathy in the cardiovascular field. This unique cardiomyopathy is characterized by transient apical ballooning and hypokinesis of the left ventricle. We describe 2 cases of ampulla cardiomyopathy associated with hemophagocytic lymphohistiocytosis (HLH). In both of the patients, ventricular dysfunction suddenly occurred during the active phase of HLH. In each case, the findings on ECG, echocardiogram and left ventriculogram were compatible with ampulla cardiomyopathy. To our knowledge, this communication is the first to report cases of ampulla cardiomyopathy associated with HLH. Our cases suggest that HLH hypercytokinemia may have a role in causing ampulla cardiomyopathy.
2547	C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion.	17215301	During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.
2548	Massive expansion of EBV+ monoclonal T cells with CD5 down regulation in EBV-associated haemophagocytic lymphohistiocytosis.	17213357	Haemophagocytic lymphohistiocytosis (HLH) comprises primary and secondary forms; the secondary form is most commonly triggered by the Epstein-Barr virus (EBV; EBV-HLH). Patients with EBV-HLH usually exhibit oligoclonal or monoclonal T cell proliferation, which may mimic T cell lymphoproliferative disorder (T-LPD). This article reports on EBV-HLH in a 17-month-old girl with an extreme surge of reactive T lymphocytosis (absolute count 167x10(9)/l) with CD5 down regulation. Bone marrow aspirate and trephine contained florid haemophagocytosis and massive infiltration of CD3+ Epstein-Barr virus-encoded RNA+ lymphocytes, as seen by double labelling. These lymphocytes were monoclonal for EBV and T cell receptor gamma chain gene rearrangement. The patient responded dramatically to intravenous immunoglobulin, interferon alpha2b, ganciclovir and prednisolone, suggesting restoration of her immune system and eradication of the clonal T cells through these immunoregulatory agents. Thus, careful clinicopathological correlation is warranted in the interpretation of immunophenotyping and clonality data in T cell proliferation in association with EBV-HLH to avoid erroneous diagnosis of T-LPD.
2549	MyoD, modularity, and myogenesis: conservation of regulators and redundancy in C. elegans.	1718286317142668	NaN
2550	Tumor necrosis factor alpha promoter polymorphism associated with increased susceptibility to secondary hemophagocytic lymphohistiocytosis in the Korean population.	17166738	Secondary hemophagocytic lymphohistiocytosis (HLH) can be associated with various diseases including infections and lymphoma. The clinical findings of HLH can be explained by an increased production of cytokines such as tumor necrosis factor alpha (TNF-alpha). As not all the patients with infection or lymphoma have secondary HLH, we investigated the relationship between susceptibility to secondary HLH and TNF-alpha promoter polymorphisms to identify genetic factors of secondary HLH. We determined the alleles of four promoter sites (-1031/-857/-308/-238) of TNF-alpha gene by using Taqman-based allelic discrimination assays in the 66 Korean patients with secondary HLH and 100 healthy Korean controls. We found that the frequency of the TNF-alpha -1031C allele, which is associated with higher-plasma TNF-alpha levels, was enriched in patients with secondary HLH compared with healthy controls (OR=2.00, 95% CI 1.20-3.30, P=0.007). In haplotype analysis of TNF-alpha polymorphisms, the haplotype H6 (CTGG) was detected only in the patient group, and the haplotype group (H2 or H5 or H6) including TNF-alpha -1031C allele was overexpressed in secondary HLH patients (OR=2.52, 95% CI 1.33-4.77, P=0.004). These results suggested that TNF-alpha -1031C allele and its associated haplotypes in Koreans may enhance susceptibility to secondary HLH.
2551	Prolonged course of familial hemophagocytic lymphohistiocytosis.	17164654	Familial hemophagocytic lymphohistiocytosis is usually diagnosed in the first 2 years of life and, if untreated, is rapidly fatal. We describe a 10-year-old boy with a 9-year history of prolonged fever and progressive hepatosplenomegaly who was diagnosed as having hemophagocytic lymphohistiocytosis 2, being homozygote to a previously described mutation in the PRF1 gene, and cured by the HLH-2004 protocol and allogenic bone marrow transplantation. This unique case emphasizes the heterogeneity of this disease and the diversity of its clinical presentations.
2552	Familial and acquired hemophagocytic lymphohistiocytosis.	17151879	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of severe hyperinflammation caused by the uncontrolled proliferation of activated lymphocytes and histiocytes secreting high amounts of inflammatory cytokines. Cardinal signs and symptoms are prolonged fever, hepatosplenomegaly and pancytopenia. Characteristic biochemical markers include elevated triglycerides, ferritin and low fibrinogen. HLH occurs on the basis of various inherited or acquired immune deficiencies. Impaired function of natural killer (NK) cells and cytotoxic T-cells (CTL) is shared by all forms of HLH. Genetic HLH occurs in familial forms (FHLH) in which HLH is the primary and only manifestation, and in association with the immune deficiencies Chédiak-Higashi syndrome 1 (CHS 1), Griscelli syndrome 2 (GS 2) and x-linked lymphoproliferative syndrome (XLP), in which HLH is a sporadic event. Most patients with acquired HLH have no known underlying immune deficiency. Both acquired and genetic forms are triggered by infections, mostly viral, or other stimuli. HLH also occurs as a complication of rheumatic diseases (macrophage activation syndrome) and of malignancies. Several genetic defects causing FHLH have recently been discovered and have elucidated the pathophysiology of HLH. The immediate aim of therapy in genetic and acquired HLH is suppression of the severe hyperinflammation, which can be achieved with immunosuppressive/immunomodulatary agents and cytostatic drugs. Patients with genetic forms have to undergo stem cell transplantation to exchange the defective immune system with normally functioning immune effector cells. In conclusion, awareness of the clinical symptoms and of the diagnostic criteria of HLH is crucial in order not to overlook HLH and to start life-saving therapy in time.
2553	Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for a unified theory of animal muscle development.	1714266817182863	Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in Caenorhabditis elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. We find that two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naïve blastomeres to muscle when overproduced ectopically in the embryo. In addition, we have used genetic null mutants to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type.
2554	A Zn-finger/FH2-domain containing protein, FOZI-1, acts redundantly with CeMyoD to specify striated body wall muscle fates in the Caenorhabditis elegans postembryonic mesoderm.	17138663	Striated muscle development in vertebrates requires the redundant functions of multiple members of the MyoD family. Invertebrates such as Drosophila and Caenorhabditis elegans contain only one MyoD homolog in each organism. Earlier observations suggest that factors outside of the MyoD family might function redundantly with MyoD in striated muscle fate specification in these organisms. However, the identity of these factors has remained elusive. Here, we describe the identification and characterization of FOZI-1, a putative transcription factor that functions redundantly with CeMyoD (HLH-1) in striated body wall muscle (BWM) fate specification in the C. elegans postembryonic mesoderm. fozi-1 encodes a novel nuclear-localized protein with motifs characteristic of both transcription factors and actin-binding proteins. We show that FOZI-1 shares the same expression pattern as CeMyoD in the postembryonic mesodermal lineage, the M lineage, and that fozi-1-null mutants exhibit similar M lineage-null defects to those found in animals lacking CeMyoD in the M lineage (e.g. loss of a fraction of M lineage-derived BWMs). Interestingly, fozi-1-null mutants with a reduced level of CeMyoD lack most, if not all, M lineage-derived BWMs. Our results indicate that FOZI-1 and the Hox factor MAB-5 function redundantly with CeMyoD in the specification of the striated BWM fate in the C. elegans postembryonic mesoderm, implicating a remarkable level of complexity for the production of a simple striated musculature in C. elegans.
2555	Dystrophin-dependent muscle degeneration requires a fully functional contractile machinery to occur in C. elegans.	17134897	In mammals, the lack of dystrophin leads to a degeneration of skeletal muscles. It has been known for many years that this pathology can be blocked by denervation or immobilization of muscles. It is not yet clear, however, whether this suppressing effect is due to the absence of fiber contraction per se, or to other mechanisms which may be induced by such treatments. We took advantage of the genetic tools available in the animal model Caenorhabditis elegans to address this question. Using RNA interference and existing mutants, we genetically impaired the excitation-contraction cascade at specific points in a dystrophin-deficient C. elegans strain which normally undergoes extensive muscle degeneration. Our data show that reducing sarcomere contraction by slightly impairing the contraction machinery is sufficient to dramatically suppress muscle degeneration. Thus, it is the physical tension exerted on the muscle fibers which is the key deleterious event in the absence of dystrophin.
2556	GCIP/CCNDBP1, a helix-loop-helix protein, suppresses tumorigenesis.	17131381	Deletions and/or loss of heterozygosity (LOH) on chromosome 15 (15q15 and 15q21) have been found in several human tumors, including carcinomas of the colorectum, breast, lung, prostate, and bladder, suggesting the presence of potential tumor suppressor gene(s) in this particular region of chromosome 15. GCIP also called CCNDBP1, DIP1, or HHM, localized at chromosome 15q15, is a recently identified helix-loop-helix leucine zipper (HLH-ZIP) protein without a basic region like the Id family of proteins. In this study, we reported that the expression of GCIP was significantly downregulated in several different human tumors, including breast tumor, prostate tumor, and colon tumors. In human colon tumors, both mRNA and protein expression levels of GCIP were decreased significantly compared to the normal tissues. Treatment of colon cancer cells SW480 with sodium butyrate (NaB), which induces colon cancer cell differentiation, can induce the upregulation of GCIP expression, suggesting that the protein functions as a negative regulator in cell proliferation. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation, while silencing of GCIP expression by siRNA can promote cell colony formation. Furthermore, overexpression of GCIP inhibited the transcriptional activity of cyclin D1 promoter and the expression of cyclin D1 protein in the cell. Finally, we demonstrate that GCIP specifically interacts with one of the class III HDAC proteins, SirT6, which is important for maintaining genome stability. Together, our data suggest a possible function of GCIP in tumor suppression.
2557	TEL/ETV6 binds to corepressor KAP1 via the HLH domain.	17118767	NaN
2558	The mechanism of discrimination between cognate and non-specific DNA by dimeric b/HLH/LZ transcription factors.	17109882	The Myc/Max/Mad proteins are basic region-helix-loop-helix-leucine zipper (b/HLH/LZ) transcription factors that regulate the transcription of numerous genes involved in cell growth and proliferation. The Max protein is the obligate heterodimeric partner of the Myc and Mad proteins. Heterodimerization and DNA binding to target gene promoters are mediated by the b/HLH/LZ domains. Max can also form a homodimeric b/HLH/LZ. The enhanced expression of Myc and binding to promoters of target genes contribute to almost every aspect of tumor biology. However, the detailed mechanism by which dimeric and heterodimeric b/HLH/LZs discriminate cognate DNA (E-Box: CACGTG) from non-specific sequences in the target gene promoters is still unknown. Here, we use the Max b/HLH/LZ homodimer as a model for this class of transcription factors in the characterization and understanding of the mechanism of discrimination between the E-Box and non-specific DNA sequences. We report the characterization of a cognate and a non-specific Max b/HLH/LZ/DNA complex by EMSA, CD and NMR. Our results support a detailed mechanism by which dimeric b/HLH/LZ transcription factors can discriminate E-Box sequences from non-specific DNA. The mechanism proceeds via the conformational selection of fitting b/HLH/LZ homodimers with the basic region only partially helical. Next, the basic region undergoes a DNA-assisted folding or induced-fit. It is this step that provides the discrimination by stabilizing and destabilizing the alpha-helical conformation of the basic region in the cognate and non-specific complexes, respectively. This leads to a low affinity complex with a higher probability of being dissociated and hence to discrimination. A description of the side-chains and nucleotides proposed to be involved in the discrimination process is provided.
2559	Immunocytochemical localization and ultrastructural study of gonadotroph cells in the female desert lizard Uromastyx acanthinura.	17098269	The pars distalis from the pituitary gland of adult female desert lizards (Uromastyx acanthinura), captured during vitellogenesis (late may) and hivernal period, was studied with immunocytochemical methods using specific antisera against human FSH (hFSH) and LH (hLH). The immunostaining with anti-hLH and anti-hFSH allowed the identification of only FSH-like containing cells. The FSH-like immunoreactive cells were affected differently by a physiological stage and showed some heterogenous cytological characteristics. During vitellogenesis, four aspects of rostral FSH-like immunoreactive cells could be recognized. The expression of FSH-like in mainly immunoreactive cells was parallel to an intense synthetic activity and to the presence of ultrastructural features indicating an intense release of the hormone. This release was considerably altered in winter, the immunoreactive cells stored an important amount of secretion granules which increased in size and undergo a crinophagic process.
2560	Perforin and granzymes have distinct roles in defensive immunity and immunopathology.	17088087	Successful control of viral infection requires the host to eliminate the infecting pathogen without causing overt immunopathology. Here we showed that perforin (Prf1) and granzymes (Gzms) have distinct roles in defensive immunity and immunopathology in a well-established model of viral infection. Both Prf1 and Gzms drastically affected the outcome of murine cytomegalovirus (MCMV) infection. Viral titres increased markedly in both Prf1(-/-) and Gzma(-/-)Gzmb(-/-) mice, but Gzma(-/-)Gzmb(-/-) mice recovered and survived infection, whereas Prf1(-/-) mice did not. Indeed, infected Prf1-deficient hosts developed a fatal hemophagocytic lymphohistiocytosis (HLH)-like syndrome. This distinction in outcome depended on accumulation of mononuclear cells and T cells in infected Prf1(-/-) mice. Importantly, blocking experiments that clearly identified tumor necrosis factor-alpha (TNF-alpha) as the principal contributor to the lethality observed in infected Prf1(-/-) mice provided support for the clinical potential of such an approach in HLH patients whose disease is triggered by viral infection.
2561	The hydraulic limitation hypothesis revisited.	17080592	We proposed the hydraulic limitation hypothesis (HLH) as a mechanism to explain universal patterns in tree height, and tree and stand biomass growth: height growth slows down as trees grow taller, maximum height is lower for trees of the same species on resource-poor sites and annual wood production declines after canopy closure for even-aged forests. Our review of 51 studies that measured one or more of the components necessary for testing the hypothesis showed that taller trees differ physiologically from shorter, younger trees. Stomatal conductance to water vapour (g(s)), photosynthesis (A) and leaf-specific hydraulic conductance (K L) are often, but not always, lower in taller trees. Additionally, leaf mass per area is often greater in taller trees, and leaf area:sapwood area ratio changes with tree height. We conclude that hydraulic limitation of gas exchange with increasing tree size is common, but not universal. Where hydraulic limitations to A do occur, no evidence supports the original expectation that hydraulic limitation of carbon assimilation is sufficient to explain observed declines in wood production. Any limit to height or height growth does not appear to be related to the so-called age-related decline in wood production of forests after canopy closure. Future work on this problem should explicitly link leaf or canopy gas exchange with tree and stand growth, and consider a more fundamental assumption: whether tree biomass growth is limited by carbon availability.
2562	Differentiation of human mesenchymal stem cells and articular chondrocytes: analysis of chondrogenic potential and expression pattern of differentiation-related transcription factors.	17072841	Mesenchymal stem cells (MSCs) are a candidate for replacing chondrocytes in cell-based repair of cartilage lesions. However, it has not been clarified if these cells can acquire the hyaline phenotype, and whether chondrocytes and MSCs show the same expression patterns of critical control genes in development. In order to study this, articular chondrocytes and iliac crest derived MSCs were allowed to differentiate in pellet mass cultures. Gene expression of markers for the cartilage phenotype, helix-loop-helix (HLH) transcription factors, and chondrogenic transcription factors were analyzed by real-time PCR. Matrix production was assayed using biochemical analysis for hydroxyproline, glycosaminoglycans, and immunohistochemistry for collagen types I and II. Significantly decreased expression of collagen type I was accompanied by increased expression of collagen types IIA and IIB during differentiation of chondrocytes, indicating differentiation towards a hyaline phenotype. Chondrogenesis in MSCs on the other hand resulted in up-regulation of collagen types I, IIA, IIB, and X, demonstrating differentiation towards cartilage of a mixed phenotype. Expression of HES1 increased significantly during chondrogenesis in chondrocytes while expression in MSCs was maintained at a low level. The HLH gene HES5 on the other hand was only detected in chondrocytes. Expression of ID1 decreased significantly in chondrocytes while the opposite was seen in MSCs. These findings suggest that chondrocytes and MSCs differentiated and formed different subtypes of cartilage, the hyaline and a mixed cartilage phenotype, respectively. Differentially regulated HLH genes indicated the possibility for HLH proteins in regulating chondrogenic differentiation. This information is important to understand the potential use of MSCs in cartilage repair.
2563	Muscle development in Ciona intestinalis requires the b-HLH myogenic regulatory factor gene Ci-MRF.	17055476	The activity of myogenic regulatory factor (MRF) genes is essential for vertebrate muscle development, whereas invertebrate muscle development is largely independent of MRF function. This difference indicates that myogenesis is controlled by distinct regulatory mechanisms in these two groups of animals. Here we used overexpression and gene knockdown to investigate the role in embryonic myogenesis of the single MRF gene of the invertebrate chordate Ciona intestinalis (Ci-MRF). Injection of Ci-MRF mRNA into eggs resulted in increased embryonic muscle-specific gene activity and revealed the myogenic activity of Ci-MRF by inducing the expression of four muscle marker genes, Acetylcholinesterase, Actin, Troponin I, and Myosin Light Chain in non-muscle lineages. Conversely, inhibiting Ci-MRF activity with antisense morpholinos down-regulated the expression of these genes. Consistent with the effects of morpholinos on muscle gene activity, larvae resulting from morpholino injection were paralyzed and their "muscle" cells lacked myofibrils. We conclude that Ci-MRF is required for larval tail muscle development and thus that an MRF-dependent myogenic regulatory network probably existed in the ancestor of tunicates and vertebrates. This possibility raises the question of whether the earliest myogenic regulatory networks were MRF-dependent or MRF-independent.
2564	Designing a new generation of anti-hCG vaccines for cancer therapy.	17049720	The heterodimeric 'pregnancy-specific' hormone human chorionic gonadotropin (hCG) has been used as the basis for a contraceptive vaccine. More recently, the observation that hCG, particularly in the form of the beta-chain expressed in the absence of alpha-chain, is aberrantly expressed in a number of different tumors has opened up a second potential application for such vaccines. Drawbacks of the currently available vaccines are that they are either relatively weakly immunogenic or that they induce antibodies that cross-react with human leuteinizing hormone (hLH). We have explored the possibility of creating mutated versions of the hCG beta-chain with improved immunologic properties.
2565	Analysis of intact human follicle-stimulating hormone preparations by reversed-phase high-performance liquid chromatography.	17049544	A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the qualitative and quantitative analysis of intact human follicle-stimulating hormone (hFSH) was established and validated for accuracy, precision and sensitivity. Human FSH is a dimeric glycoprotein hormone widely used as a diagnostic analyte and as a therapeutic product in reproductive medicine. The technique developed preserves the protein integrity, allowing the analysis of the intact heterodimeric form rather than just of its subunits, as is the case for the majority of the conditions currently employed. This methodology has also been employed for comparing the relative hydrophobicity of pituitary, urinary and two Chinese hamster ovary (CHO)-derived hFSH preparations, as well as of two other related glycoprotein hormones of the anterior pituitary: human thyroid-stimulating hormone (hTSH) and human luteinizing hormone (hLH). The least hydrophobic of the three glycohormones analyzed was hFSH, followed by hTSH and hLH. A significant difference (p<0.005) was observed in t(R) between the pituitary and recombinant hFSH preparations, reflecting structural differences in their carbohydrate moieties. Two main isoforms were detected in urinary hFSH, including a form which was significantly different (p<0.005) from the pituitary and recombinant preparations. The linearity of the dose-response curve (r=0.9965, n=15) for this RP-HPLC methodology, as well as an inter-assay precision of less than 4% for the quantification of different hFSH preparations and a sensitivity of the order of 40 ng, were demonstrated. The chromatographic behaviour and relative hydrophobicity of the individual subunits of the pituitary and recombinant preparations were also analyzed. Furthermore, the molecular mass of individual hFSH subunits and of the heterodimer were simultaneously determined by matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS). The present methodology represents, in our opinion, an essential tool for the characterization and quality control of this hormone, that is not yet described in the main pharmacopoeias.
2566	Gland-specific expression of C. elegans hlh-6 requires the combinatorial action of three distinct promoter elements.	17049341	The pharyngeal glands of Caenorhabditis elegans are one of five cell types in the pharynx. The transcription factor HLH-6 is required for gland development and function, and is specifically expressed in pharyngeal glands. As a first step to understanding specification of pharyngeal glands, we analyzed the promoter of hlh-6 to identify the elements required for gland-specific expression. Our experiments identified three distinct regulatory elements required for hlh-6 expression: a PHA-4-binding site and two new elements, HRL1 and HRL2 (for hlh-6 regulatory elements 1 and 2). The three elements employ a simple logic for producing cell-type-specific expression: the PHA-4 site restricts expression to the pharynx, HRL2 restricts expression in both a position and lineage-dependent manner, and HRL1 restricts expression to a subset of cell types. In isolation, these three elements have little or no enhancer activity but in combination they produce robust, gland-specific expression. These findings describe a combinatorial code for gland-specific expression and suggest that similar codes may be employed for specification of other pharyngeal cell types.
2567	Modeling fire-induced smoke spread and carbon monoxide transportation in a long channel: Fire Dynamics Simulator comparisons with measured data.	17049158	Smoke and toxic gases, such as carbon monoxide, are the most fatal factors in fires. This paper models fire-induced smoke spread and carbon monoxide transportation in an 88m long channel by Fire Dynamics Simulator (FDS) with large eddy simulation (LES). FDS is now a well-founded fire dynamics computational fluid dynamic (CFD) program, which was developed by National Institute of Standards and Technology (NIST). Two full scale experiments with fire sizes of 0.75 and 1.6MW were conducted in this channel to validate the program. The spread of the fire-induced smoke flow together with the smoke temperature distribution along the channel, and the carbon monoxide concentration at an assigned position were measured. The FDS simulation results were compared with experimental data with fairly good agreement demonstrated. The validation work is then extended to numerically study the carbon monoxide concentration distribution, both vertically and longitudinally, in this long channel. Results showed that carbon monoxide concentration increase linearly with the height above the floor and decreases exponentially with the distance away from the fire source.
2568	Spontaneous Escherichia coli meningitis associated with hemophagocytic lymphohistiocytosis.	16959624	Spontaneous Escherichia coli meningitis has not been previously reported in association with hemophagocytic lymphohistiocytosis (HLH). A previously healthy 72-year-old woman was admitted due to fever, nuchal rigidity, disturbed consciousness and splenomegaly. Anemia, thrombocytopenia and hyperferritinemia developed on the 8th day of hospitalization. Cultures of cerebrospinal fluid and blood grew E. coli. Abundant macrophages overwhelmed erythrocytes in the bone marrow aspirate, confirming the presence of hemophagocytosis. E. coli meningitis was managed with a 40-day course of antibiotic treatment. However, the severity of anemia and thrombocytopenia progressed despite intensive transfusion therapy. The patient died of HLH on the 60th day of hospitalization.
2569	Newborn infant with hemophagocytic lymphohistiocytosis and generalized skin eruptions.	16958808	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease resulting from abnormal proliferation of histiocytes in tissues and organs. The incidence of HLH is 1:50,000-300,000. Cutaneous eruptions have been reported in 6-65% of the cases. It's important to differentiate the eruptions from other systemic diseases. We present an infant with prominent skin manifestations of HLH. On the 11th day of life, she was admitted to our hospital with complaint of a generalized rash that had started the previous day. The eruptions consisted of irregularly shaped maculopapular erythematous rash and purpura. Bone marrow aspiration on the 25th day of life revealed hemophagocytosis with increased macrophages and histiocytes, consistent with HLH. Treatment was started with dexamethasone followed by induction chemotherapy with etoposide. All skin manifestations resolved in a few days. Although the clinical features are nonspecific, HLH should be kept in mind as an accompanying disease in neonates presenting with skin eruptions.
2570	Fulminant neonatal liver failure in siblings: probable congenital hemophagocytic lymphohistiocytosis.	16944969	Familial hemophagocytic lymphohistiocytosis (HLH) is an autosomal recessive disorder of immune regulation characterized by fever, splenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. Although presentation usually occurs during the first 2 years of life, congenital presentation is rare. We report siblings with a presumptive diagnosis of familial HLH who presented with hydrops fetalis and severe hepatic involvement ultimately resulting in their deaths. This report emphasizes the difficulty of confirming the diagnosis of HLH. However, establishing the diagnosis has important implications for genetic counseling and family planning. HLH should be considered in the setting of perinatal liver failure. The immunologic basis of the disease is incompletely understood but testing for natural killer cell function, and perforin defects may be helpful in establishing a diagnosis. HLH can be treated with chemotherapy, immunotherapy, and stem cell transplantation.
2571	HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis.	16937360	In HLH-94, the first prospective international treatment study for hemophagocytic lymphohistiocytosis (HLH), diagnosis was based on five criteria (fever, splenomegaly, bicytopenia, hypertriglyceridemia and/or hypofibrinogenemia, and hemophagocytosis). In HLH-2004 three additional criteria are introduced; low/absent NK-cell-activity, hyperferritinemia, and high-soluble interleukin-2-receptor levels. Altogether five of these eight criteria must be fulfilled, unless family history or molecular diagnosis is consistent with HLH. HLH-2004 chemo-immunotherapy includes etoposide, dexamethasone, cyclosporine A upfront and, in selected patients, intrathecal therapy with methotrexate and corticosteroids. Subsequent hematopoietic stem cell transplantation (HSCT) is recommended for patients with familial disease or molecular diagnosis, and patients with severe and persistent, or reactivated, disease. In order to hopefully further improve diagnosis, therapy and biological understanding, participation in HLH studies is encouraged.
2572	Drosophila CK2 regulates lateral-inhibition during eye and bristle development.	16930955	Lateral inhibition is critical for cell fate determination and involves the functions of Notch (N) and its effectors, the Enhancer of Split Complex, E(spl)C repressors. Although E(spl) proteins mediate the repressive effects of N in diverse contexts, the role of phosphorylation was unclear. The studies we describe implicate a common role for the highly conserved Ser/Thr protein kinase CK2 during eye and bristle development. Compromising the functions of the catalytic (alpha) subunit of CK2 elicits a rough eye and defects in the interommatidial bristles (IOBs). These phenotypes are exacerbated by mutations in CK2 and suppressed by an increase in the dosage of this protein kinase. The appearance of the rough eye correlates, in time and space, to the specification and refinement of the 'founding' R8 photoreceptor. Consistent with this observation, compromising CK2 elicits supernumerary R8's at the posterior margin of the morphogenetic furrow (MF), a phenotype characteristic of loss of E(spl)C and impaired lateral inhibition. We also show that compromising CK2 elicits ectopic and split bristles. The former reflects the specification of excess bristle SOPs, while the latter suggests roles during asymmetric divisions that drive morphogenesis of this sensory organ. In addition, these phenotypes are exacerbated by mutations in CK2 or E(spl), indicating genetic interactions between these two loci. Given the centrality of E(spl) to the repressive effects of N, our studies suggest conserved roles for this protein kinase during lateral inhibition. Candidates for this regulation are the E(spl) repressors, the terminal effectors of this pathway.
2573	The hemophagocytic syndrome: titrating continuous hemofiltration to the degree of lactic acidosis.	16928655	In 3 cases of severe multiple organ failure due to hemophagocytic lymphohistiocytosis (HLH) in children, the authors demonstrate the utility of continuous hemofiltration in attenuating the consequences of excess cytokine activity, with therapy titrated to the degree of lactic acidosis. HLH was diagnosed in 3 encephalopathic children with multiple organ failure, elevated ferritin (49,396-237,582 pmol/L; or 21,983-105,733 ng/mL), elevated serum triglyceride, and depressed cell lines. One had a known malignancy, one had EBV-associated lymphoproliferative disease, and one was previously healthy. Continuous hemofiltration was initiated, with the ultrafiltrate production rate and countercurrent dialysate flow titrated to metabolic acidosis as reflected by the serum lactate (maximum 3.5 mmol/L or 31.6 mg/dL). Hemofiltration was titrated upward until lactic acidosis resolved, through clearance of lactate or interruption of excess cytokine-driven activity; maximum prescription was 2000 mL/h ultrafiltrate production plus 2500 mL/h dialysate flow. Stability was achieved with hemofiltration, then substantial resolution occurred with treatment according to the HLH-94 protocol (dexamethasone, cyclosporin, VP-16, intrathecal methotrexate). One child succumbed to candidiasis. Another made a full recovery. A third succumbed to his primary malignancy. HLH should be suspected in unexplained or unresolving multiple organ failure. Titration of hemofiltration based on measurable parameters of cellular metabolism (e.g., lactate, base deficit) may stabilize the child with metabolic acidosis long enough to allow proper diagnosis and institution of definitive therapy. Hemofiltration is not a panacea but rather a stabilizing mechanism, with poorly understood effects on interstitial water and solute flux, that facilitates recovery over weeks, not days.
2574	Possible implication of the transcriptional regulator Id3 in PACAP-induced pro-survival signaling during PC12 cell differentiation.	16928405	PACAP inhibits cell proliferation and promotes cell survival and neurite outgrowth of pheochromocytoma PC12 cells. Transcriptome analysis of PACAP-treated PC12 cells allowed to identify potential genes implicated in this differentiation process. Among the genes whose expression is up-regulated by PACAP, we identified the Inhibitor of DNA binding 3 (Id3). Id3 is a member of the helix-loop-helix (HLH) family of transcription factors which acts as a negative dominant inhibitor of basic HLH factors. Time-course studies revealed that Id3 is an early PACAP response gene (8-fold after 1 h of stimulation), and that the up-regulation of its expression persists over 12 h after the onset of PACAP treatment. The stimulatory effect of PACAP on Id3 mRNA levels was mimicked by adenylate cyclase/PKA activators like forskolin and dibutyryl cyclic AMP. Moreover, PACAP-induced Id3 gene expression was inhibited by phosphatidylinositol 3'-OH-kinase and p38 MAP kinase blockers. Northern blot analysis of Id3 distribution in rat tissues showed a strong expression of this gene in the adrenal medulla. Overexpression of Id3 increased the number of living PC12 cells, in basal condition and after exposure to oxidative stress. These results indicate that Id3 is a cAMP-responsive gene whose up-regulation could be involved in PACAP-induced pro-survival signaling during sympathoadrenal cell differentiation.
2575	Molecular switches and developmental potential of adult stem cells.	16903417	Stem cell commitment and differentiation entails the successive loss of self-renewal and developmental potential, and results in the final restriction to a terminally differentiated mature cell type. Hematopoiesis, the development of blood cells from hematopoietic stem cells in bone marrow, is particularly well studied, and at different branching points within the hematopoietic system multiple developmental intermediates have been identified. Here we describe a Flt3+ CD11b+ multipotent progenitor that can be amplified in vitro by a specific cytokine combination to high cell numbers, and following adoptive transfer into syngeneic mice, it generates dendritic cells but also additional mature cell types. By employing gene expression profiling with DNA microarrays and knockout mouse models, we demonstrate that the helix-loop-helix (HLH) transcription factor Id2 (inhibitor of DNA binding/differentiation 2) acts as a molecular switch in development of Langerhans cells (LCs), the cutaneous contingent of dendritic cells (DCs), and of specific DC subsets and B cells.
2576	[Hemophagocytic syndrome: diagnostic problems].	16892901	Hemophagocytic syndrome (HS) is a rare but life-threatening disease caused by inappropriate activation of T-lymphocytes and histiocytes, hipercytokinemia and hemophagocytosis. The most common symptoms are fever, hepatosplenomegaly, unspecific neurological abnormalities, pancytopenia, coagulopathy, hiperferritinemia and lipid abnormalities. HS is classified into two forms: primary, inherited (Familial Hamophagocytic Lymphohistiocytosis--FHL) and secondary (associated with infection, malignancy, autoimmune disease). In spite of the fact that diagnostic guidelines are available it often remains unrecognised. Prognosis of HS depends on the form of disease and in case of secondary HS on the underlying disease. Development of the treatment protocols (HLH-94, HLH-2004) which combine immunochemiotherapy with hematopoietic stem cell transplantation has strongly improved prognosis in HS especially in the primary form. Three-year overall survival for children with HS is now over 50%. Early diagnosis and appropriate therapy is crucial for effectiveness of the treatment. Popularisation of the knowledge about the syndrome, diagnostic guidelines and treatment protocols can contribute to more frequent appropriate recognition of HS and to improvement of the treatment results.
2577	Decreased expression of myogenic transcription factors and myosin heavy chains in Caenorhabditis elegans muscles developed during spaceflight.	16888068	The molecular mechanisms underlying muscle atrophy during spaceflight are not well understood. We have analyzed the effects of a 10-day spaceflight on Caenorhabditis elegans muscle development. DNA microarray, real-time quantitative PCR, and quantitative western blot analyses revealed that the amount of MHC in both body-wall and pharyngeal muscle decrease in response to spaceflight. Decreased transcription of the body-wall myogenic transcription factor HLH-1 (CeMyoD) and of the three pharyngeal myogenic transcription factors, PEB-1, CEH-22 and PHA-4 were also observed. Upon return to Earth animals displayed reduced rates of movement, indicating a functional defect. These results demonstrate that C. elegans muscle development is altered in response to spaceflight. This altered development occurs at the level of gene transcription and was observed in the presence of innervation, not simply in isolated cells. This important finding coupled with past observations of decreased levels of the same myogenic transcription factions in vertebrates after spaceflight raises the possibility that altered muscle development is a contributing factor to spaceflight-induced muscle atrophy in vertebrates.
2578	A novel type of PTD, common helix-loop-helix motif, could efficiently mediate protein transduction into mammalian cells.	16870135	Protein transduction domains (PTDs), such as HIV TAT PTD, have been widely used as delivery tools into living cells. Here we reported for the first time that the helix-loop-helix (HLH) domain of basic helix-loop-helix (bHLH) family was a novel type of PTD. Efficient internalization has been obtained with HLH domains derived from bHLH proteins, NeuroD/BETA2, Neurogenin3, and Mitf, in various cell types including stable cell lines and primary cells. Cellular uptake of HLH PTD was barely or slightly inhibited by the metabolic, phagocytosis, clathrin- or caveolar-endocytosis formation inhibitors, but significantly and substantially reduced by heparin and macropinocytosis inhibitor, which suggested important roles of cell surface glycosaminoglycans and macropinocytosis during the protein transduction. Furthermore, NeuroD proteins still preserved transcription activation after internalized into cells. Our results demonstrated a new motif of PTD different from previous models as cationic residues cluster or amphipathic helix. The HLH domain is also the characteristic of the bHLH family, which implied a large number of PTDs could be discovered in this family to fit different purposes, and some of them could be directly recruited to penetrate cell membrane according to their crucial roles in development such as NeuroD and Ngn3.
2579	Patients of African ancestry with hemophagocytic lymphohistiocytosis share a common haplotype of PRF1 with a 50delT mutation.	16860143	Mutations of the perforin gene (PRF1) are present in a proportion of patients with hemophagocytic lymphohistiocytosis (HLH). We found that all identified infants with HLH of African descent (17 from USA, 4 from Europe) have 50delT-PRF1 (16 homozygotes, 5 compound heterozygotes), accounting for the most frequently observed PRF1 mutation. Two additional patients with HLH, self-reporting as Hispanic, carried 50delT, but no Caucasians were identified with 50delT. To test the hypothesis that this mutation represents a single haplotype, DNA from 23 patients with HLH and 30 African-American control subjects was sequenced for the PRF1 gene, including portions of the intron containing known single nucleotide polymorphisms (SNPs). The same groups were genotyped at 3 microsatellites proximal to PRF1. The SNP profiles of patients with 50delT-PRF1 were identical, and 5 novel SNPs were identified among African-American control subjects. Patients with 50delT-PRF1 were also found to have had an earlier age of disease onset than patients with other PRF1 mutations. Extent of haplotype sharing and variability of microsatellite alleles in 50delT-PRF1 chromosomes suggest that this mutation arose approximately 1000 to 4000 years ago and is restricted to patients of African descent.
2580	Successful father-to-son stem cell transplantation in a child with hemophagocytic lymphohistiocytosis using a reduced-intensity conditioning regimen.	16856932	Hemophagocytic lymphohistiocytosis (HLH) is an uncommon disorder, usually lethal without allogeneic stem cell transplantation (SCT).We report a 9-month-old boy, the first child of consanguineous parents, diagnosed with HLH and neurological involvement demonstrated by magnetic resonance imaging (MRI), who received an allogeneic SCT from his HLA genetically matched father. Transplant was performed after a reduced-intensity conditioning (RIC) regimen consisting of cyclophosphamide, fludarabine, and melphalan. Graft vs. host disease (GVHD) prophylaxis included cyclosporine a and methotrexate.An absolute neutrophil count of 0.5 x 10(9)/L was documented on day +20 and a platelet count >20 x 10(9)/L was shown by day 33. Full donor chimerism was showed on day +175. A follow-up brain MRI was reported normal. Twenty months after SCT, the child shows no evidence of HLH or GVHD activity, and has a normal psychomotor development.Given the reduced toxicity of SCT with RIC, it could represent an attractive transplant method for children with HLH, in whom myeloablation plays no role in disease eradication, and in whom mixed chimerism may be enough to cure the disease.
2581	Precursor B-cell acute lymphoblastic leukemia presenting with hemophagocytic lymphohistiocytosis.	16856156	Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome which can be an inherited congenital disorder or can develop secondary to malignancy, infection, or autoimmune disease. Secondary HLH due to malignancy occurs most commonly with T or NK-cell lymphoid neoplasms. HLH with B-cell malignancies is less common and HLH has rarely been described in association with precursor B-cell acute lymphoblastic leukemia (B-ALL). We report three cases of HLH associated with B-ALL and review 17 cases of ALL-associated HLH previously reported in the literature.
2582	Macrophage activation syndrome in juvenile idiopathic arthritis.	16801165	Macrophage activation syndrome (MAS) is a rare and potentially lethal complication of chronic rheumatic diseases of childhood, in particular of systemic-onset juvenile idiopathic arthritis (s-JIA), resulting from uncontrolled activation and proliferation of T lymphocytes and macrophages. The onset, acute and dramatic, may mimic a flare of the underlying disease or a severe sepsis. Diagnosis is difficult and, until now, no specific criteria have been developed. Laboratory data show pancytopenia, coagulopathy, low ESR and low concentrations of serum albumin, and high levels of ferritin, liver enzymes and triglycerides. Activated macrophages are found in various organs, particularly in bone marrow. Most hypotheses on the mechanism underlying MAS are based on the data obtained in primary haemophagocytic lymphohistiocytosis (HLH), a genetic disease very similar to MAS. Prompt diagnosis is essential because prognosis is highly related to early treatment. The first approach was to use intravenous methylprednisolone pulse therapy; cyclosporin A was proposed in patients resistant to steroids. We describe nine patients affected by haemophagocytosis: seven patients developed MAS and two patients developed HLH. A child with s-JIA developed three episodes of MAS. After the third episode, as there was no improvement with pulses of methylprednisolone and cyclosporine, he was successfully given etanercept.Our data, together with a similar, published observation, suggest that the TNF inhibitor etanercept is potentially useful for obtaining remission in children not responding to steroids and cyclosporin A.
2583	KIDARI, encoding a non-DNA Binding bHLH protein, represses light signal transduction in Arabidopsis thaliana.	16786307	Through activation tagging mutagenesis, we isolated a kidari-D (kdr-D) mutant, which exhibited a defect in blue and far-red light mediated photomorphogenesis. Under continuous blue light, the kdr-D mutant showed long hypocotyl phenotype, whereas it showed normal cotyledon opening and expansion. In addition, the kdr-D showed slightly longer hypocotyl under continuous far-red light, suggesting that KDR functions in a branch of cry signaling and mediates a cross-talk between cry and phyA. In the kdr-D mutant, a gene encoding a putative basic/Helix-Loop-Helix (bHLH) protein was overexpressed by the insertion of 35S enhancer into 10 kb upstream of the gene. Consistently, overexpression of this gene recapitulated the phenotype of kdr-D. KDR is composed of 94 amino acids with non-DNA binding HLH domain, a structure found in human Inhibitor of DNA binding 1 (Id-1) which functions as a negative regulator of bHLH proteins through heterodimerization with them. The KDR specifically interacted with HFR1, a bHLH protein regulating photomorphogenesis, in yeast two hybrid assay and the kdr-D was epistatic to 35S::HFR1 in the hypocotyl phenotype. Thus, it shows that KDR functions as a negative regulator of HFR1, similar to Id-1 in human. The KDR exhibited circadian expression pattern with an increase during the day. Taken together, our results suggest that KDR attenuates light mediated responses in day light condition through inhibition of the activity of bHLH proteins involved in light signaling.
2584	Diffusion-weighted cranial MR imaging findings in a patient with hemophagocytic syndrome.	16775287	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder of the immune system that is associated with frequent involvement of the central nervous system (CNS). The MR imaging and CT findings of the CNS infiltration have been reported in the past; however, the diffusion-weighted imaging (DWI) findings have not been previously described. We present MR imaging findings in a case of secondary HLH with CNS involvement, with an emphasis on the DWI findings.
2585	A key role for the HLH transcription factor EBF2COE2,O/E-3 in Purkinje neuron migration and cerebellar cortical topography.	16774995	Early B-cell factor 2 (EBF2) is one of four mammalian members of an atypical helix-loop-helix transcription factor family (COE). COE proteins have been implicated in various aspects of nervous and immune system development. We and others have generated and described mice carrying a null mutation of Ebf2, a gene previously characterized in the context of Xenopus laevis primary neurogenesis and neuronal differentiation. In addition to deficits in neuroendocrine and olfactory development, and peripheral nerve maturation, Ebf2 null mice feature an ataxic gait and obvious motor deficits associated with clear-cut abnormalities of cerebellar development. The number of Purkinje cells (PCs) in the Ebf2 null is markedly decreased, resulting in a small cerebellum with notable foliation defects, particularly in the anterior vermis. We show that this stems from the defective migration of a molecularly defined PC subset that subsequently dies by apoptosis. Part of the striped cerebellar topography is disrupted due to cell death and, in addition, many of the surviving PCs, that would normally adopt a zebrin II-negative phenotype, transdifferentiate to Zebrin II-positive, an unprecedented finding suggesting that Ebf2 is required for the establishment of a proper cerebellar cortical map.
2586	A case of rhesus hemolytic disease with hemophagocytosis and severe iron overload due to multiple transfusions.	16772878	A newborn with cholestatic hepatic disease and hemophagocytic lymphohistiocytosis due to rhesus hemolytic disease (RHD) is reported.A 34 weeks' gestation baby with RHD, who had received multiple intrauterine transfusions (IUT), developed cholestatic hepatic disease and secondary hemophagocytic lymphohistiocytosis (HLH). Her serum ferritin level increased to 5,527 ng/mL, and liver biopsy showed severe iron overload. Treatment with intravenous desferrioxamine resulted in a marked decrease in serum ferritin levels and normalization of liver functionWe suggest that patients who have undergone IUT be evaluated for hyperferritinemia. If hyperferritinemia is noted, chelation therapy should be considered. As another rare finding, HLH can complicate the course of RHD.
2587	Consanguineous marriage and congenital heart defects: a case-control study in the neonatal period.	16763961	The independent effect of consanguinity on the prevalence of congenital heart defects (CHDs), all and specific types, was investigated in newborns admitted to nine hospitals located in Beirut, Lebanon and members of the National Collaborative Perinatal Neonatal Network (NCPNN). Cases were 173 newborns admitted to the Neonatal Intensive Care Units (NICU) of participating hospitals during the 3-year period from January 1, 2000 to December 31, 2002 and diagnosed during their hospital stay as having one or more CHD. Cases with chromosomal abnormalities were excluded. Cases with more than one CHD were assigned one principal malformation. Controls consisted of a random sample of 865 newborns without a CHD admitted to the NICU during the same period. After controlling for confounders, first cousin consanguinity remained significantly associated with an increased risk of CHD: infants born to first cousin marriages had a 1.8 times higher risk of having a CHD diagnosed at birth compared to those born to unrelated parents (95% CI: 1.1-3.1). In particular, first-cousin marriage was a significant risk factor for ventricular septal defect (VSD), atrial septal defect (ASD), hypoplastic left heart (HLH), and single ventricle (SV). No association was found with d-transposition of the great arteries, coarctation, pulmonary atresia (PA), atrioventricular septal defect (AVSD), and tetralogy of Fallot (TOF). The results of this study suggest a familial factor in the multifactorial etiology of CHDs. Additional epidemiologic and family-based genetic studies are needed to understand the complex cause of CHDs.
2588	FLT3 is fused to ETV6 in a myeloproliferative disorder with hypereosinophilia and a t(12;13)(p13;q12) translocation.	16761019	The FMS-like tyrosine kinase 3 (FLT3) gene, belonging to the receptor tyrosine kinase (TK) subclass III family, plays an important role in normal hematopoiesis and is one of the most frequently mutated genes in hematologic malignancies as well as an attractive target for directed inhibition. Activating mutations of this gene, including internal tandem duplication in the juxtamembrane (JM) domain and point mutations in the TK domain, are found in approximately one-third of patients with acute myeloid leukemia and in a smaller subset of patients with acute lymphoblastic leukemia. We report here that FLT3 may contribute to leukemogenesis in a patient with myeloproliferative disorder and a t(12;13)(p13;q12) translocation through generating a fusion gene with the ETS variant gene 6 (ETV6) gene. ETV6 has been reported to fuse to various partner genes, including TK and transcription factors. Both ETV6/FLT3 and reciprocal FLT3/ETV6 transcripts were detected in the patient mRNA by reverse transcriptase-polymerase chain reaction. At the protein level, however, only ETV6/FLT3 products were expressed. Among them, one retains the helix-loop-helix (HLH) oligomerization domain of ETV6 and the JM as well as TK domain of FLT3. FLT3 receptor in leukemic cells might be inappropriately activated through dimerization by HLH domain of ETV6, which consequently interfered with proliferation and differentiation of hematopoietic cells.
2589	A short Id2 protein fragment containing the nuclear export signal forms amyloid-like fibrils.	16756960	The negative regulator of DNA-binding/cell-differentiation Id2 is a small protein containing a central helix-loop-helix (HLH) motif and a C-terminal nuclear export signal (NES). Whereas the former is essential for Id2 dimerization and nuclear localization, the latter is responsible for the transport of Id2 from the nucleus to the cytoplasm. Whereas the isolated Id2 HLH motif is highly helical, large C-terminal Id2 fragments including the NES sequence are either unordered or aggregation-prone. To study the conformational properties of the isolated NES region, we synthesized the Id2 segment 103-124. The latter was insoluble in water and only temporarily soluble in water/alcohol mixtures, where it formed quickly precipitating beta-sheets. Introduction of a positively charged N-terminal tail prevented aggressive precipitation and led to aggregates consisting of long fibrils that bound thioflavin T. These results show an interesting structural aspect of the Id2 NES region, which might be of significance for both protein folding and function.
2590	Synthesis and conformational analysis of Id2 protein fragments: impact of chain length and point mutations on the structural HLH motif.	16733829	The Id proteins are negative regulators of several basic-helix-loop-helix (HLH) transcription factors, including the ubiquitous E factors and the tissue-specific myogenin-regulating factors. Id1 through Id4 contain highly identical HLH domains but different N- and C-terminal extensions. Beside the heterodimerization with the parent HLH factors, Id2 was shown to additionally interact with the retinoblastoma protein and to be overexpressed in neuroblastoma. Thus, Id2 represents an interesting target for cancer therapy based on the inhibition of protein-protein interactions. Here we present the synthesis and circular dichroism (CD) analysis of peptides derived from point mutations and N-/C-terminal truncations of Id2. The helix character of the HLH domain (residues 36-76) was reduced upon substitution of Met39/-62 and Cys42 with Nle and Ser, respectively, suggesting a structural role of these side chains. The largest sequence that could be obtained by stepwise solid-phase peptide synthesis (SPPS) with Fmoc strategy spanned the entire HLH motif (with Cys42 replaced by Ser) and part of the C-terminus (residues 77-110). This 75-residue long fragment was less helical than the isolated HLH domain and had propensity to aggregate, which was correlated with the presence of the flanking residues C-terminal to helix-2. By CD analysis of an equimolar mixture of the sequence 36-110 with the N-terminus 1-35, noncovalent interactions between the two peptides were detected, which, however, changed upon aging. In contrast, the mixture of the HLH sequence 36-76 with the N-terminus was characterized by a stabilized helix structure that was maintained also upon aging. Presumably, the N-terminal region interacted with the folded HLH motif in a specific manner, whereas only unspecific, weak contacts occurred with the partly unfolded HLH domain and/or the immediate flanking residues 77-110.
2591	The expression pattern of genes involved in early neurogenesis suggests distinct and conserved functions in the diplopod Glomeris marginata.	16724224	We have shown recently that the expression and function of proneural genes is conserved in chelicerates and myriapods, although groups of neural precursors are specified in the ventral neuroectoderm of these arthropod groups, rather than single cells as in insects and crustaceans. We present additional evidence that the pattern of neurogenesis seen in chelicerates and in previously analyzed myriapod species is representative of both arthropod groups, by analysing the formation of neural precursors in the diplopod Archispirostreptus sp. This raises the question as to what extent the genetic network has been modified to result in different modes of neurogenesis in the arthropod group. To find out which components of the neural genetic network might account for the different mode of neural precursor formation in chelicerates and myriapods, we identified genes in the diplopod Glomeris marginata that are known to be involved in early neurogenesis in Drosophila and studied their expression pattern. In Drosophila, early neurogenesis is controlled by proneural genes that encode HLH transcription factors. These genes belong to two major subfamilies, the achaete-scute group and the atonal group. Different proneural proteins activate both a common neural programme and distinct neuronal subtype-specific target genes. We show that the expression pattern of homologs of the Drosophila proneural genes daughterless, atonal, and Sox B1 are partially conserved in Glomeris mariginata. While the expression of the pan-neural gene snail is conserved in the ventral neuroectoderm of G. marginata, we found an additional expression domain in the ventral midline. We conclude that, although the components of the genetic network involved in specification of neural precursors seem to be conserved in chelicerates, myriapods, and Drosophila, the function of some of the genes might have changed during evolution.
2592	Variations of the perforin gene in patients with autoimmunity/lymphoproliferation and defective Fas function.	16720836	Mutations decreasing function of the Fas death receptor cause the autoimmune lymphoproliferative syndrome (ALPS) with autoimmune manifestations, spleen/lymph node enlargement, and expansion of CD4/CD8-negative T cells. Dianzani Autoimmune Lymphoproliferative Disease (DALD) is a variant lacking this expansion. Perforin is involved in cell-mediated cytotoxicity and its biallelic mutations cause familial hemophagocytic lymphohistiocytosis (HLH). We previously described an ALPS patient carrying heterozygous mutations of the Fas and perforin genes and suggested that they concurred in ALPS. This work extends the analysis to 14 ALPS, 28 DALD, and 816 controls, and detects an N252S amino acid substitution in 2 ALPS, and an A91V amino acid substitution in 6 DALD. N252S conferred an OR = 62.7 (P = .0016) for ALPS and A91V conferred an OR = 3 (P = .016) for DALD. Copresence of A91V and variations of the osteopontin gene previously associated with DALD conferred an OR = 17 (P = .0007) for DALD. In one N252S patient, NK activity was strikingly defective in early childhood, but became normal in late childhood. A91V patients displayed lower NK activity than controls. These data suggest that perforin variations are a susceptibility factor for ALPS/DALD development in subjects with defective Fas function and may influence disease expression.
2593	Identification of a basic helix-loop-helix transcription factor expressed in mammary gland alveolar cells and required for maintenance of the differentiated state.	16645041	The development of mammary glands relies on complicated signaling pathways that control cell proliferation, differentiation, and apoptotic events through transcriptional regulatory circuits. A key family of transcription factors used in mammary gland development is the helix-loop-helix/basic helix-loop-helix (HLH/bHLH) protein family. In this study, we identify Mist1 as a tissue-restricted Class II bHLH transcription factor expressed in lactating mammary glands. Mouse and human mammary glands accumulated Mist1 protein exclusively in secretory alveolar cells, and Mist1 transcripts were differentially expressed in mouse SCp2 cells induced to differentiate by addition of lactogenic hormones. Mist1 null (Mist1(KO)) lactating mammary glands were defective in normal lobuloalveolar organization, exhibiting shedding of cells into the alveolus lumen and premature activation of the signal transducer and activator of transcription 3 signaling pathway. These cells also failed to maintain expression of the gap junction proteins connexin26 and connexin32, leading to the loss of gap junctions. Our findings suggest that loss of Mist1 impairs the maintenance of the fully differentiated alveolar state and, for the first time, places Mist1 within the hierarchy of known HLH/bHLH proteins that control mammary epithelial cell development.
2594	Molecular characterization, structure and developmental expression of Megane bHLH factor.	16644143	We report here the full-length sequence identification, molecular characterization, detailed demarcation expression analysis relevant to morphological marker genes and mapping of a bHLH transcription gene, referred to as Megane (Mgn). Mgn protein is structurally related to the neurogenic Drosophila hairy and Enhancer of split (h/E(spl)) proteins. The unique structural properties of Mgn factor in several characteristic residues define the gene as related to h/E(spl), but distinguish it from previously identified mammalian members of the family. Mgn is a single copy gene on mouse chromosome 8 and encodes a 27kDa protein that functions in the nucleus. First expression of Mgn is detected at mouse embryonic day 9.5 within the most rostral part of the cephalic flexure of the developing midbrain. Later, Mgn expression extends into other alar areas of the midbrain and forebrain, developmentally controlled in a regional specific pattern.
2595	Regulation of IkappaB kinase (IKK) complex by IKKgamma-dependent phosphorylation of the T-loop and C terminus of IKKbeta.	16597623	The mechanistic relationship of phosphorylation of the C terminus of IKKbeta with phosphorylation of its T-loop kinase domain within the IKK complex remained unclear. We investigated the regulatory role of the serine cluster residing immediately adjacent to the HLH domain and of the serines in the NEMO/IKKgamma-binding domain (NBD/gammaBD) in the C-terminal portion of IKKbeta in MEFs deficient in IKKbeta and IKKalpha and in yeast reconstitution system. We show that phosphorylation events at the C terminus of IKKbeta can be divided into autophosphorylation of the serine cluster adjacent to the HLH domain and phosphorylation of the NBD/gammaBD. Autophosphorylation of the serine cluster occurs immediately after IKK activation and requires IKKgamma. In MEFs, this autophosphorylation does not have the down-regulatory function on the IKK complex that was previously described (1). On the other hand, phosphorylation of the NBD/gammaBD regulates IKKgamma-dependent phosphorylation of the T-loop activation domain in IKKbeta and, hence, IKK complex activation. Our study suggests that, within the IKK complex, modulation of the NBD/gammaBD by IKKgamma is upstream to the T-loop phosphorylation.
2596	Identification of follicle-stimulating hormone-selective beta-strands in the N-terminal hormone-binding exodomain of human gonadotropin receptors.	16574743	Glycoprotein hormone receptors contain large N-terminal extracellular domains (ECDs) that distinguish these receptors from most other G protein-coupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N- and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which beta-strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R) counterparts. Introduction of hFSH-R beta-strand 1 into hLH-R conferred responsiveness to hFSH, whereas hLH-R mutants harboring one of the other hFSH-R beta-strands displayed none or very limited sensitivity to hFSH. However, combined substitution of hFSH-R beta-strand 1 and some of the other hFSH-R beta-strands further increased the sensitivity of the mutant hLH-R to hFSH. The apparent contribution of multiple hFSH-R beta-strands in providing a selective hormone binding interface corresponds well with their position in relation to hFSH as recently determined in the crystal structure of hFSH in complex with part of the hFSH-R ECD.
2597	Lethal hemophagocytic lymphohistiocytosis in Hermansky-Pudlak syndrome type II.	16551969	Griscelli syndrome (GS) was diagnosed in a 2-year-old patient with oculocutaneous albinism and immunodeficiency, but sequencing of RAB27a revealed only a heterozygous mutation. Due to impaired natural killer (NK) and T-cell cytotoxicity implying a high risk of developing hemophagocytic lymphohistiocytosis (HLH), he was prepared for hematopoietic stem cell transplantation (HSCT). Unexpectedly, a severe bleeding episode occurred that led to the demonstration of disturbed platelet aggregation, reduced plateletdense granules, and impaired platelet degranulation. In combination with neutropenia, this suggested the diagnosis of Hermansky-Pudlak syndrome type II (HPSII) and a novel homozygous mutation in AP3B1 was detected. None of the 3 reported HPSII patients had developed HLH, and our patient seroconverted to Epstein-Barr virus (EBV) without clinical symptoms. HSCT was therefore withheld, and granulocyte-colony-stimulating factor (G-CSF) therapy was initiated and prevented further bacterial infections. At 3 years of age, however, the patient developed, without an obvious trigger, fulminant HLH that was resistant to therapy. This patient shows that careful clinical and molecular diagnosis is essential to differentiate the complex disorders of lysosomal trafficking. HPSII belongs to the group of familial hemophagocytic syndromes and may represent an indication for HSCT.
2598	Hematopoietic stem cell transplantation in hemophagocytic lymphohistiocytosis: a single-center report of 48 patients.	16549504	Familial hemophagocytic lymphohistiocytosis (FHLH) is a genetically determined disorder characterized by the early onset of fever, hepatosplenomegaly, central nervous system disease, thrombocytopenia, coagulation disorders, and hemophagocytosis. It is caused by genetic defects that impair T cell-mediated and natural cytotoxicity. Chemotherapy- or immunotherapy-based treatments can achieve remission. Hematopoietic stem cell transplantation (HSCT), however, is the only curative option, but optimal modalities and long-term outcome are not yet well known.We retrospectively analyzed the outcome of HSCT that was performed in 48 consecutive patients who had FHLH and were treated in a single center between 1982 and 2004.The overall survival was 58.5% with a median follow-up of 5.8 years and extending to 20 years. A combination of active disease and haploidentical HSCT had a poor prognosis because in this situation, HLH disease is more frequently associated with graft failure. Twelve patients received 2 transplants because of graft failure (n = 7) or secondary graft loss that led to HLH relapse (n = 5). Transplant-related toxicity essentially consisted in veno-occlusive disease, which occurred in 28% of transplants and was associated with young age, haploidentical transplantation, and the use of antithymocyte globulin (ATG) in the conditioning regimen. A sustained remission was achieved in all patients with a donor chimerism > or = 20% of leukocytes. Long-term sequelae were limited, because only 2 (7%) of 28 patients experienced a mild neurologic disorder.This survey demonstrates the long-term efficacy of HSCT as a cure of FHLH. HSCT preserves quality of life. It shows that HSCT should be performed as early as a complete remission has been achieved. Additional studies are required to improve the procedure and reduce its toxic effects.
2599	Cytotoxic therapy for severe avian influenza A (H5N1) infection.	16530581	The mortality rate in documented avian influenza A virus subtype H5N1 infection is still high, which is currently reported by WHO at about 50%. Post-mortem analyses in affected patients have revealed haemophagocytosis similar to that found in patients with haemophagocytic lymphohistiocytosis (HLH); such haemophagocytosis could be a very prominent post-mortem feature in H5N1 infection. There are also clinical similarities between H5N1 infection and HLH, such as massive hypercytokinaemia, cytopenia, and acute encephalitis. Importantly, patients with another severe viral infection that may be complicated by secondary HLH, severe Epstein-Barr-virus-associated HLH, have significantly better survival if specific HLH therapy (including the cytotoxic and pro-apoptotic drug etoposide) is initiated early, with survival reported to rise from about 50% to 90%. With this notable improvement in survival, specific HLH treatment, including cytotoxic therapy, could be considered in patients with severe avian influenza A infection complicated by secondary HLH.
2600	Significance of Id-1 up-regulation and its association with EGFR in bladder cancer cell invasion.	16525633	Epidermal growth factor receptor (EGFR) is suggested to be one of the positive factors in the invasive progression of bladder cancer. Id-1 (inhibitor of differentiation or DNA binding), a helix-loop-helix (HLH) transcription factor, was recently identified as a key factor in the EGFR signalling pathway. The aim of this study was to investigate the role of Id-1 in bladder cancer progression and its relation-ship with EGFR. Using clinical specimens from different stages of bladder cancer, immunohistochemical staining was performed to determine if Id-1 expression was positively associated with tumour staging and EGFR expression. The direct role of Id-1 in cancer cell invasion was also investigated through ectopically expressing the Id-1 gene in a RT112 bladder cancer cell line by wound closure and collagen invasion assays. To explore the therapeutic potential of targeting the Id-1 gene in the treatment of invasive bladder cancer, we studied if inactivation of the Id-1 gene through small RNA interference could lead to the suppression of invasion in a MGHU1 bladder cancer cell line. Our results showed that the up-regulation of Id-1 was associated with increased EGFR expression, clinical staging and the invasion ability of bladder cancer cells. Inactivation of Id-1 may be a potential therapeutic target to inhibit the invasion by bladder cancer cells.
2601	Twist1 dimer selection regulates cranial suture patterning and fusion.	16502419	Saethre-Chotzen syndrome is associated with haploinsufficiency of the basic-helix-loop-helix (bHLH) transcription factor TWIST1 and is characterized by premature closure of the cranial sutures, termed craniosynostosis; however, the mechanisms underlying this defect are unclear. Twist1 has been shown to play both positive and negative roles in mesenchymal specification and differentiation, and here we show that the activity of Twist1 is dependent on its dimer partner. Twist1 forms both homodimers (T/T) and heterodimers with E2A E proteins (T/E) and the relative level of Twist1 to the HLH inhibitor Id proteins determines which dimer forms. On the basis of the expression patterns of Twist1 and Id1 within the cranial sutures, we hypothesized that Twist1 forms homodimers in the osteogenic fronts and T/E heterodimers in the mid-sutures. In support of this hypothesis, we have found that genes regulated by T/T homodimers, such as FGFR2 and periostin, are expressed in the osteogenic fronts, whereas genes regulated by T/E heterodimers, such as thrombospondin-1, are expressed in the mid-sutures. The ratio between these dimers is altered in the sutures of Twist1+/- mice, favoring an increase in homodimers and an expansion of the osteogenic fronts. Of interest, the T/T to T/E ratio is greater in the coronal versus the sagittal suture, and this finding may contribute to making the coronal suture more susceptible to fusion due to TWIST haploinsufficiency. Importantly, we were able to inhibit suture fusion in Twist1+/- mice by modulating the balance between these dimers toward T/E formation, by either increasing the expression of E2A E12 or by decreasing Id expression. Therefore, we have identified dimer partner selection as an important mediator of Twist1 function and provide a mechanistic understanding of craniosynostosis due to TWIST haploinsufficiency.
2602	Expression of GCIP in transgenic mice decreases susceptibility to chemical hepatocarcinogenesis.	16501603	Transcription factors with helix-loop-helix (HLH) motif play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, proliferation, and tumorigenesis. To examine whether the newly identified HLH protein GCIP/CCNDBP1 modulates cell fate determination and plays a role in hepatocyte growth, proliferation, and hepatocarcinogenesis, we generated transgenic mice with human GCIP gene driven by a liver-specific albumin promoter. We demonstrated that in GCIP transgenic mice, the overall liver growth and regeneration occurred normally after liver injury induced by carbon tetrachloride (CCl4). In the diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis, we demonstrated that overexpression of GCIP in mouse liver suppressed DEN-induced hepatocarcinogenesis at an early stage of tumor development. The number of hepatic adenomas at 24 weeks was significantly lower or not detected in GCIP transgenic male mice compared to the control mice under the same treatment. Although GCIP has little inhibition on the number of hepatic tumors at later stages (40 weeks), hepatocellular tumors in GCIP transgenic mice are smaller and well-differentiated compared to the poorly differentiated tumors in wild-type mice. Furthermore, we demonstrate that GCIP functions as a transcriptional suppressor, regulates the expression of cyclin D1, and inhibits anchorage-independent cell growth and colony formation in HepG2 cells, suggesting a significant role of GCIP in tumor initiation and development.
2603	An exclusive case of juvenile myelomonocytic leukemia in association with Kikuchi's disease and hemophagocytic lymphohistiocytosis and a review of the literature.	16487587	We present a case of juvenile myelomonocytic leukemia (JMML) accompanied by immune-mediated hemophagocytic lymphohistiocytosis (HLH) and Kikuchi's disease, both as a paraneoplastic phenomenon. As this combination, to the best of our knowledge, has not been described before, consensus on preferable treatment is lacking. Our patient was treated with prednisolone according to the few described cases of HLH and Kikuchi's disease in non-JMML patients, resulting in disappearance of the clinical symptoms.
2604	Identification of novel target genes of CeTwist and CeE/DA.	16480708	Twist, a basic helix-loop-helix (bHLH) transcription factor, plays an important role in mesoderm development in many organisms, including C. elegans where CeTwist is required to direct cell fate specifications of a subset of mesodermal cells. Although several target genes of CeTwist have been identified, how this protein accomplishes its function is unclear. In addition, several human genes whose mutations cause different syndromes of craniosynostosis (premature fusion of cranial sutures) have homologues in the CeTwist pathway. Identification of novel target genes of CeTwist will shed more light on the functions of CeTwist in mesoderm development, and the corresponding human homologues will be good candidates for related syndromes with unidentified mutated genes. In our study, both CeTwist and its heterodimeric partner, CeE/DA, were overexpressed from the inducible heat-shock promoter, and potential target genes were detected with Affymetrix oligonucleotide microarrays. Using transcriptional GFP reporters, we found 11 genes were expressed in cells coincident with known CeTwist target gene products. Based on subsequent validation experiments, 9 genes were defined as novel CeTwist and CeE/DA targets. Human homologues of two of these genes might be involved in craniofacial diseases, which further validates C. elegans as a good model organism for providing insights into these disorders.
2605	Hemophagocytic lymphohistiocytosis: diagnosis, pathophysiology, treatment, and future perspectives.	16448985	Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease in which the immune system becomes overactive due to its inability to effectively respond to infections and/or shut down the immune response to such infections. The discovery of genetic defects in the secretory pathway of natural killer (NK) cells and cytotoxic T cells in some patients with this disease has raised important questions of the role of cytotoxic cells in the control of infections and in immune regulation. This review will give a brief overview of the clinical presentation and accepted treatment of HLH. Furthermore, it will give an in-depth review into the known genetic defects and current knowledge of the pathophysiology of this disorder, and will highlight recent evidence suggesting that cytotoxic defects in CD4+ T regulatory cells may contribute to the pathogenesis of HLH.
2606	A novel perforin gene mutation in a Japanese family with hemophagocytic lymphohistiocytosis.	16443553	A 4-month-old girl with clinical features of hemophagocytic lymphohistiocytosis (HLH) was successfully treated with immunochemotherapy but died at the age of 1 year and 3 months, before hematopoietic stem cell transplantation could be performed. Her family history showed death during infancy of the eldest sister, suggesting a diagnosis of familial HLH (FHL). Direct sequencing of the DNA extracted from the patient's spleen tissue obtained at autopsy revealed a novel perforin gene mutation: a homozygous 1289G insertion (Asp430 frameshift and termination at amino acid residue 457), which has not previously been reported in FHL patients.
2607	Role of the basic helix-loop-helix protein ITF2 in the hormonal regulation of Sertoli cell differentiation.	16425294	Sertoli cells are a post-mitotic terminally differentiated cell population that forms the seminiferous tubules in the adult testis and provides the microenvironment and structural support for developing germ cells. During pubertal development, Sertoli cells are responsive to follicle-stimulating hormone (FSH) to promote the expression of differentiated gene products. The basic helix-loop-helix (bHLH) and inhibitors of differentiation (Id) transcription factors are involved in the differentiation of a variety of cell lineages during development. Both bHLH and Id transcription factors have been identified in Sertoli cells. A yeast two-hybrid screen was conducted using a rat Sertoli cell cDNA library to identify bHLH dimerization partners for the Id1 transcription factor. The ubiquitous bHLH protein ITF2 (i.e., E2-2) was identified as one of the interacting partners. The current study investigates the expression and function of ITF2 in Sertoli cells. ITF2 was found to be ubiquitously expressed in all testicular cell types including germ cells, peritubular myoid cells, and Sertoli cells. Stimulation of cultured Sertoli cells with FSH or dibutryl cAMP resulted in a transient decrease in expression of ITF2 mRNA levels followed by a rise in expression with FSH treatment. ITF2 expression was at its highest in mid-pubertal 20-day-old rat Sertoli cells. ITF2 was found to directly bind to negative acting Id HLH proteins and positive acting bHLH proteins such as scleraxis. Transient overexpression of ITF2 protein in cultured Sertoli cells stimulated transferrin promoter activity, which is a marker of Sertoli cell differentiation. Co-transfections of ITF2 and Id proteins sequestered the inhibitory effects of the Id family of proteins. Observations suggest ITF2 can enhance FSH actions through suppressing the inhibitory actions of the Id family of proteins and increasing the actions of stimulatory bHLH proteins (i.e., scleraxis) in Sertoli cells.
2608	Accounting for strain-specific differences during RTG target gene regulation in Saccharomyces cerevisiae.	16423076	Mitochondrial dysfunction results in the expression, via the retrograde response pathway, of a concise set of genes (RTG target genes) that encode enzymes involved in the anapleurotic production of alpha-ketoglutarate. Inhibiting the rapamycin-sensitive TOR kinases, important regulators of cell growth, similarly results in RTG target gene expression under rich nutrient conditions. Retrograde and TOR-dependent regulation of RTG target genes requires a number of shared components, including the heterodimeric bZip/HLH transcription factors Rtg1p and Rtg3p, as well as their upstream regulator Mks1p. Two unresolved discrepancies exist with regard to the mechanism of RTG target gene control: (1) deletion of MKS1 results in constitutive expression of RTG target genes in most but not all strain backgrounds; and (2) RTG target gene expression has been correlated with both decreased as well as increased Rtg3p phosphorylation. Here we have addressed both of these issues. First, we demonstrate that the mks1 deletion strain used in a previous study by Shamji and coworkers contains a nonsense mutation within codon Ser 231 in RTG3 that likely accounts for the inactivity of the RTG system in this strain. Second, we confirm results by Butow and coworkers that Rtg3p is dephosphorylated as a primary response to induction of the pathway. Hyper-phosphorylation of this protein appears to be a secondary consequence of rapamycin treatment and is influenced both by strain background as well as by specific supplied nutrients. That hyper-phosphorylation of Rtg3p is also caused by heat shock suggests that it may reflect a more generalized response to cell stress. Together these results contribute toward a uniform view of RTG target gene regulation.
2609	Evaluation of apoptosis in Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	16419109	Epstein-Barr virus (EBV) is known to be a causative agent of hemophagocytic lymphohistiocytosis (HLH). To investigate association of apoptosis in the pathogenesis of EBV-associated HLH, the serum EBV loads, and serum concentrations of soluble tumor necrosis factor receptor 1 (sTNF-R1), soluble Fas ligand, and cytochrome c were examined in 15 patients with EBV-associated HLH and 24 patients with infectious mononucleosis (IM). Levels of sTNF-R1 are known to reflect the biological activity of TNF-alpha and cytochrome c is a specific marker of apoptosis. EBV loads, and concentrations of sTNF-R1 and cytochrome c were significantly higher in patients with EBV-associated HLH than in patients with IM. On the other hand, there were no statistically significant differences in the concentrations of soluble Fas ligand. In patients with EBV-associated HLH, EBV loads, concentrations of sTNF-R1, and cytochrome c were correlated with each other. These results suggest that apoptosis, which is dependent on the EBV load and could be mediated by TNF-alpha, plays a major role in the pathophysiology of EBV-associated HLH.
2610	Fetal and neonatal histiocytoses.	16416419	The histiocytoses are a group of disorders of the monophagocytic system having a variety of clinical and pathological findings. They occur less often during the perinatal period than later in life. Their biologic behavior, response to therapy, and histologic types are not the same.The study consisted of 221 fetuses and neonates collected from the literature and from personal files.Langerhans' cell histiocytosis (LCH), the hemophagocytic lymphohistiocytoses (HLH), and juvenile xanthogranuloma (JXG), in order of rank, were the main histiocytoses occurring in the perinatal period. HLH accounted for the highest mortality (74%) followed by disseminated LCH (52%) and JXG (11%). All neonates with LCH and JXG limited to the skin and/or subcutaneous tissue survived with or without treatment.This study suggests that there is an increased incidence of spontaneous regression of certain histiocytic lesions in neonates as compared to older individuals. Cutaneous forms JXG and LCH had the highest incidence of regression followed by infection associated HLH.
2611	A cluster of hypoplastic left heart malformation in Baltimore, Maryland.	16391975	Congenital cardiovascular malformations (CCVMs) of the left side of the heart show familial recurrence of various forms of obstructive malformations, including hypoplastic left heart (HLH), interrupted aortic arch, coarctation of the aorta, and aortic stenosis. In a previous population-based study in the Baltimore-Washington region, these malformations were associated with parental reports of occupational or leisure solvent exposure, overt diabetes, and family history of CCVM in first-degree relatives. Spatial analysis in this well-characterized study population may augment self-reported data by revealing additional associations with potential environmental risk factors. We used spatial analysis to identify clusters of HLH as a group. The study population included all live-born cases of hypoplastic left heart syndrome diagnosed in the first year of life between 1981 and 1989 and a random sample of unaffected infant controls matched by year and hospital of birth. The nested case-control cohort in this spatial analysis included 77 HLH cases and 1894 controls in Maryland and the District of Columbia. Nonparametric and regression analyses included personal variables from the interview data set as well as spatial variables. A region of Baltimore was identified that contained HLH at twice the expected frequency based on the distribution of population younger than 5 years of age. The region included 30 of 77 geocoded cases of HLH in the cohort and is significant by spatial scanning at p = 0.056. Within this region, male cases of HLH were disproportionately present compared to females. This cluster is in a region of Baltimore with industrial release of solvents, dioxin, and polychlorinated biphenyls in air. Outside the cluster, HLH is associated with family history of CCVM in a first-degree relative, maternal exposure to miscellaneous solvents, paternal anesthesia, maternal art painting, aspirin ingestion, and maternal diabetes. Inside the cluster, father's painting and exposure to sympathomimetic drugs were associated risk factors. Spatial analysis of HLH cases delineated an urban region with increased prevalence of this left heart malformation. Within this region, excess male cases of HLH occurred, and industrial release to air of solvents, dioxin, and polychlorinated biphenyl compounds was documented. We propose that both genetic and environmental factors contribute to the phenotype of HLH.
2612	Success with infliximab in treating refractory hemophagocytic lymphohistiocytosis.	16369976	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder characterized by fever, pancytopenia, hepatosplenomegaly, liver dysfunction, and hemophagocytosis. A 29-year-old woman, diagnosed with systemic lupus erythematosus in 1996, developed HLH in early June 2002. HLH remained refractory during 1.5 months of treatment including corticosteroid, cyclosporine, plasma exchange, vincristine, and etoposide. Infliximab (5 mg/kg/day) was then administered twice. After the second administration, the patient attained remission. Because HLH itself is not a neoplasm but an uncontrolled immune reaction, blocking cytokines involved in the reaction should have therapeutic potentials. For HLH patients not responding to conventional therapy, anticytokine treatment with infliximab may represent one of promising options.
2613	Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma.	1636953518418090	B cell differentiation is controlled by a complex network of lineage-restricted transcription factors. How perturbations to this network alter B cell fate remains poorly understood. Here we show that classical Hodgkin lymphoma tumor cells, which originate from mature B cells, have lost the B cell phenotype as a result of aberrant expression of transcriptional regulators. The B cell-specific transcription factor program was disrupted by overexpression of the helix-loop-helix proteins ABF-1 and Id2. Both factors antagonized the function of the B cell-determining transcription factor E2A. As a result, expression of genes specific to B cells was lost and expression of genes not normally associated with the B lineage was upregulated. These data demonstrate the plasticity of mature human lymphoid cells and offer an explanation for the unique classical Hodgkin lymphoma phenotype.
2614	Mutations of perforin and Munc13-4 do not mark HLH by NK defects.	1636587016365863	NaN
2615	Correlation between phenotypic heterogeneity and gene mutational characteristics in familial hemophagocytic lymphohistiocytosis (FHL).	1636586316365870	Classification of familial hemophagocytic lymphohistiocytosis (FHL) into FHL2, FHL3, and other subtypes based on genetic abnormalities has recently become possible. We studied the phenotypic differences among these subtypes in Japan.Forty patients clinically diagnosed with FHL were analyzed. Perforin abnormality was screened by flow cytometric analysis and/or DNA sequencing in these patients, and those without perforin abnormalities were further examined for the presence of mutations in the Munc13-4 gene by DNA sequencing. The correlation between clinical features and genetic subtypes was investigated.Of the 40 HLH patients, 11 showed perforin gene mutations (classified as FHL2) and ten had Munc13-4 gene mutations (FHL3), but neither mutation was noted in 19 patients (non-FHL2/3). Although the majority of the patients developed the disease before the age of 1 year, the onset in three FHL2 patients with missense mutations was late (7, 11, and 12 years). Incidence of deficient natural killer cell activity was higher in FHL2 patients (9/9 FHL2, 4/9 FHL3, and 6/17 non-FHL2/3; P = 0.005). The serum levels of ferritin and soluble interleukin-2 receptor were significantly higher in FHL2 patients with nonsense perforin mutations compared to other subgroups (P < or = 0.05). Epstein-Barr virus infection was involved in 8 of the 40 HLH patients: one FHL2, one FHL3, and six non-FHL2/3.Although clinical features of FHL3 appear to be homogeneous, the heterogeneous clinical features of FHL2 depend upon the nature of perforin gene mutations. Characterization of the non-FHL2/3 group with regard to FHL1 or other novel gene mutations remains to be conducted.
2616	Sensorineural hearing loss in a case of familial hemophagocytic lymphohistiocytosis.	16358309	Severe sensorineural hearing loss (bilateral >80 dB) was diagnosed in a case of familial hemophagocytic lymphohistiocytosis (FHL). The female patient developed HLH at 3 months of age and underwent allogeneic cord blood transplantation at 11 months of age following 7 months of immuno-chemotherapy. The type 2 FHL patient had a homozygous perforin gene mutation of 1090-1091delCT, and was noted to have hearing loss at 3.5 years of age. Retrospective evaluation did not clarify the exact causes of hearing loss. Reports on Kawasaki disease, suggesting a correlation between severe inflammatory status in infancy and the development of sensorineural hearing loss, may shed some light on this rare complication in this case of FHL. Considering the markedly improved prognosis of FHL due to recent advances made in the molecular diagnosis and in the management including allogeneic hematopoietic stem cell transplantation, auditor by screening might be warranted for surviving FHL patients.
2617	Frequency of the allelic variant (Trp8Arg/Ile15Thr) of the luteinizing hormone gene in a Brazilian cohort of healthy subjects and in patients with hypogonadotropic hypogonadism.	16358135	To evaluate the frequency of allelic variant Trp8Arg/Ile15Thr in the luteinizing hormone beta-subunit gene in a Brazilian population of healthy subjects and in patients with hypogonadotropic hypogonadism.Two hundred and two adults (115 women) with normal sexual function and 48 patients (24 women) with hypogonadotropic hypogonadism underwent a molecular study of the the luteinizing hormone beta-subunit gene using a polymerase chain reaction technique followed by enzymatic digestion with the restriction enzymes Nco I (for detection of the Trp8Arg point mutation) and Fok I (for detection of the Ile15Thr point mutation). Basal luteinizing hormone and FSH, testosterone, or estradiol levels were measured in 37 healthy subjects (21 women) and in 27 hypogonadotropic hypogonadism patients (13 women) by immunofluorometric methods (hLH-Spec and hFSH-Spec, AutoDELFIA, Wallac Oy, Turku, Finland).The genetic variant of the luteinizing hormone beta-subunit gene was present at a similar frequency in healthy subjects (14.4%) compared to patients with hypogonadotropic hypogonadism (16.6%). There was no effect of the allelic variant of the luteinizing hormone beta-subunit gene on luteinizing hormone levels in patients with hypogonadotropic hypogonadism as compared to healthy subjects.This study indicates that the allelic variant Trp8Arg/Ile15Thr of the luteinizing hormone beta-subunit gene is a common polymorphism in the Brazilian population (14.4%). The same frequency of this luteinizing hormone variant in the groups with hypogonadotropic hypogonadism and in the healthy subjects excludes a relationship between this variant and hypogonadotropic hypogonadism.
2618	Hyperlipemia and oxidation of LDL induce vascular smooth muscle cell growth: an effect mediated by the HLH factor Id3.	16340216	Hyperlipemia and oxidized LDL (ox-LDL) are important independent cardiovascular risk factors. Ox-LDL has been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. However, the effects of hyperlipemia and the molecular mechanisms mediating hyperlipemia and ox-LDL effects on VSMC growth are poorly understood. The helix-loop-helix (HLH) transcription factor, Id3, is a redox-sensitive gene expressed in VSMC in response to mitogen stimulation and vascular injury. Accordingly, we hypothesize that Id3 is an important mediator of ox-LDL and hyperlipemia-induced VSMC growth. Aortas harvested from hyperlipemic pigs demonstrated significantly more Id3 than normolipemic controls. Primary VSMC were stimulated with ox-LDL, native LDL, sera from hyperlipemic pigs, or normolipemic pigs. VSMC exposed to hyperlipemic sera demonstrated increased Id3 expression, VSMC growth and S-phase entry and decreased p21cip1 expression and transcription. Cells stimulated with ox-LDL demonstrated similar findings of increased growth and Id3 expression and decreased p21cip1 expression. Moreover, the effects of ox-LDL on growth were abolished in cells devoid of the Id3 gene. Results provide evidence that the HLH factor Id3 mediates the mitogenic effect of hyperlipemic sera and ox-LDL in VSMC via inhibition of p21cip1 expression, subsequently increasing DNA synthesis and proliferation.
2619	NH/pi interaction in a spin-crossover complex [FeII(HLMe)2](BPh4)2.2CH3CN (BPh4-=Tetraphenylborate; HLMe=2-Methylimidazol-4-yl-methylideneamino-2-ethylpyridine).	16323911	Three FeII complexes, [Fe(HLR)2](BPh4)2.solvent (R=H, Me, Ph), were synthesized, where BPh4-=tetraphenylborate and HLR=2-substituted-imidazol-4-yl-methylideneamino-2-ethylpyridine. The magnetic susceptibility measurements in 5-300 K revealed that [Fe(HLH)2](BPh4)2.H2O, [Fe(HLMe)2](BPh4)2.2CH3CN, and [Fe(HLPh)2](BPh4)2.CH3CN are low-spin (LS), spin-crossover (SC), and high-spin (HS) FeII complexes, respectively, indicating that the spin state can be effectively tuned by the bulkiness of the substituent. Complex shows a steep SC around 250 K, where it assumes a cyclic structure of {[Fe(HLMe)2]BPh4}2 constructed by four NH/pi bonds between the imidazole group and the phenyl ring of BPh4- in the HS state and a deformed structure with NH/pi bonds and linear CH3CN...HN hydrogen bonds at the terminals in the LS state.
2620	Id helix-loop-helix proteins negatively regulate TRANCE-mediated osteoclast differentiation.	16322470	Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE) induces osteoclast formation from monocyte/macrophage lineage cells via various transcription factors, including the Mi transcription factor (Mitf). Here, we show that inhibitors of differentiation/DNA binding (Ids), helix-loop-helix (HLH) transcription factors, negatively regulate TRANCE-induced osteoclast differentiation. Expression levels of Id1, Id2, and Id3 genes are significantly reduced by TRANCE during osteoclastogenesis. Interestingly, overexpression of the 3 Id genes in bone marrow-derived monocyte/macrophage lineage cells (BMMs) inhibits the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts, but it does not alter the ability of BMMs to either phagocytose or differentiate into dendritic cells (DCs). Overexpression of Id2 in BMMs attenuates the gene induction of nuclear factor of activated T cells c1 (NFATc1) and osteoclast-associated receptor (OSCAR) during TRANCE-mediated osteoclastogenesis. Furthermore, Id proteins interact with Mitf, a basic HLH (bHLH) transcription factor, and inhibit its transactivation of OSCAR, which is a costimulatory receptor expressed by osteoclast precursors, by attenuating the DNA binding ability of Mitf to the E-box site of the OSCAR promoter. Taken together, our results reveal both a new facet of negative regulation, mediated by Id proteins, as well as the mechanism whereby TRANCE signaling overcomes it, allowing osteoclastogenesis to proceed.
2621	Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2.	16311606	Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by progressive cyst formation and ultimate loss of renal function. Increased cell proliferation is a key feature of the disease. Here, we show that the ADPKD protein polycystin-2 (PC2) regulates the cell cycle through direct interaction with Id2, a member of the helix-loop-helix (HLH) protein family that is known to regulate cell proliferation and differentiation. Id2 expression suppresses the induction of a cyclin-dependent kinase inhibitor, p21, by either polycystin-1 (PC1) or PC2. The PC2-Id2 interaction is regulated by PC1-dependent phosphorylation of PC2. Enhanced Id2 nuclear localization is seen in human and mouse cystic kidneys. Inhibition of Id2 expression by RNA interference corrects the hyperproliferative phenotype of PC1 mutant cells. We propose that Id2 has a crucial role in cell-cycle regulation that is mediated by PC1 and PC2.
2622	Guillain-Barré syndrome and hemophagocytic lymphohistiocytosis in a patient with severe chronic active Epstein-Barr virus infection syndrome.	16311154	Epstein-Barr virus (EBV) infection causes a wide range of neurologic and hematologic manifestations. We report a 72-year-old Japanese male patient with severe chronic active EBV infection syndrome (SCAEBV) who presented with Guillain-Barré syndrome (GBS) and developed hemophagocytic lymphohistiocytosis (HLH) several months after the onset of GBS. He showed acute onset of distal muscle weakness, ophthalmoplegia and bulbar palsy. Results of nerve conduction study revealed acute motor-sensory axonal neuropathy (AMSAN). His serum was positive for anti-LM1 IgG and anti-GM1b IgM. Titers of antibodies to EBV-related antigens indicated chronic reactivated EBV infection. Treatment with IVIg resolved the acute ophthalmoplegia, but there was no notable improvement in the AMSAN and bulbar palsy despite repeated. Finally, he developed refractory HLH resulting in a fatal outcome. In the present patient, it seems that SCAEBV was associated with the development of GBS and fatal HLH via parainfectious autoimmunity and direct infectious immune mechanisms, respectively.
2623	Life-threatening hemophagocytic syndromes: current outcomes with hematopoietic stem cell transplantation.	16305623	Life-threatening hemophagocytic syndromes represent a subset of genetic disorders of inflammation. Many are rapidly lethal and can only be definitively treated at the present time with allogeneic hematopoietic stem cell transplantation (HSCT). In this report, current results with allogeneic transplantation for Hemophagocytic Lymphohistiocytosis (HLH) are described. HLH typically presents symptomatically during infancy and early childhood and can be identified by a constellation of numerous physical findings and laboratory tests indicative of overwhelming inflammation. The majority of patients with familial HLH lack natural killer (NK) cell function; in approximately 50% of cases the specific underlying genetic cause can now be discerned. Effective treatment consists of initial combination therapy with proapoptotic chemotherapy (typically etoposide) and anti-inflammatory therapies (principally steroids) in addition to aggressive supportive care, followed by allogeneic HSCT from the best available donor. Over the past 25 yr, through collaborative worldwide efforts, survival of children with HLH and related disorders has improved from 5% at 1 yr after diagnosis to greater than 50% 3-5 yr after diagnosis.
2624	Familial and acquired hemophagocytic lymphohistiocytosis.	16304363	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition characterized by uncontrolled hyperinflammation on the basis of various inherited or acquired immune deficiencies. Cardinal symptoms are prolonged fever, hepatosplenomegaly and cytopenias. Central nervous system (CNS) symptoms are common. Biochemical markers include elevated triglyceride and ferritin, high levels of the alpha chain of the soluble interleukin-2 receptor and low fibrinogen. Impaired function of natural killer (NK) cells and cytotoxic T-cells (CTL) is a characteristic of all forms of HLH. Genetic HLH occurs in familial forms (FHLH), in which HLH is the primary and only manifestation, and in association with the immune deficiencies Chédiak-Higashi syndrome (CHS), Griscelli syndrome (GS) and X-linked lymphoproliferative syndrome (XLP), in which secondary HLH occurs sporadically. Most patients with acquired HLH have no known underlying immune deficiency. Both acquired and genetic forms are triggered by infections, mostly viruses, or other stimuli. HLH also occurs as a complication of rheumatic diseases (macrophage activation syndrome) and of malignancies. The recent discovery of several genetic defects causing FHLH as well as the identification of the genes responsible for CHS, GS and XLP have underscored the role of granule (perforin/granzymes)-mediated cytotoxicity in both the killing of infected cells and the termination of the immune response. The immediate aim of therapy is suppression of the increased inflammatory response by immunosuppressive/immunomodulatory agents and cytotoxic drugs. Genetic cases can only be cured with stem cell transplantation. Awareness of the clinical symptoms and of diagnostic criteria for HLH is crucial to starting life-saving therapy in time.
2625	The Enhancer of split and Achaete-Scute complexes of Drosophilids derived from simple ur-complexes preserved in mosquito and honeybee.	16293187	In Drosophila melanogaster the Enhancer of split-Complex [E(spl)-C] consists of seven highly related genes encoding basic helix-loop-helix (bHLH) repressors and intermingled, four genes that belong to the Bearded (Brd) family. Both gene classes are targets of the Notch signalling pathway. The Achaete-Scute-Complex [AS-C] comprises four genes encoding bHLH activators. The question arose how these complexes evolved with regard to gene number in the evolution of insects concentrating on Diptera and the Hymenoptera Apis mellifera.In Drosophilids both gene complexes are highly conserved, spanning roughly 40 million years of evolution. However, in species more diverged like Anopheles or Apis we find dramatic differences. Here, the E(spl)-C consists of one bHLH (mbeta) and one Brd family member (malpha) in a head to head arrangement. Interestingly in Apis but not in Anopheles, there are two more E(spl) bHLH like genes within 250 kb, which may reflect duplication events in the honeybee that occurred independently of that in Diptera. The AS-C may have arisen from a single sc/l'sc like gene which is well conserved in Apis and Anopheles and a second ase like gene that is highly diverged, however, located within 50 kb.E(spl)-C and AS-C presumably evolved by gene duplication to the nowadays complex composition in Drosophilids in order to govern the accurate expression patterns typical for these highly evolved insects. The ancestral ur-complexes, however, consisted most likely of just two genes: E(spl)-C contains one bHLH member of mbeta type and one Brd family member of malpha type and AS-C contains one sc/l'sc and a highly diverged ase like gene.
2626	Mutation spectrum in children with primary hemophagocytic lymphohistiocytosis: molecular and functional analyses of PRF1, UNC13D, STX11, and RAB27A.	16278825	Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal-recessive disease that affects young children. It presents as a severe hyperinflammatory syndrome with activated macrophages and T lymphocytes. Mutations in the perforin 1 gene (PRF1) were found in FHL-2 in 15-50% of all cases. Defective granule exocytosis caused by mutations in the hMunc13-4 gene (UNC13D) has been described in FHL-3. FHL-4 patients have mutations in STX11, a t-SNARE involved in intracellular trafficking. We analyzed a large group of 63 unrelated patients with FHL of different geographic origins (Turkey:32; Germany:23; others:8) for mutations in STX11, PRF1, and UNC13D. We identified mutations in 38 samples (20 in PRF1, 12 in UNC13D, and six in STX11). Of 32 patients from Turkey, 14 had mutations in PRF1, six had mutations in UNC13D, and six had mutations in STX11. The mutation Trp374X in PRF1 was found in 12 patients from Turkey and was associated with a very early onset of the disease below the age of 3 months in all cases. In contrast, three of 23 and four of 23 patients from Germany, and three of eight and two of eight from other origins showed mutations in PRF1 and UNC13D, respectively, but none in STX11. Thus, FHL-2, FHL-3, and FHL-4 account for 80% of the HLH cases of Turkish origin, and for 30% of German patients. Furthermore, we identified mutations in RAB27A in three patients with FHL-related Griscelli syndrome type 2. In functional studies using a mammalian two-hybrid system we found that missense mutations Ala87Pro in Rab27a and Leu403Pro in hMunc13-4 each prevented the formation of a stable hMunc13-4/Rab27a complex in vitro. Our findings demonstrate extensive genetic and allelic heterogeneity in FHL and delineate an approach for functionally characterizing missense mutations in RAB27A and UNC13D.
2627	Gastric antral vascular ectasia in 2-yr-old girl undergoing unrelated cord blood stem cell transplantation.	16269052	Gastrointestinal bleeding is a common complication after hematopoietic stem cell transplantation (HSCT) and is often related to acute graft-vs.-host disease (aGVHD). Gastric antral vascular ectasia (GAVE), recently recognized as a complication after HSCT, is a rare cause of severe gastrointestinal bleeding, which has only been reported in adult patients so far. We report a 2-yr-old girl who developed GAVE after unrelated cord blood stem cell transplantation (CBSCT) as treatment of intractable Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). Her conditioning regimen for CBSCT consisted of etoposide, busulfan, and cyclophosphamide. She was doing well after CBSCT without recurrence and developed only grade I aGVHD. She suddenly developed coffee ground emesis, tarry stools and severe anemia 76 days after CBSCT. As antacids were ineffective, esophagogastroduodenoscopy was performed and revealed GAVE on day 97. Endoscopic coagulation therapy was performed twice; subsequently, she needed no further transfusions and there was no clinical recurrence of GAVE.
2628	Histiocytosis following T-acute lymphoblastic leukemia: a BFM study.	16263575	The coincidence of T-cell acute lymphoblastic leukemia (T-ALL) and histiocytic disorders, including hemophagocytic lymphohistiocytosis (T-ALL/HLH) and Langerhans cell histiocytosis (T-ALL/LCH), is very seldom and is usually associated with a dismal prognosis. Retrospective statistical analysis of all T-ALL patients, who have been registered in the BFM-ALL trials from 1981 - 2001 and who have subsequently developed a LCH/HLH, in order to identify any common risk factors pre-disposing to the synchronous occurrence of both disorders. Six out of 971 T-ALL patients had either HLH or LCH ( approximately 0.03% of treated T-ALL/year). The mean age at diagnosis of T-ALL/HLH/LCH was significantly lower than in the remaining T-ALL group (4.05 +/- 0.59 vs 8.82 +/- 0.14 years; p = 0.000). The mean initial leukocyte count was higher than in the non-HLH/LCH group (270,700 +/- 60,677 microl(-1) vs 134,141 +/- 5,663 microl(-1); p = 0.074). No hemophagocytosis was seen in the initial bone marrow (BM) smears. Five of 6 patients obtained a good prednisone response (GPR) at day 8 in peripheral blood with <5% blasts at day 15 in BM and all cases were in complete remission (CR) at day 33. The mean time until development of the histiocytosis was 17.95 months (range 2.5 - 33 months). Four patients developed a HLH and 2 a LCH. All patients with HLH showed a multi-organ involvement, while the LCH patients had only local disease. Only the LCH patients survived, while all patients with HLH died. The authors recommend a close follow-up for at least 3 years after diagnosis in younger T-ALL patients with high initial leukocyte count.
2629	The basic domain of ATH5 mediates neuron-specific promoter activity during retina development.	16260616	In the developing retina, the gene encoding the beta3 subunit of the neuronal nicotinic receptor, a specific marker of retinal ganglion cells, is under the direct control of the atonal homolog 5 (ATH5) basic helix-loop-helix (bHLH) transcription factor. Although quite short (143 bp in length), the beta3 promoter has the remarkable capacity to discriminate between ATH5 and the other neuronal bHLH proteins expressed in the developing nervous system. We have identified three amino acids within the basic domain that confer specificity to the ATH5 protein. These residues do not mediate direct DNA binding but are required for interaction between ATH5 and chromatin-associated proteins during retina development. When misexpressed in neurons, the myogenic bHLH factor MyoD is also able to activate the beta3 gene. This, however, is achieved not by binding of the protein to the promoter but by dimerization of MyoD with a partner, a process that depends not on the basic domain but on the HLH domain. By sequestering an E-box-binding protein, MyoD relieves the active repression that blocks the beta3 promoter in most neurons. The mechanisms used by bHLH proteins to activate beta3 thus highlight how ATH5 is selected by the beta3 promoter and coordinates the derepression and transcriptional activation of the beta3 gene during the specification of retinal ganglion cells.
2630	Zebrafish id2 developmental expression pattern contains evolutionary conserved and species-specific characteristics.	16252281	The inhibitor of differentiation or inhibitor of DNA binding (Id) family are members of the helix-loop-helix (HLH) group of transcription factors that play important roles in cell proliferation, differentiation, cell cycle control, and apoptosis. They modulate the formation of active class A-class B basic HLH (bHLH) complexes. Ids lack the amino-terminal associated basic region necessary for DNA binding, thus sequestering the class A factors, inhibiting the formation of active class A-class B heterodimers and, therefore, are considered to act as dominant-negative regulators of differentiation pathways. We isolated zebrafish id2, and its expression during development was characterized. id2, in addition to regions of expression detected in Xenopus and mice, is also expressed in the tegmentum; midbrain-hindbrain boundary; cerebellum; rhombomeres 2,3,4,6; notochord; and corpuscles of Stannius. Furthermore, we show that expression of id2 is repressed in mind bomb mutants, suggesting a role of Notch upstream of Id2.
2631	Cobalamin C disease presenting with hemophagocytic lymphohistiocytosis.	16251179	Cobalamin C disease is a rare genetic condition resulting in methylmalonic aciduria, homocystinuria, and hematologic abnormalities. Clinical characteristics include ophthalmologic findings and neurological abnormalities, such as microcephaly, seizure, and mental retardation. The authors report on a 4-month-old patient initially diagnosed with hemophagocytic lymphohistiocytosis (HLH), who was later diagnosed with cobalamin C disease.
2632	Helix-loop-helix proteins and lymphocyte development.	16239924	Helix-loop-helix (HLH) proteins are transcriptional regulators that control a wide variety of developmental pathways in both invertebrate and vertebrate organisms. Results obtained in the past decade have shown that HLH proteins also contribute to the development of lymphoid lineages. A subset of HLH proteins, the 'E proteins', seems to be particularly important for proper lymphoid development. Members of the E protein family include E12, E47, E2-2 and HEB. The E proteins contribute to B lineage- and T lineage-specific gene expression programs, regulate lymphocyte survival and cellular proliferation, activate the rearrangement of antigen receptor genes and control progression through critical developmental checkpoints. This review discusses HLH proteins in lymphocyte development and homeostasis.
2633	Stem cell transplantation with reduced-intensity conditioning for hemophagocytic lymphohistiocytosis.	16219800	Allogeneic stem cell transplantation (SCT) is curative for hemophagocytic lymphohistiocytosis (HLH). However, patients frequently have significant morbidity before transplantation and there is high transplant-related mortality (TRM). Because first-degree HLH is caused by immune dysregulation, a reduced-intensity conditioned (RIC) regimen might be sufficient for cure while decreasing the TRM. Twelve patients with HLH underwent RIC SCT from a matched family/unrelated or haploidentical donor. Eleven were conditioned with fludarabine/melphalan with additional busulphan for haploidentical grafts. One received fludarabine and 2-Gy total body irradiation (TBI). All patients showed engraftment at a median of 14 days. Nine of 12 (75%) are alive and in complete remission (CR) a median of 30 months (range, 9-73 months) after SCT. Two patients died from pneumonitis and one from hepatic rupture. Four patients developed acute graft-versus-host disease (GVHD) and 3 have chronic GVHD. Three of 9 survivors have mixed chimerism but remain free of disease. In summary, RIC compares favorably to conventional SCT with long-term disease control in surviving patients despite a significant incidence of mixed chimerism.
2634	Familial haemophagocytic lymphohistiocytosis: survival of a premature twin with immuno-chemotherapy and bone marrow transplantation from an HLA-identical unrelated donor.	1618882519764921	We describe a premature twin born at 30 wk of gestational age, affected with familial haemophagocytic lymphohistiocytosis. Two different mutations were identified in his DNA: one inherited from the mother and one from the father. Haemophagocytosis had been confirmed in his twin brother, who died soon after birth, as well as in the re-evaluation of the autopsy of his older sister, who died 1 y earlier. At 26 d of age, chemotherapy and immune-suppressive treatment were started according to the HLH-94 protocol. At 6 mo of age, a bone marrow transplant from an HLA-identical, unrelated volunteer was performed. Now at 32 mo of age, the infant is healthy and without signs of graft-versus-host disease.This case report shows that immuno-chemotherapy and allogenic bone marrow transplant are feasible even in premature infants affected with familial haemophagocytic lymphohistiocytosis, which should be ruled out in unknown bleeding disorders of neonates.
2635	Vitamin D-dependent rickets type 2 with characteristic radiographic changes in a 4-month-old kitten.	16182185	This report describes the presenting signs, biochemical abnormalities, and radiographic changes in a 4-month-old kitten with vitamin D-dependent rickets type 2. Details of therapy are described and possible reasons for treatment failure are discussed.
2636	The use of recombinant human LH (lutropin alfa) in the late stimulation phase of assisted reproduction cycles: a double-blind, randomized, prospective study.	16172149	The effect of recombinant human LH (r-hLH; lutropin alfa) in women undergoing controlled ovarian stimulation with recombinant human FSH (r-hFSH) prior to IVF was investigated.After down-regulation with the GnRH agonist, buserelin, 114 normo-ovulatory women (aged 18-37 years) received r-hFSH alone until the lead follicle reached a diameter of 14 mm. Patients were then randomized in a double-blind fashion to receive r-hFSH in addition to r-hLH, 75 IU s.c., or placebo daily for a maximum of 10 days prior to oocyte retrieval and IVF. The primary end-point was the number of metaphase II oocytes.There were no significant differences between treatment groups for the primary end-point. Serum estradiol concentrations on the day of HCG administration were significantly higher in the group receiving r-hLH plus r-hFSH than in the group receiving r-hFSH alone (P = 0.0001), but there were no significant differences between the groups in dose and duration of r-hFSH treatment required, oocyte maturation, fertilization rate, pregnancy rate and live birth rate.In this patient population, the addition of r-hLH during the late follicular phase of a long GnRH agonist and r-hFSH stimulation cycle provides no further benefit in terms of oocyte maturation or other end-points.
2637	Toward the elucidation of the structural determinants responsible for the molecular recognition between Mad1 and Max.	16171401	Mad1 is a member of the Mad family. This family is part of the larger Myc/Max/Mad b-HLH-LZ eukaryotic transcription-factor network. Mad1 forms a specific heterodimer with Max and acts as a transcriptional repressor when bound to an E-box sequence (CACGTG) found in the promoter of c-Myc target genes. Mad1 cannot form a complex with DNA by itself under physiological conditions. A global model for the molecular recognition has emerged in which the Mad1 b-HLH-LZ homodimer is destabilized and the Mad/Max b-HLH-LZ heterodimer is favored. The detailed structural determinants responsible for the molecular recognition remain largely unknown. In this study, we focus on the elucidation of the structural determinants responsible for the destabilization of the Mad1 b-HLH-LZ homodimer. Conserved acidic residues at the dimerization interface (position a) of the LZ of all Max-interacting proteins have been hypothesized to be involved in the destabilization of the homodimeric states. In Mad1, this position corresponds to residue Asp 112. As reported for the complete gene product of Mad1, we show that wild-type b-HLH-LZ does not homodimerize or bind DNA under physiological conditions. On the other hand, the single mutation of Asp 112 to an Asn enables the b-HLH-LZ to dimerize and bind DNA. Our results suggest that Asp 112 is implicated in the destabilization of Mad1 b-HLH-LZ homodimer. Interestingly, this side chain is observed to form a salt bridge at the interface of the LZ domain in the crystal structure of Mad1/Max heterodimeric b-HLH-LZ bound to DNA [Nair, S. K., and Burley, S. K. (2003) Cell 112, 193-205]. This clearly suggests that Asp 112 plays a crucial role in the molecular recognition between Max and Mad1.
2638	Structural and thermodynamical characterization of the complete p21 gene product of Max.	16171389	The b-HLH-LZ family of transcription factors contains numerous proteins including the Myc and Mad families of proteins. Max heterodimerizes with other members to bind the E-Box DNA sequence in target gene promoters. Max is the only protein in this network that recognizes and binds E-Box DNA sequences as a homodimer in vitro and represses transcription of Myc target genes in vivo. Key information such as the structure of p21 Max, the complete gene product, and its KD in the absence of DNA are still unknown. Here, we report the characterization of the secondary and quaternary structures, the dimerization and DNA binding of p21 Max and a thermodynamically stable mutant. The helical content of p21 Max indicates that its N-terminal and C-terminal regions are unstructured in the absence of DNA. NMR experiments further support the location of folded and unfolded domains. We also show that p21 Max has an apparent KD (37 degrees C) of 7 x 10(-6), a value 10-100 times smaller than the b-HLH-LZ itself. We demonstrate that electrostatic repulsions are responsible for the higher KD of the b-HLH-LZ. Finally, we show that a p21 Max double mutant forms a very stable dimer with a KD (37 degrees C) of 3 x 10(-10) and that the protein/DNA complex depicts a higher temperature of denaturation than p21 Max/DNA complex. Our results indicate that Max could homodimerize, bind DNA, and repress transcription in vivo and that its mutant could be more efficient at repressing the expression of c-Myc target genes.
2639	Viral mutations enhance the Max binding properties of the vMyc b-HLH-LZ domain.	16166655	Interaction with Max via the helix-loop-helix/leucine zipper (HLH-LZ) domain is essential for Myc to function as a transcription factor. Myc is commonly upregulated in tumours, however, its activity can also be potentiated by virally derived mutations. vMyc, derived from the virus, MC29 gag-Myc, differs from its cellular counterpart by five amino acids. The N-terminal mutation stabilizes the protein, however, the significance of the other mutations is not known. We now show that vMyc can sustain longer deletions in the LZ domain than cMyc before complete loss in transforming activity, implicating the viral mutations in contributing to Myc:Max complex formation. We confirmed this both in vitro and in vivo, with loss of Max binding correlating with a loss in the biological activity of Myc. A specific viral mutation, isoleucine383>leucine (I383>L) in helix 2 of the HLH domain, extends the LZ domain from four to five heptad repeats. Significantly, introduction of I383>L into a Myc mutant that is defective for Max binding substantially restored its ability to complex with Max in vitro and in vivo. We therefore propose that this virally derived mutation is functional by significantly contributing to establishing a more hydrophobic interface between the LZs of Myc and Max.
2640	Combinatorial profiles of oligodendrocyte-selective classes of transcriptional regulators differentially modulate myelin basic protein gene expression.	16148239	Recent studies suggest that specific neural basic helix-loop-helix (HLH; i.e., Olig1 and Olig2, Mash1), associated inhibitory HLH (i.e., Id2 and Id4), high-mobility group domain (i.e., Sox10), and homeodomain (i.e., Nkx2.2) transcription factors are involved in oligodendrocyte (OL) lineage specification and progressive stages of maturation including myelination. However, the developmental interplay among these lineage-selective determinants, in a cell- and maturational stage-specific context, has not yet been defined. We show here in vivo and in vitro developmental expression profiles for these distinct classes of transcriptional regulators of OLs. We show that progressive stages of OL lineage maturation are characterized by dynamic changes in the subcellular distribution of these transcription factors and by different permutations of combinatorial transcriptional codes. Transient transfections of these precise combinatorial codes with a luciferase reporter gene driven by the myelin basic protein promoter define how changes in the molecular composition of these transcriptional complexes modulate myelin gene expression. Our overall findings suggest that the dynamic interplay between developmental stage-specific classes of transcriptional activators and associated inhibitory factors orchestrate myelin gene expression during terminal maturation of the mammalian CNS.
2641	The effect of polymorphisms in the enhancer of split gene complex on bristle number variation in a large wild-caught cohort of Drosophila melanogaster.	16143618	The Enhancer of split complex [E(spl)-C] in Drosophila encompasses a variety of functional elements controlling bristle patterning and on the basis of prior work is a strong candidate for harboring alleles having subtle effects on bristle number variation. Here we extend earlier studies identifying associations between complex phenotypes and polymorphisms segregating among inbred laboratory lines of Drosophila and test the influence of E(spl)-C on bristle number variation in a natural cohort. We describe results from an association mapping study using 203 polymorphisms spread throughout the E(spl)-C genotyped in 2000 wild-caught Drosophila melanogaster. Despite power to detect associations accounting for as little as 2% of segregating variation for bristle number, and saturating the region with single-nucleotide polymorphisms (SNPs), we identified no single SNP marker showing a significant (additive over loci) effect after correcting for multiple tests. Using a newly developed test we conservatively identify six regions of the E(spl)-C in which the insertion of transposable elements as a class contributes to variation in bristle number, apparently in a sex- or trait-limited fashion. Finally, we carry out all possible 20,503 two-way tests for epistasis and identify a slight excess of marginally significant interactions, although none survive multiple-testing correction. It may not be straightforward to extend the results of laboratory-based association studies to natural populations.
2642	Assembly of b/HLH/z proteins c-Myc, Max, and Mad1 with cognate DNA: importance of protein-protein and protein-DNA interactions.	16128587	Among the best characterized of the transcription factors are the b/HLH/z proteins: USF, Max, Myc, and Mad. These proteins bind to the DNA E-box, a six base pair sequence, CACGTG. Max and Myc form a heterodimer that has strong oncogenic potential but can also repress transcription, while Mad and Max form a heterodimer that acts as a transcription repressor. We have used fluorescence anisotropy to measure protein-protein and protein-DNA affinity. The specific binding between MLP DNA and Max (K = 2.2 +/- 0.5 nM) is about 10-fold higher affinity than LCR DNA and about 100-fold higher than for a nonspecific DNA. USF has a similar binding affinity as Max to MLP DNA (K = 15 +/- 10 nM), but Max binds more tightly to LCR and nonspecific DNA. A series of oligonucleotides designated E-box, half-E-box, and non-E-box were constructed to examine the effects of DNA sequence. The binding results indicate that for Max protein most of the binding energy can be attributed to individual elements with little cooperativity among the two halves of the E-box. Further studies measured the equilibria for the entire thermodynamic cycle of monomer-dimer-DNA interactions. Surprisingly, the affinity of the Max monomer-DNA for the second monomer was greatly reduced (K for the first monomer in the nanomolar range and for the second monomer in the micromolar range). Looked at from the perspective of the Max protein, the binding of DNA to Max significantly reduces the affinity of the Max protein for the second monomer, whether the second monomer is Myc, Mad, or Max. These data suggest the importance of protein-protein interactions in assembly of a transcription initiation complex.
2643	Fate redirection of hippocampal astrocytes toward neuronal lineage by aggregate culture.	16112216	Mammalian cells that have been committed to a certain cell lineage cannot be directed to other lineages. However, some astrocytes in the mammalian brains have been reported to represent plasticity to redirect to other cell lineages. We found that mouse hippocampal astrocytes cultured in aggregate forms of "astrosphere", redirected to MAP2-positive immature neurons. In astrospheres, basic HLH factors positively regulating neuronal differentiation were up-regulated and Id3 inhibiting basic HLH factors was down-regulated. Ectopic Id3 induction repressed redirection of astrocytes to a neuronal lineage, suggesting that astrosphere formation induced plasticity of astrocytes by changing the gene expression patterns.
2644	The HMX homeodomain protein MLS-2 regulates cleavage orientation, cell proliferation and cell fate specification in the C. elegans postembryonic mesoderm.	16107479	The proper formation of a complex multicellular organism requires the precise coordination of many cellular events, including cell proliferation, cell fate specification and differentiation. The C. elegans postembryonic mesodermal lineage, the M lineage, allows us to study mechanisms coordinating these events at single cell resolution. We have identified an HMX homeodomain protein MLS-2 in a screen for factors required for M lineage patterning. The MLS-2 protein is present in nuclei of undifferentiated cells in the early M lineage and in a subset of head neurons. In the M lineage, MLS-2 activity appears to be tightly regulated at the fourth round of cell division, coincident with the transition from proliferation to differentiation. A predicted null allele of mls-2, cc615, causes reduced cell proliferation in the M lineage, whereas a semi-dominant, gain-of-function allele, tm252, results in increased cell proliferation. Loss or overexpression of mls-2 also affects cleavage orientation and cell fate specification in the M lineage. We show that the increased cell proliferation in mls-2(tm252) mutants requires CYE-1, a G1 cell cycle regulator. Furthermore, the C. elegans Myod homolog HLH-1 acts downstream of mls-2 to specify M-derived coelomocyte cell fates. Thus MLS-2 functions in a cell type-specific manner to regulate both cell proliferation and cell fate specification.
2645	Hemophagocytic lymphohistiocytosis (HLH).	160529611546227516052953	NaN
2646	Successful bone marrow transplantation for life threatening xanthogranuloma disseminatum in neurofibromatosis type-1.	16048609	A 2-yr and 9-month-old female patient with neurofibromatosis type-1 presented with hepatomegaly, anemia, thrombocytopenia, and croupy cough and diagnosed with xanthogranuloma disseminatum (XD). She failed chemotherapy consisting of steroids, 6-mercaptopurine and methotrexate. A partial response to HLH-94 therapy that included etoposide and cyclosporine A was initially observed. However, she continued to have significant organ dysfunction without further improvement at 6 months of therapy. She then received matched unrelated donor bone marrow transplantation (BMT) following carmustine, etoposide, cytarabine and melphelan conditioning with complete resolution of symptoms. BMT is an option in therapy-resistant, life threatening XD cases.
2647	Prolonged severe pancytopenia preceding the cutaneous lesions of juvenile xanthogranuloma.	16047346	We report a case of juvenile xanthogranuloma (JXG) having progressive pancytopenia for 6 months until the proliferating skin lesions. A 2-month-old infant presented recurrent fever, anemia, and hepatosplenomegaly mimicking hemophagocytic lymphohistiocytosis (HLH) or juvenile myelomonocytic leukemia (JMML). At 8 months of age, the biopsy of a growing papule on the elbow made the diagnosis. Bone marrow (BM) specimens showed clustering foamy cells including hemophagocytosis by histiocytes. Treatment with etoposide followed by vinblastine plus prednisolone (PSL) therapy improved the disease. Although JXG is a benign non-Langerhans cell histiocytosis, the multisystem-visceral form should be considered as a potential aggressive disease when associated with BM failure in early infancy.
2648	Predicted consequences of site-directed mutagenesis and the impact of species variation on prion protein misfolding through the N-terminal domain.	16034619	Variant Creutzfeldt-Jacob disease (vCJD) is considered to afflict humans through the acquisition of variant isomers and misfolding of the normal cellular prion polypeptide, PrP(C). Although the exact mechanism of the misfolding is not been yet clearly understood, this paper provides four additional pieces of evidence in support of the hypothesis that misfolding within PrP(C) involves N-terminal residues, up to and including Asn178. Structural predictions for N-terminal residues between Leu4 and Gly124 revealed that Leu4-Leu19 might adopt a helical conformation. Furthermore, measurement of C(alpha) distance variations, as determined from available NMR solution structures of wild type, as well as the biologically significant Val166, Asn170 and Lys220 variants of PrP(C), revealed previously unreported global and local conformational differences may occur in PrP(C) as a result of these amino-acid substitutions. Notably, three regions, His140-Tyr150 and Met166-Phe175 showed deviations greater than 3 A in their C(alpha)-coordinates (cf wild type) indicating that the majority of the N-terminal domain is likely to contribute to the misfolding of PrP(C). Minor variations in the orientation of amino acids Thr193-Glu200, located towards the C terminus of the protein, were also noted. This most likely indicates the presence of a hinge mechanism, inherent to a Helix-Loop-helix (HLH) motif formed by amino acids within alpha2, LIII and alpha3, in order to accommodate reorientation of the motif in response to misalignment of the N-terminal domain. An unexpected 3 angstroms deviation from the coordinates of the wild type polypeptide, absent from either Val166, Asn170 variants was observed over the region Arg154-Tyr155 within the Val166 form of PrP(C). This may contribute to the explanation as to why patients carrying the Val166 isoform of PrP(C) may be more susceptible to vCJD.
2649	Id1 gene expression and regulation in human thyroid tissue.	16029118	The Id genes encode helix-loop-helix (HLH) transcription proteins that lack a DNA binding basic domain. Id proteins (Id1, Id2, Id3, and Id4) function as dominant negative regulators of basic HLH transcription factors by forming inactive heterodimers. Recent studies suggest that the Id proteins participate in the regulation of cell growth, differentiation, tumorigenesis and angiogenesis in a variety of cell types. This report summarizes studies done in our laboratory on the expression and regulation of the Id1 gene in human thyroid tissue, the result of activation of the major mitogen signal transduction pathways on Id1 gene expression, and the effect of the Id1 gene on thyroid cancer cell growth and differentiation.
2650	Molecular characterization of HLH-17, a C. elegans bHLH protein required for normal larval development.	16014321	The basic helix-loop-helix (bHLH) transcription factor family regulates numerous developmental events in eukaryotic cells. In the model system, C. elegans, thirty-seven bHLH proteins have been identified via genome-wide sequence analysis and fourteen have been genetically characterized to date. These proteins influence cell fate specification of neural lineages and differentiation of myogenic lineages and have distinct roles in somatic gonadogenesis. We report here on the molecular characterization of HLH-17, a protein whose putative bHLH domain is homologous to the mammalian bHLH proteins BETA3 and bHLHB5. The gene hlh-17 is transcriptionally active at all developmental stages, with the highest steady state accumulation of hlh-17 mRNA during embryogenesis. An upstream hlh-17 sequence drives expression of GFP in the sheath cells of the cephalic sensilla. Finally, animals that are defective in HLH-17 via RNAi display egg-laying defects, while those carrying null mutations in hlh-17 do not develop beyond the L2 stage and are less attracted to potassium and sodium ions. We propose that hlh-17 affects the ability of C. elegans to respond to food cues, with possible downstream effects on insulin-signaling genes involved in the normal development and reproductive viability of the worm.
2651	Epstein-Barr virus LMP1 inhibits the expression of SAP gene and upregulates Th1 cytokines in the pathogenesis of hemophagocytic syndrome.	16002423	The primary infection of Epstein-Barr virus (EBV) may result in fatal infectious mononucleosis or hemophagocytic syndrome (HPS) in 2 diseases; that is, X-linked lymphoproliferative disorder (XLP) and hemophagocytic lymphohistiocytosis (HLH). XLP is linked to mutations of the SAP/SH2D1A gene with dysregulated T-cell activation in response to EBV infection. Patients with sporadic HLH, however, usually have no mutation of the SAP/SH2D1A gene, and EBV latent membrane protein-1 (LMP1) can up-regulate Th1 cytokines in EBV-infected T cells. Since both diseases share common manifestations of HPS, it is important to clarify whether a cross-talk exists between signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) and LMP1-mediated pathways to explain the common pathogenesis of HPS. In this study, no mutation of the SAP/SH2D1A gene at exon 2/3 was detected in 7 HLH cases. Interestingly, EBV LMP1 could transcriptionally inhibit the expression of SAP/SH2D1A and activate downstream molecules ERK and interferon-gamma (IFN-gamma). LMP1-mediated SAP/ERK/IFN-gamma signals appear to act via the TNF receptor-associated factor (TRAF)2,5/nuclear factor kappaB (NF-kappaB) pathway, since dominant-negative TRAF2/5 and NF-kappaB inhibitor could rescue SAP expression and downregulate IFN-gamma. Although HLH is genetically distinct from XLP, our data suggest that both diseases share a common signal pathway, through either the mutation or LMP1-mediated suppression of the SAP gene, leading to overt T-cell activation and enhanced Th1 cytokine secretion in response to EBV infection.
2652	Overexpressed Id-1 is associated with a high risk of hepatocellular carcinoma development in patients with cirrhosis without transcriptional repression of p16.	15999366	Inhibitor of differentiation/DNA binding protein 1 (Id-1) plays a pivotal role in the regulation of cell proliferation and carcinogenesis via inhibiting basic helix-loop-helix (HLH) transcription factors. Recently, Id-1 was found to repress p16 in tumorous tissue specimens including hepatocellular carcinoma (HCC), but its relevance in precancerous liver tissues is unknown.Id-1 expression in the liver tissue specimens of 112 patients with cirrhosis without HCC was studied by immunohistochemical analysis. Correlations were investigated between Id-1 expression and clinicopathologic features, the status of p16, and the risk of HCC occurrence.A high expression of Id-1 was observed in 42 patients (38%). The level of Id-1 expression was not associated with clinical standard parameters or the status of p16 in cirrhotic tissue specimens. The cumulative incidence of HCC development was significantly higher in a group of patients with high Id-1 expression (P = 0.0008). Multivariate analysis revealed that increased Id-1 expression is an independent significant factor for the risk of HCC development in patients with cirrhosis (relative risk = 2.75, P = 0.003).The results of the current study suggested that increased expression of Id-1 may play an important role in the early step of hepatocarcinogenesis, and might serve as a useful marker for determining patients with cirrhosis with a high risk of HCC occurrence.
2653	A basic helix-loop-helix transcription factor regulates cell elongation and seed germination.	15995349	Plants are sessile and rely on a wide variety of growth hormones to adjust growth and development in response to internal and external stimuli. We have identified a gene, designated NAN, encoding a basic helix-loop-helix (bHLH) transcription factor that regulates cell elongation and seed germination in plants. NAN has an HLH motif in its C-terminal region but does not have any other discernible homologies to bHLH proteins. A bipartite nuclear localization signal is located close to the HLH motif. An Arabidopsis mutant, nan-1D, in which NAN is activated by the insertion of the 35S enhancer, exhibits growth retardation with short hypocotyls and curled leaves. It is also characterized by reduced seed germination and apical hook formation, symptomatic of GA deficiency or disrupted GA signaling. The phenotypic effects of nan-1D were increased by treatment with paclobutrazol (PAC), an inhibitor of gibberellic acid (GA) biosynthesis. NAN is constitutively expressed throughout the life cycle. Our observations indicate that NAN has a housekeeping role in plant growth and development, particularly in seed germination and cell elongation, and that it may modulate GA signaling.
2654	Tissue-specific and ubiquitous basic helix-loop-helix transcription factors in human placental trophoblasts.	15993702	Tissue-specific class B basic helix-loop-helix (bHLH) transcription factors, dimerising with ubiquitously produced class A bHLH proteins, play a major role in murine trophoblast development. Here, we investigated expression patterns of class A and B bHLH factors in the human placenta and different trophoblast culture systems. Semi-quantitative RT-PCR and RNase protection assay revealed expression of the tissue-restricted factors Hash-2, I-mfa and Stra13 in placentae of early and late pregnancy, in purified villous trophoblasts as well as in invasive trophoblasts isolated from first trimester villous explant cultures. Accordingly, RNA in situ hybridisation localised Hash-2, I-mfa and Stra13 to the trophoblast epithelium, cell columns and extravillous trophoblasts invading maternal decidua. Villous stromal cells in situ and cultivated placental fibroblasts also produced I-mfa and Stra13 but failed to express Hash-2. The widely expressed class A proteins, E12/E47 were absent from all placental cell types while ITF-2 was restricted to placental stromal cells of early and late gestation. In contrast, HEB was identified in all trophoblast cell types using RT-PCR, Western blotting and immunohistochemistry. The negative HLH-regulators Id-1 and Id-2 lacking the DNA-binding domain, were detected in villous stromal cells and different cytotrophoblast subtypes but were absent from the syncytium. The data suggest that a complex interplay of activators (Hash-2, HEB) and repressors (Stra13, I-mfa) could be involved in extravillous trophoblast differentiation whereas downregulation of Id proteins could play a role in syncytialisation.
2655	Identification of a heterodimer-specific epitope present in human chorionic gonadotrophin (hCG) using a monoclonal antibody that can distinguish between hCG and human LH.	15956355	Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implantation and maintenance of the pregnancy. Active or passive immunoneutralization of hCG results in termination of pregnancy and this forms the basis of the hCG-based female contraceptive vaccine. However, the beta subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the beta subunit of pituitary human LH (hLH), which is required for ovulation and maintenance of the corpus luteum function during the menstrual cycle. Immunization against hCG or its beta subunit leads to generation of antibodies that can neutralize hLH due to many shared epitopes and hence may cause abnormal menstrual cycles. Therefore, it is essential to identify epitopes that are different in the two hormones. In the present study, we report a monoclonal antibody (MAb) specific for hCG that shows no binding to the isolated subunits. Interestingly, the MAb also does not bind hLH at all. The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCG beta that is absent in hLH beta. The MAb, either as IgG or its recombinant single-chain variable region fragment, inhibited the response to hCG, but not to hLH. Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception.
2656	Characterization of a novel class II bHLH transcription factor from the black widow spider, Latrodectus hesperus, with silk-gland restricted patterns of expression.	15941389	Members of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including lymphopoiesis, myogenesis, neurogenesis, and sex determination. Screening a cDNA library prepared from silk-producing glands of the black widow spider, we have identified a new bHLH transcription factor named SGSF. Within the bHLH region, SGSF showed considerable conservation with other HLH proteins, including Drosophila melanogaster achaete and scute, as well as three HLH proteins identified by gene prediction programs. The expression pattern of SGSF was restricted to a subset of silk-producing glands, which include the tubuliform and major ampullate glands. SGSF was capable of binding an E-box element as a heterodimer with the E protein, E47, but was unable to bind this motif as a homodimer. SGSF was demonstrated to be a nuclear transcription factor capable of attenuating the transactivation of E47 homodimers in mammalian cells. SGSF represents the first example of a silk gland-restricted bHLH protein, and its expression pattern suggests that SGSF plays a role in regulating differentiation of cells in the spider that control silk gland formation or egg case silk gene expression.
2657	[Enigmatic diseases in children. Still a lot of questions left in spite of great research progress on LCH and HLH].	15929429	NaN
2658	Subtyping of natural killer cell cytotoxicity deficiencies in haemophagocytic lymphohistocytosis provides therapeutic guidance.	15916689	The familial form of haemophagocytic lymphohistiocytosis (HLH) is a fatal disease, with allogeneic stem cell transplantation (SCT) being the only curative treatment. In contrast, patients with secondary (infection-associated) HLH usually do not require SCT. Since it often is difficult to distinguish primary and secondary HLH, we wanted to identify a tool that provides guidance on whether SCT is required. The clinical outcome of 65 HLH patients was analysed in relation to the recently reported four types of defects in natural killer (NK)-cell cytotoxicity in HLH. None (0%) of the 36 patients with NK-cell deficiency type 3 attained a sustained (1-year) remission after stopping therapy without receiving SCT, in contrast to 45% (13/29) non-type 3 patients (P < 0.001). Most type 3 patients (22/36) underwent SCT (14/22, 64% are alive), whereas 11 of 14 that did not receive SCT died, and the three others had received HLH-therapy during the last year of follow-up. Of 54 patients analysed for perforin expression and/or mutation, the five with perforin deficiency were all type 3 patients. The data suggests that HLH patients with NK-cell deficiency type 3 will probably require SCT to survive. Thus, NK-cell deficiency classification may provide valuable guidance in judging whether an HLH-patient needs SCT.
2659	Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis.	15916685	Haemophagocytic lymphohistiocytosis (HLH) poses major therapeutic challenges, and the primary inherited form, familial haemophagocytic lymphohistiocytosis (FHL), is usually fatal. We evaluated, including Cox regression analysis, survival in 86 children (29 familial) that received HLH-94-therapy (etoposide, dexamethasone, ciclosporin) followed by allogeneic stem cell transplantation (SCT) between 1995 and 2000. The overall estimated 3-year-survival post-SCT was 64% [confidence interval (CI) = +/-10%] (n = 86); 71 +/- 18% in those patients with a matched related donor (MRD, n = 24), 70 +/- 16% with a matched unrelated donor (MUD, n = 33), 50 +/- 24% with a family haploidentical donor (haploidentical, n = 16), and 54 +/- 27% with a mismatched unrelated donor (MMUD, n = 13). After adjustment for potential confounding factors, estimated odds ratios (OR) for mortality were 1.93 (CI =0.61-6.19) for MUD, 3.31 (1.02-10.76) for haploidentical, and 3.01 (0.91-9.97) for MMUD, compared with MRD. In children with active disease after 2-months of therapy (n = 43) the OR was 2.75 (1.26-5.99), compared with inactive disease (n = 43). In children with active disease at SCT (n = 37), the OR was 1.80 (0.80-4.06) compared with inactive disease (n = 49), after adjustment for disease activity at 2-months. Mortality was predominantly transplant-related. Most HLH patients survived SCT using MRD or MUD, and survival with partially mismatched donors was also acceptable. Patients that responded well to initial pretransplant-induction therapy fared best, but some persisting HLH activity should not automatically preclude performing SCT.
2660	Persistent cervical lymphadenopathy in an adolescent with Epstein-Barr induced hemophagocytic syndrome: manifestations of a rare but often fatal disease.	15911025	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a non-malignant proliferative disorder characterized by histiocytic proliferation and hemophagocytosis following Epstein-Barr virus infection. Though quite rare, this condition represents an often fatal disease primarily affecting the pediatric population. We discuss the case of an adolescent female who presented initially with persistent cervical lymphadenopathy and the typical findings of tonsillar hypertrophy, pharyngitis, and splenomegaly associated with infectious mononucleosis. This case study outlines the pathogenesis, common clinical findings, diagnostic criteria, and a review of the HLH-94 treatment protocol. Early recognition and treatment is emphasized because of the fulminant course of the disorder.
2661	Synthetic lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes identifies conserved genetic interactions.	15892873	Phenotypic robustness is evidenced when single-gene mutations do not result in an obvious phenotype. It has been suggested that such phenotypic stability results from 'buffering' activities of homologous genes as well as non-homologous genes acting in parallel pathways. One approach to characterizing mechanisms of phenotypic robustness is to identify genetic interactions, specifically, double mutants where buffering is compromised. To identify interactions among genes implicated in posterior patterning of the Caenorhabditis elegans embryo, we measured synthetic lethality following RNA interference of 22 genes in 15 mutant strains. A pair of homologous T-box transcription factors (tbx-8 and tbx-9) is found to interact in both C. elegans and C. briggsae, indicating that their compensatory function is conserved. Furthermore, a muscle module is defined by transitive interactions between the MyoD homolog hlh-1, another basic helix-loop-helix transcription factor, hnd-1, and the MADS-box transcription factor unc-120. Genetic interactions within a homologous set of genes involved in vertebrate myogenesis indicate broad conservation of the muscle module and suggest that other genetic modules identified in C. elegans will be conserved.
2662	Human homologue of maid is a useful marker protein in hepatocarcinogenesis.	15887118	Human homologue of maid (HHM) is a helix-loop-helix (HLH) transcriptional regulatory protein that is involved in the hepatic stem cell development and differentiation. We analyzed the potential involvement of HHM in hepatocarcinogenesis.We analyzed HHM expression in the choline-deficient L-amino acid defined (CDAA) diet model of rat hepatocarcinogenesis and in human adenomatous hyperplasia (AH) and hepatocellular carcinoma (HCC) biopsy samples. We assessed the effects of HHM on cell proliferation. We screened proteins that bind to HHM protein using a yeast 2-hybrid screen.High HHM expression was seen in foci and HCC induced in the rat CDAA diet model. HHM protein was expressed in 23 of 32 AH samples (72%), 19 of 28 well-differentiated HCC samples (68%), and 9 of 18 poorly-moderately differentiated HCC samples (50%). Over-expressed HHM enhanced the S phase. HHM interference RNA significantly inhibited cell proliferation. A yeast 2-hybrid screen identified Jun activation domain-binding protein 1 (Jab1) as a binding partner for HHM. We confirmed HHM and Jab1 binding by immunoprecipitation and immunofluorescent histochemistry. The expression of Jab1 was found in human AH and HCC samples. We found an association between levels of expression of HHM and those of Jab1 in AH and HCC tissues examined (P = .027 by chi2 test).High-level HHM expression was found from the very early stages of hepatocarcinogenesis, suggesting that HHM may be a useful marker protein to detect.
2663	Synthesis and conformational properties of protein fragments based on the Id family of DNA-binding and cell-differentiation inhibitors.	15880794	Id proteins are dominant negative regulators of the helix-loop-helix (HLH) transcription factors and are important during development, especially by preventing cell differentiation while inducing cell proliferation. In contrast, they are poorly expressed in healthy adults but are found in several tumor types. The Id HLH motif is responsible for the inhibitory activity, whereas not much is known about the role of the N- and C-termini. In the presented work, synthetic peptides reproducing the HLH, the N-terminal region, and the C-terminal region of the Id proteins were characterized by CD. The four HLH sequences built highly stable helical conformations, whereas the N- and C-termini were unstructured, with the exception of an alanine-rich fragment preceding the Id4 HLH motif. Deletion of the loop connecting the two helices led to helix destabilization for all four Id HLH peptides. In addition, modifications of the amino acid composition within the hydrophobic face of the helices of the Id1 HLH peptide induced conformational changes, mostly associated with loss of helix content. Moreover, a fragment containing the helix-2 and the C-terminus of the Id1 protein did not show any helical character. Therefore, both the helix propensity and stability of the HLH domain were shown to be strongly dependent on favorable interhelical contacts. In contrast, it is suggested that the regions beyond this domain could rather play a destabilizing role, for example, by increasing the flexibility of the folded protein.
2664	Development of hemophagocytic lymphohistiocytosis in triplets infected with HHV-8.	15840696	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder of immune dysregulation, characterized by end-organ damage from lymphocytic infiltration and macrophage activation. All known mutations associated with the HLH occur in genes critical in the perforin-granzyme pathway. Herein, we report HLH occurring in 2 female triplet infants who also had associated human herpesvirus type 8 (HHV-8) infections. The subjects had identical novel compound-heterozygous mutations in the Perforin alleles, resulting in undetectable perforin expression and NK-cell cytotoxicity. Both infants also had evidence of infection with HHV-8. These reports are, to our knowledge, the first cases of HLH in triplets and the first reported cases of HHV-8 infection associated with HLH in non-renal transplant and non-HIV-infected subjects.
2665	Successful treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with HLH-94 protocol.	15831988	Hemophagocytic lymphohistiocytosis (HLH) is a rare, fatal disorder of children, affecting predominantly the mononuclear phagocytic system. Previous reports indicate that Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (EBV-HLH) can also be fatal in many cases, although the prognosis for EBV-HLH is better than for the familial form of hemophagocytic lymphohistiocytosis. We treated four patients with EBV-HLH using immunochemotherapy including steroid, etoposide (VP-16), and cyclosporin, according to the HLH-94 protocol. All patients experienced persistent fever, cytopenia, and hypertriglyceridemia. Serological testing for EBV showed reactivated EBV infections in all patients. EBV DNA detected by PCR and EBV-encoded small RNA measured by in situ hybridization were confirmed in the patients' bone marrow specimens. Hemophagocytosis was shown in bone marrow aspirates and liver biopsy specimen. Complete remission was achieved in all patients after induction and continuation therapy for 4-10 months (median, 7 months) and was maintained for 15-27 months (median, 19 months) without the need for bone marrow transplantation. These results suggest that EBV-HLH can be effectively controlled by immunochemotherapy using the HLH-94 protocol.
2666	The effect of nutrition during pregnancy on the in vitro production of embryos from resulting lambs.	15823357	It is possible to produce offspring from FSH-treated lambs using in vitro maturation and fertilisation procedures but a major constraint is the high embryo wastage after transfer. It is postulated that this wastage is associated, at least in part, with the quality of the harvested oocytes. The aim of this study was thus to determine if nutrition during pregnancy influenced the quality of oocytes collected from resulting lambs. The study was a 2x2x2 factorial that examined the effect of a low (L; 0.7x maintenance) or high (H; 1.5x maintenance) diet provided during three periods (-82 to 70, 71-100 and 101-126 days relative to the date of conception). There were eight treatments namely LLL, LLH, LHL, LHH, HLL, HLH, HHL and HHH. Oocytes were harvested from 9-week-old lambs, matured and fertilised in vitro and the percentages of oocytes and embryos that developed into blastocysts were recorded. There were significant differences between treatments in oocyte and embryo yields and these resulted from complex interactions between diet and the stage of pregnancy. The efficiency of producing blastocysts from oocytes was highest when a H diet was provided between 71 and 110 and/or 101-126 days of pregnancy. These results demonstrate the need to manage nutrition during pregnancy in programs aimed at producing offspring from juvenile animals.
2667	Haemophagocytic lymphohistiocytosis in Malaysian children.	15790325	To study the clinical presentation, therapy and outcome of children diagnosed with both primary and secondary haemophagocytic lymphohistiocytosis (HLH) at the University of Malaya Medical Centre.All patients diagnosed with HLH between 1998 and 2004 were studied. Clinico-pathological data of these patients were prospectively collected and analysed.Thirteen consecutive patients (eight boys) with a median age of 28 months were seen. All patients presented with high-grade unremitting fever and almost all, with hepatosplenomegaly and cytopenias. Neurological manifestations, which ranged from irritability to seizures and coma, were seen in 10 (77%) patients. Other common presenting features include liver dysfunction (46%) and skin rash (38%). All patients were treated using the HLH-94 protocol chemotherapy which consisted of a combination of etoposide, dexamethasone and cyclosporine. Complete response was seen in seven patients while two required bone marrow transplantation and one developed secondary acute myeloid leukaemia. Two patients died before treatment could be commenced. Overall mortality rate in our series was 46%.Haemophagocytic lymphohistiocytosis is an uncommon disease with a high fatality rate. Due to its protean clinical manifestations, it may be underdiagnosed. Early detection and prompt institution of appropriate therapy is necessary to improve the outcome in affected patients.
2668	Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells.	15772131	Neural crest cells, a population of proliferative, migratory, tissue-invasive stem cells, are a defining feature of vertebrate embryos. These cells arise at the neural plate border during a time in development when precursors of the central nervous system and the epidermis are responding to the extracellular signals that will ultimately dictate their fates. Neural crest progenitors, by contrast, must be maintained in a multipotent state until after neural tube closure. Although the molecular mechanisms governing this process have yet to be fully elucidated, recent work has suggested that Myc functions to prevent premature cell fate decisions in neural crest forming regions of the early ectoderm. Here, we show that the small HLH protein Id3 is a Myc target that plays an essential role in the formation and maintenance of neural crest stem cells. A morpholino-mediated 'knockdown' of Id3 protein results in embryos that lack neural crest. Moreover, forced expression of Id3 maintains the expression of markers of the neural crest progenitor state beyond the time when they would normally be downregulated and blocks the differentiation of neural crest derivatives. These results shed new light on the mechanisms governing the formation and maintenance of a developmentally and clinically important cell population.
2669	The myogenic potency of HLH-1 reveals wide-spread developmental plasticity in early C. elegans embryos.	15772130	In vertebrates, striated muscle development depends on both the expression of members of the myogenic regulatory factor family (MRFs) and on extrinsic cellular cues, including Wnt signaling. The 81 embryonically born body wall muscle cells in C. elegans are comparable to the striated muscle of vertebrates. These muscle cells all express the gene hlh-1, encoding HLH-1 (CeMyoD) which is the only MRF-related factor in the nematode. However, genetic studies have shown that body wall muscle development occurs in the absence of HLH-1 activity, making the role of this factor in nematode myogenesis unclear. By ectopically expressing hlh-1 in early blastomeres of the C. elegans embryo, we show that CeMyoD is a bona fide MRF that can convert almost all cells to a muscle-like fate, regardless of their lineage of origin. The window during which ectopic HLH-1 can function is surprisingly broad, spanning the first 3 hours of development when cell lineages are normally established and non-muscle cell fate markers begin to be expressed. We have begun to explore the maternal factors controlling zygotic hlh-1 expression. We find that the Caudal-related homeobox factor PAL-1 can activate hlh-1 in blastomeres that either lack POP-1/TCF or that have down-regulated POP-1/TCF in response to Wnt/MAP kinase signaling. The potent myogenic activity of HLH-1 highlights the remarkable developmental plasticity of early C. elegans blastomeres and reveals the evolutionary conservation of MyoD function.
2670	A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis.	15755897	Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms.
2671	A single amino acid change, A91V, leads to conformational changes that can impair processing to the active form of perforin.	15741215	Mutations in the perforin gene have been found in patients with hemophagocytic lymphohistiocytosis (HLH), a rare autosomal recessive disease. We describe a patient expressing perforin with amino acid changes A91V and W374X. The ability of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to lyse target cells is greatly reduced. Here we demonstrate that perforin from this patient is not recognized using an antibody raised against native perforin (deltaG9), but is readily detected using an antibody raised against a peptide epitope (2d4), suggesting that the epitope recognized by deltaG9 is destroyed by the change at A91V. Immunoblotting reveals no protein corresponding to the truncated transcript encoded by W374X, revealing that only perforin with the A91V change is expressed in CTLs from the patient. Patient CTLs show bands corresponding to the immature and intermediate forms of perforin, but the mature active form of perforin is absent or barely detectable. The conformational changes and impaired cleavage of A91V perforin are likely to explain the reduced cytotoxicity in CTLs and NK cells from this patient and are likely to contribute to the pathogenesis of HLH.
2672	Occurrence of haemophagocytic lymphohistiocytosis at less than 1 year of age: analysis of 96 patients.	15731905	We analysed data of 96 infants (under 1 year of age) with haemophagocytic lymphohistiocytosis (HLH) from the registry of an HLH study conducted during 1986-2002 in Japan. The cases were classified into five groups. The diagnosis of familial HLH (FHL) as group 1 (n = 27) was made with positive family history and/or recent molecular test for perforin and Munc13-4 mutations. Neonatal enterovirus- or herpes simplex virus-associated HLH as group 2a (n = 7), Epstein-Barr virus-associated HLH (n = 12) as group 2b, adenovirus- or cytomegalovirus-associated HLH as group 3 (n = 9) were mostly diagnosed by viral isolation or by the detection of viral genome. Juvenile rheumatoid arthritis-associated macrophage activation syndrome was classified as group 4 (n = 4) and the remaining without known triggers as group 5 (n = 37). The peak onset age was 1-2 months for group 1, 1-2 weeks for group 2a, 12 months for group 2b, none for group 3, 9 months for group 4 and 2 months for group 5. Future novel diagnostic measures are required to define the precise nature of HLH in group 5.These data may provide useful information for neonatologists/ paediatricians in the differential diagnosis of haemophagocytic lymphohistiocytosis in early infancy.
2673	A proportion of patients with lymphoma may harbor mutations of the perforin gene.	15728124	Perforin mutations have been demonstrated in a proportion of patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH). In the present study, we evaluated whether some patients with lymphoma sharing clinical characteristics with HLH might harbor mutations of the perforin gene. We analyzed 29 patients and found that 4 patients, who developed either Hodgkin or non-Hodgkin lymphoma, had biallelic mutations of the perforin gene. One of these 4 patients, a 19-year-old female with T-cell lymphoma, had a brother carrying the same mutations who developed HLH. In 2 of the 4 patients with biallelic mutations of the perforin gene, we evaluated perforin expression by flow cytometry and natural killer (NK) activity and both were found to be absent. Moreover, we documented the presence of monoallelic mutations of the perforin gene in 4 more patients. One of these 4 latter patients also carried a mutation of the Fas gene. These data indicate that perforin deficiency, either alone or in combination with other mutations of genes involved in lymphocyte survival or functional activity, may be present in patients with lymphoma. These findings suggest that perforin also plays a key role in the mechanisms of immune surveillance that prevent tumor growth and/or development.
2674	Review of hemophagocytic lymphohistiocytosis (HLH) in children with focus on Japanese experiences.	15718147	Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever and hepatosplenomegaly associated with pancytopenia, hypertriglyceridemia and hypofibrinogenemia. Increased levels of cytokines and impaired natural killer activity are biological markers of HLH. HLH can be classified into two distinct forms, including primary HLH, also referred to as familial hemophagocytic lymphohistiocytosis (FHL), and secondary HLH. Although FHL is an autosomal recessive disorder typically occurring in infancy, it is important to clarify that the disease may also occur in older patients. It is now considered that FHL is a disorder of T-cell function; moreover, clonal proliferation of T lymphocytes is observed in a few FHL patients, and cytotoxicity of these T lymphocytes for target cells is usually impaired. In 1999, perforin gene (PRF1) mutation was identified as a cause of 20-30% of FHL (FHL2) cases. In Japan, two specific mutations of PRF1 were also detected. Furthermore, in 2003, MUNC13-4 mutations were identified in some non-FHL2 patients (FHL3). Identification of other genes responsible for remaining cases is a major concern. Hematopoietic stem cell transplantation (HSCT) has been established as the only accepted curative therapy for FHL. Thus, appropriate diagnosis and prompt treatment with HSCT are necessary for FHL patients. Genetic analysis for PRF1 and MUNC13-4 and functional assay of cytotoxic T lymphocytes are recommended to be performed in each patient. In those patients displaying impaired cytotoxic function but lacking genetic defects, samples should be employed for identification of unknown genes. In the near future, an entire pathogenesis should be clarified in order to establish appropriate therapies including immunotherapy, HSCT and gene therapy.
2675	Negative acting HLH proteins Id 1, Id 2, Id 3, and Id 4 are expressed in prostate epithelial cells.	15717313	The four known Id proteins, Id 1, Id 2, Id 3, and Id 4 are largely considered as dominant negative helix-loop-helix (HLH) proteins. They can dimerize with basic helix loop proteins (bHLH) but the dimers fail to bind the consensus E box response element (CANNTG). Alternatively, members of the Id family, for example, Id 2 can also bind to non-bHLH proteins such as retinoblastoma (Rb) and ETS-TCF to modulate their activities. Consistent with their role as promoters of proliferation, subset of Id genes for example, Id 1 and Id 2 are expressed in many cancers including that of the prostate. However, their expression and function in the normal prostate is unknown.The present study was designed to evaluate the expression profile and functional significance of all Id isoforms in normal rat prostate epithelial cells. The data suggests that all four Id isoforms are expressed in normal cells, albeit at different levels.Agents that promote growth, for example, serum increase the levels of Id 1, Id 2, and Id 3. The hormones and mitogens such as testosterone and hepatocyte growth factor (HGF) that promote prostate epithelial cell differentiation stimulate Id 4 and Id 2, respectively.In prostate epithelial cells, Id 1 may be specifically involved in promoting proliferation whereas Id 4 and Id 2 may have defined roles in regulating differentiated functions in response to androgens and local paracrine factors such as HGF.
2676	Inheritance analysis of congenital left ventricular outflow tract obstruction malformations: Segregation, multiplex relative risk, and heritability.	15690347	The left ventricular outflow tract (LVOTO) malformations, aortic valve stenosis (AVS), coarctation of the aorta (COA), and hypoplastic left heart (HLH) constitute a mechanistically defined subgroup of congenital heart defects that have substantial evidence for a genetic component. Evidence from echocardiography studies has shown that bicuspid aortic valve (BAV) is found frequently in relatives of children with LVOTO defects. However, formal inheritance analysis has not been performed. We ascertained 124 families by an index case with AVS, COA, or HLH. A total of 413 relatives were enrolled in the study, of which 351 had detailed echocardiography exams for structural heart defects and measurements of a variety of aortic arch, left ventricle, and valve structures. LVOTO malformations were noted in 30 relatives (18 BAV, 5 HLH, 3 COA, and 3 AVS), along with significant congenital heart defects (CHD) in 2 others (32/413; 7.7%). Relative risk for first-degree relatives in this group was 36.9, with a heritability of 0.71-0.90. Formal segregation analysis suggests that one or more minor loci with rare dominant alleles may be operative in a subset of families. Multiplex relative risk analysis, which estimates number of loci, had the highest maximum likelihood score in a model with 2 loci (range of 1-6 in the lod-1 support interval). Heritability of several aortic arch measurements and aortic valve was significant. These data support a complex but most likely oligogenic pattern of inheritance. A combination of linkage and association study designs is likely to enable LVOTO risk gene identification. This data can also provide families with important information for screening asymptomatic relatives for potentially harmful cardiac defects.
2677	The differential binding affinities of the luteinizing hormone (LH)/choriogonadotropin receptor for LH and choriogonadotropin are dictated by different extracellular domain residues.	15677709	The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the beta-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the beta-sheets of LRR1-9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the beta-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.
2678	SAP controls the cytolytic activity of CD8+ T cells against EBV-infected cells.	15677558	The adaptor protein SAP regulates signaling through signaling lymphocytic activation molecule (SLAM)-family receptors expressed on T and natural killer (NK) cells. In patients affected by X-linked lymphoproliferative (XLP) disease, mutations in the SH2D1A gene result in defective lytic activity. However, the mechanism by which SAP controls cytotoxic activity remains unclear. T-cell-receptor (TCR) activation of CD8(+) cytotoxic T cells (CTLs) results in down-regulation of SAP, suggesting that this protein is involved in early activation events. Here, we show that SAP-deficient CTLs from patients with XLP and hemophagocytic lymphohistiocytosis (HLH) display a specific lytic defect against autologous and allogeneic Epstein-Barr virus (EBV)-positive B cells. This defect is associated with the defective polarization of 2B4, perforin, and lipid rafts at the contact area of CTLs with EBV-positive targets. Blockade of 2B4 in normal CTLs reproduces the defects in lysis and polarization observed in SAP-deficient CTLs. Expression and regulation of the SLAM-family receptors SLAM, CD84, and 2B4, as well as the lytic effectors perforin and granzyme-B are normal in SAP-deficient CTLs. In addition, TCR stimulation leads to normal proliferation and production of interleukin 2 (IL-2), IL-4, and interferon-gamma (IFN-gamma). These results demonstrate that the SAP/2B4 pathway plays a key role in CTL lytic activity against EBV-positive targets by promoting the polarization of the lytic machinery.
2679	[Onset and maintenance of the luteal phase with GnRH agonists or hCG in cycles under GnRH antagonists].	15643682	Corpus luteum function seems to be impaired in IVF cycles with COH (Controlled Ovarian Hyperstimulation) and GnRH antagonist. Support of corpus luteum function remains mandatory after ovarian stimulation for IVF with GnRH antagonist cotreatment. GnRH antagonists do not have any impact on the luteal phase of IVF/ICSI cycles when hormonal support is given. A new treatment option for patients undergoing ovarian stimulation is the gonadotrophin-releasing hormone (GnRH) antagonist protocol, with the possibility to trigger a mid-cycle LH surge using a single bolus of GnRH agonist, reducing the risk of developing ovarian hyperstimulation syndrome (OHSS) in high responders. In IVF cycle with COH and GnRH antagonist, support of corpus luteum function is essential, whatever molecule to trigger LH surge (hCG, r-hLH, GnRH agonist). Adequate luteal support (progesterone and estrogens) compensates for luteolysis and assures good clinical outcome.
2680	Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome.	15642140	Macrophage activation syndrome (MAS) has been reported in association with many rheumatic diseases, most commonly in systemic juvenile rheumatoid arthritis (sJRA). Clinically, MAS is similar to hemophagocytic lymphohistiocytosis (HLH), a genetic disorder with absent or depressed natural killer (NK) function. We have previously reported that, as in HLH, patients with MAS have profoundly decreased NK activity, suggesting that this abnormality might be relevant to the pathogenesis of the syndrome. Here we examined the extent of NK dysfunction across the spectrum of diseases that comprise juvenile rheumatoid arthritis (JRA). Peripheral blood mononuclear cells (PBMC) were collected from patients with pauciarticular (n = 4), polyarticular (n = 16), and systemic (n = 20) forms of JRA. NK cytolytic activity was measured after co-incubation of PBMC with the NK-sensitive K562 cell line. NK cells (CD56+/T cell receptor [TCR]-alphabeta-), NK T cells (CD56+/TCR-alphabeta+), and CD8+ T cells were also assessed for perforin and granzyme B expression by flow cytometry. Overall, NK cytolytic activity was significantly lower in patients with sJRA than in other JRA patients and controls. In a subgroup of patients with predominantly sJRA, NK cell activity was profoundly decreased: in 10 of 20 patients with sJRA and in only 1 of 20 patients with other JRA, levels of NK activity were below two standard deviations of pediatric controls (P = 0.002). Some decrease in perforin expression in NK cells and cytotoxic T lymphocytes was seen in patients within each of the JRA groups with no statistically significant differences. There was a profound decrease in the proportion of circulating CD56bright NK cells in three sJRA patients, a pattern similar to that previously observed in MAS and HLH. In conclusion, a subgroup of patients with JRA who have not yet had an episode of MAS showed decreased NK function and an absence of circulating CD56bright population, similar to the abnormalities observed in patients with MAS and HLH. This phenomenon was particularly common in the systemic form of JRA, a clinical entity strongly associated with MAS.
2681	Rapid detection of intracellular SH2D1A protein in cytotoxic lymphocytes from patients with X-linked lymphoproliferative disease and their family members.	15632210	Mutations in the SH2D1A gene have been described in most patients with the clinical syndrome of X-linked lymphoproliferative disease (XLP). The diagnosis of XLP is still difficult given its clinical heterogeneity and the lack of a readily available rapid diagnostic laboratory test, particularly in patients without a family history of XLP. XLP should always be a consideration in males with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). Four-color flow cytometric analysis was used to establish normal patterns of SH2D1A protein expression in lymphocyte subsets for healthy subjects. Three of 4 patients with XLP, as confirmed by the detection of mutations in the SH2D1A gene, had minimal intracellular SH2D1A protein in all cytotoxic cell types. The remaining patient lacked intracellular SH2D1A protein in CD56+ natural killer (NK) and T lymphocytes and had an abnormal bimodal pattern in CD8+ T cells. Carriers of SH2D1A mutations had decreased SH2D1A protein staining patterns compared with healthy controls. Eleven males with clinical syndromes consistent with XLP, predominantly EBV-HLH, had patterns of SH2D1A protein expression similar to those of healthy controls. Four-color flow cytometry provides diagnostic information that may speed the identification of this fatal disease, differentiating it from other causes of EBV-HLH.
2682	[Comparative properties of myogenesis in invertebrates and in lower and higher vertebrates].	15624775	Development of the muscle system in invertebrates (on the example of nematode Caenorhabditis elegans and Drosophila) and vertebrates (fish, birds, and mammals) demonstrates a number of common patterns at the level of molecular and genetic control mechanisms but also a number of distinctive features. C. elegans muscle is formed from several cells of different origin at the earliest developmental stages, while inductive interactions play a critical role in Drosophila: the mesoderm, which is a source of muscle formation, receives the inductive signals from the neighboring ectoderm as expression of Dpp and Hh genes. In vertebrates, the induction and commitment of cells to the myogenic lineage are promoted by morphogenetic signals from the neighboring notochord (as expression of Shh gene) and neural tube (as expression of Wnt and Shh genes). The inductive signals entering the mesoderm are related to the subsequent activation of genes encoding protein transcription factors of bHLH gene family and analogs. These include hlh-1 and CeTwi in C. elegans; Twi and nau in Drosophila; and MyoD, Myf5, myogenin, and MRF4 in vertebrates. The diversity of myosin isoforms in different animals is provided by either gene duplication or alternative splicing of particular genes.
2683	Isolated central nervous system hemophagocytic lymphohistiocytosis: case report.	1561760216528177	The first case of histologically proven hemophagocytic lymphohistiocytosis (HLH) isolated to the central nervous system (CNS) is reported. HLH affecting the CNS mimics several neurological disorders and may be misdiagnosed. The diagnostic and therapeutic problems of this disease are discussed.We report a case of a 5-year-old girl with a 2-month history of right hemiparesis. The initial magnetic resonance imaging scan mimicked the appearance of malignant glioma or cerebral infarction. By use of neuroimaging alone, it was extremely difficult to reach an appropriate diagnosis.Pathological examination of a surgical specimen of the lesion revealed histological characteristics typical of HLH. Because of the absence of both physical and blood chemical findings of systemic HLH, the patient was diagnosed as having HLH isolated in the CNS without systemic HLH. Radiotherapy with corticosteroid administration led to complete resolution of the CNS lesions, but the duration of remission was only 3 months. The patient died secondary to refractory progression of the CNS lesion.Radiotherapy with corticosteroid administration led to a complete resolution of the lesions, although for only a transitory remission. Although HLH is extremely rare, the existence of this disease isolated in the CNS should be documented, and further case accumulation and therapeutic investigations are needed to clarify the pathophysiological characteristics of this disease.
2684	New tool for an old problem: can RNAi efficiently resolve the issue of genetic redundancy?	15612040	RNA interference (RNAi) has become a generally accepted tool for inhibiting gene expression in many laboratory organisms. Nagel et al.,(1) in a recent paper, give an example of how this tool can also be used to address the question of genetic redundancy. Their focus was on the redundancy in Drosophila melanogaster of the Enhancer of split gene complex [E(spl)-C] which comprises seven highly related genes. Their somewhat conflicting findings are probably the typical scenario for most RNAi experiments: some expected results and some surprises.
2685	Urinary gonadotrophins but not recombinant gonadotrophins reduce expression of VEGF120 and its receptors flt-1 and flk-1 in the mouse uterus during the peri-implantation period.	15591083	Ovarian stimulation in humans might affect the perinatal outcome and be considered as a stress factor in the implantation process. In this study we compared the effects of recombinant and urinary gonadotrophins during the mouse peri-implantation period.Adult female CD1 mice were treated as follows (a) urinary hFSH and urinary hCG, (b) recombinant hFSH and recombinant hLH and (c) saline. The effects of the gonadotrophins on the expression of vascular endothelial growth factor120 (VEG120) and its receptors and the corticotrophin releasing hormone (CRH) system during the peri-implantation period were studied. The specific effects of the different gonadotrophins on the onset of implantation were also studied.Urinary gonadotrophin treatment caused lower levels of VEGF120, flt-1 and flk-1 mRNA levels, reduced the size of the embryo implantation site, delayed implantation and prolonged the gestational period. Both urinary hFSH and urinary hCG contributed to the adverse effects. Levels of CRH and CRHR1 expression were not influenced. Recombinant gonadotrophin treatment did not alter any of the parameters studied.Our results show that the VEGF system of the mouse uterus during the peri-implantation period is adversely affected by urinary gonadotrophins but not by recombinant gonadotrophins. The CRH system was not affected by the two types of gonadotrophins.
2686	Comparative efficacy and safety of cetrorelix with or without mid-cycle recombinant LH and leuprolide acetate for inhibition of premature LH surges in assisted reproduction.	15588464	An open label, randomized, multi-centre study was performed to compare cetrorelix and leuprolide acetate for prevention of premature LH surge and to assess whether patients treated with cetrorelix benefit from addition of recombinant human (r-h)LH. Normo-ovulatory women (n = 74) undergoing ovarian stimulation prior to intracytoplasmic sperm injection were treated with leuprolide acetate (n = 25) before ovarian stimulation with recombinant human FSH (r-hFSH) or with cetrorelix 3 mg on stimulation day 7 (with (n = 25) or without (n = 24) r-hLH 150 IU on days 7-10). The main outcome measures were the number of metaphase II (MII) oocytes retrieved; secondary efficacy end-points; adverse events (AE) and other safety measures. There were no significant differences between groups for MII oocytes retrieved, duration of stimulation, total r-hFSH dose and pregnancy rates. The group treated with cetrorelix alone had a significantly lower concentration of oestradiol per follicle compared with the other groups. The majority of AE were mild to moderate in severity. Cetrorelix and leuprolide acetate appear to have comparable efficacy and safety, although cetrorelix has the advantage of typically requiring only one injection.
2687	Id1 gene expression is up-regulated in hyperplastic and neoplastic thyroid tissue and regulates growth and differentiation in thyroid cancer cells.	1557976616207896	The Id (inhibitor of DNA binding) proteins are a family of helix-loop-helix (HLH) proteins (Id1, Id2, Id3, and Id4) that lack the basic domain necessary for DNA binding. The Id1 protein enhances cell proliferation and inhibits cellular differentiation in a variety of cell types. We have previously demonstrated that the Id1 gene is up-regulated in papillary and medullary thyroid cancers. In this study we characterized the expression and distribution of the Id1 protein in normal, hyperplastic, and neoplastic human thyroid tissue. We also evaluated the effect of the Id1 gene on thyroid cancer cell growth and markers of thyroid cell differentiation. We used semiquantitative immunohistochemistry to characterize Id1 protein expression in normal, hyperplastic (multinodular goiter and Graves' disease), and neoplastic thyroid tissue from 103 patients. Normal thyroid tissue had the lowest level of Id1 protein expression (P < 0.0001). Anaplastic thyroid cancer had the highest level (vs. benign and malignant thyroid tissues, P < 0.01). Id1 protein expression was higher in malignant thyroid tissue than in hyperplastic thyroid tissue (P < 0.02). We found no significant association between the level of Id1 protein expression and patient age, sex, tumor-node-metastasis stage, tumor size, primary tumor vs. lymph node metastasis, primary tumor vs. recurrent tumors, and extent of tumor differentiation. Inhibiting Id1 mRNA expression in thyroid cancer cell lines using Id1 antisense oligonucleotides resulted in growth inhibition (P < 0.03) and decreased thyroglobulin and sodium-iodine symporter mRNA expression (P < 0.02). In conclusion, Id1 is overexpressed in hyperplastic and neoplastic thyroid tissue and directly regulates the growth of thyroid cancer cells of follicular cell origin, but is not a marker of aggressive phenotype in differentiated thyroid cancer.
2688	Hemophagocytic lymphohistiocytosis: a rare cause of pancytopenia.	15540522	A 36-year-old man with fever and pancytopenia due to Hemophagocytic Lymphohistiocytosis is reported. The patient was started on the HLH-94 based treatment. Two weeks after the initiation of therapy the patient's pancytopenia had resolved and he was discharged to complete treatment as an outpatient. The initial clinical presentation, diagnostic criteria, pathophysiology and treatment will be discussed.
2689	A role for Id proteins in mammary gland physiology and tumorigenesis.	15530557	Id helix-loop-helix (HLH) proteins are regulators of cell growth and differentiation in embryonic and adult tissues. They are members of the basic HLH family of transcription factors but lack a DNA binding domain. By binding to basic HLH transcription factors, Id proteins regulate gene expression. Id1 and Id3 have extensive sequence homology and similar patterns of expression during embryogenesis and in adult tissues. They are also expressed at high levels in the endothelial cells of tumor-infiltrating blood vessels, and breast tumors spontaneously arising in MMTV-neu mice demonstrate impaired angiogenesis when growing in an Id1- and/or Id3-deficient background. These lesions are typically cystic with a small rim of viable tumor cells surrounding an acellular necrotic core. Id2 plays a critical role in breast differentiation and lactation. Id4 regulates BRCA1 expression and may be involved in hormone-dependent regulation of BRCA1 homeostasis. Thus, all four members of the Id protein family play pivotal roles in distinct aspects of normal and malignant breast biology, the subject of this review.
2690	Changes in cAMP-dependent protein kinase (PKA) and progesterone secretion in luteinizing human granulosa cells.	15525572	Luteinization of follicular granulosa cells leads to an increase in progesterone secretion that is regulated by luteinizing hormone (LH). LH acts mainly by elevating intracellular cyclic 3',5'-adenosine monophosphate (cAMP) and activating cAMP-dependent protein kinase (PKA). In this study, we have examined the role of PKA in relation to progesterone output by luteinizing human granulosa cells. Human granulosa cells were obtained by percoll gradient centrifugation of follicular aspirates of patients undergoing oocyte retrieval for assisted conception. Cells were cultured in serum-supplemented medium for up to 3 days in the presence and/or absence of human (h)LH and other cAMP-elevating agents. Spent medium was assayed for cAMP and progesterone content by specific RIA. Cell lysates were collected and assessed for PKA regulatory (R)IIalpha/catalytic (C)alpha expression by Western blotting. Although basal progesterone secretion increased progressively throughout culture, cAMP levels remained unchanged. Under basal conditions, PKA RIIalpha/Calpha expression appeared to increase throughout the 3-day culture period. In the presence of hLH and other cAMP-elevating agents, progesterone secretion increased in a dose-dependent manner coincident with an increase in cAMP. However, despite the increase in both progesterone secretion and cAMP accumulation, there was a dose-dependent decrease in both PKA RIIalpha and Calpha expression. Thus, data presented in this study show that increases in progesterone secretion in luteinizing human granulosa cells can be dissociated from increases in PKA expression. This notion implies that progesterone secretion may be regulated by PKA-dependent as well as PKA-independent mechanisms.
2691	Construction and activity of a novel GHRH analog, Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys.	15525469	To construct another growth hormone releasing hormone (GHRH) analog, Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys peptide and to compare its activity with that of Pro-Pro-hGHRH(1-44)OH peptide.The pro-pro-hGHRH(1-44)-gly-gly-cys DNA fragment was synthesized by polymerase chain reaction. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptide was purified to homogeneity by cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25 and Sephadex G-25 column chromatography. The peptide molecular mass was determined by electrospray ionization mass spectroscopy (ESI-MS). Its purity was determined by SDS-PAGE. The concentration of growth hormone (GH) stimulated by GHRH and its analogs was determined with antiserum kit against human GH. The other human hormones as hTSH, hFSH, hLH, and hPRL were determined with a paramagnetic particle chemiluminescent immunoassay kit.The molecular weight of Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys was 5455.4 kDa which was coincident with the theoretical calculations. All the three peptides at 5 mg/L stimulated GH release from the human fetal pituitary but only the difference between Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys group and blank group was significant (P<0.05). Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys 0.01, 0.1, and 1 mg/L and Pro-Pro-hGHRH (1-44)OH 0.1 and 1 mg/L stimulated GH release from rat pituitary in a concentration-dependent manner (P<0.05, P<0.01). At the same concentration Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys stimulated more GH release than Pro-Pro-hGHRH (1-44)OH. Pro-Pro-hGHRH (1-44)OH 0.01 mg/L and hGHRH (1-40)OH 2 mg/L did not stimulate GH release from rat pituitary (P>0.05). Pro-Pro-hGHRH (1-44)OH and Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys peptides at 5 mg/L did not stimulate hTSH, hFSH, hLH, and hPRL release from human fetal pituitary in vitro.Pro-Pro-hGHRH (1-44)-Gly-Gly-Cys had a better activity than that of Pro-Pro-hGHRH(1-44)OH. Variation at C-terminus of GHRH could modulate its GH releasing activity.
2692	Transforming property of TEL-FGFR3 mediated through PI3-K in a T-cell lymphoma that subsequently progressed to AML.	15514005	We previously reported a novel fusion between TEL and FGFR3 in a patient with peripheral T-cell lymphoma with t(4; 12)(p16;p13). Disease in this patient subsequently progressed to acute myelogenous leukemia (AML) with the same translocation. Sequence analysis of TEL-FGFR3 fusion transcripts suggested that these diseases originated from the same multipotent stem cell. To determine the transforming property of TEL-FGFR3, we established transfectants of this chimeric fusion gene and investigated the major signal pathways of TEL-FGFR3-induced transformation using various signal transduction inhibitors including SU5402 (fibroblast growth factor tyrosine kinase [FGFR TK] inhibitor). Our results indicated that (1) the expression of TEL-FGFR3 but not DeltaHLH-TEL-FGFR3 resulted in efficient focus formation in NIH/3T3 cells and conferred interleukin 3 independence to Ba/F3 cells by a constitutive tyrosine kinase activity probably through oligomerization by the HLH domain of TEL; (2) although effector proteins including classical mitogen-activated protein kinase (MAPK), p38 MAPK, phosphatidylinositol 3-kinase (PI3-K), mammalian target or rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT-3) and STAT-5 were activated in TEL-FGFR3 transformants, the growth of the transformants was inhibited by SU5402 (concentration that inhibits 50% [IC5)]=5 microM) and the PI3-K inhibitor, LY294002 (IC5)=10 microM) and wortmannin (IC50=5 microM), but not by U0126, SB203580, or rapamycin; and (3) injection of TEL-FGFR3 transformants induced lethal leukemia into syngeneic mice. Taken together, the leukemogenic potential of TEL-FGFR3 may be mediated in part through PI3-K.
2693	Role of the Sc C terminus in transcriptional activation and E(spl) repressor recruitment.	15507447	Neurogenesis in all animals is triggered by the activity of a group of basic helix-loop-helix transcription factors, the proneural proteins, whose expression endows ectodermal regions with neural potential. The eventual commitment to a neural precursor fate involves the interplay of these proneural transcriptional activators with a number of other transcription factors that fine tune transcriptional responses at target genes. Most prominent among the factors antagonizing proneural protein activity are the HES basic helix-loop-helix proteins. We have previously shown that two HES proteins of Drosophila, E(spl)mgamma and E(spl)m7, interact with the proneural protein Sc and thereby get recruited onto Sc target genes to repress transcription. Using in vivo and in vitro assays we have now discovered an important dual role for the Sc C-terminal domain. On one hand it acts as a transcription activation domain, and on the other it is used to recruit E(spl) proteins. In vivo, the Sc C-terminal domain is required for E(spl) recruitment in an enhancer context-dependent fashion, suggesting that in some enhancers alternative interaction surfaces can be used to recruit E(spl) proteins.
2694	High frequency of QPY allele and linkage disequilibrium of granzyme-B in Epstein-Barr-virus-associated hemophagocytic lymphohistiocytosis.	15496206	Mediation of Epstein-Barr virus (EBV)-specific cytotoxicity in T lymphocyte via the perforin/granzyme pathway has been demonstrated; therefore, a study involving cytolytic molecules was essential for the clarification of hemophagocytic lymphohistiocytosis (HLH) pathogenesis. This investigation, which analysed the frequency of three allelic mutations of granzyme-B (55Q/R, 95P/A and 247Y/H) in patients with EBV-HLH and infectious mononucleosis, identified the high prevalence of the QPY haplotype in EBV-HLH patients in comparison with healthy controls. A > G polymorphism was also detected in intron 5; furthermore, nearly complete linkage disequilibrium was observed among these polymorphisms. The recessive role of the QPY haplotype of granzyme-B might be responsible for the pathogenesis of EBV-HLH. Cytotoxicity and DNA fragmentation of cytotoxic T lymphocytes did not differ among patients characterized by the QPY/QPY, RAH/RAH and QPY/RAH genotypes. This finding suggested that DNA fragmentation in target cells is mediated not only by granzyme-B but also by other molecules, including other granzymes or Fas.
2695	Sickle cell crisis associated with hemophagocytic lymphohistiocytosis.	15495257	Sickle-beta(+) (beta(+)) thalassemia is a double heterozygous genetic disorder characterized by both a qualitative and quantitative abnormality. We present a case of an African American male who was first diagnosed with sickle cell disease (SCD) at the age 23 years when he presented with generalized bone pain, fever, and hepatosplenomegaly. Laboratory findings included thrombocytopenia, microcytic anemia, and markedly elevated ferritin. He was subsequently diagnosed with a sickle-beta thalassemia hemoglobinopathy. Findings in the bone marrow aspirate and biopsy were consistent with hemophagocytic lymphohistiocytosis (HLH). HLH resolved with the resolution of sickle cell bone pain crisis without use of immunosuppressive therapy. To the best of our knowledge this is the first documented case of HLH associated with sickle cell bone pain crisis.
2696	Timed artificial insemination with estradiol cypionate or insemination at estrus in high-producing dairy cows.	15483154	A total of 799 Holstein cows from 3 herds were randomly assigned at 37 +/- 3 d in milk (DIM) to timed artificial insemination (AI) or insemination at detected estrus. Cows were presynchronized with injections of PGF(2alpha) at 37 and 51 DIM. At 65 DIM, cows received an injection of GnRH, followed 7 d later by PGF(2alpha). Cows in the estrus-detected group were inseminated after being observed in estrus during the 7 d after the last PGF(2alpha). Cows in the timed AI group received an injection of 1 mg of estradiol cypionate (ECP) 24 h after the last PGF(2alpha). If detected in estrus <or= 24 h after ECP, cows were inseminated then or at a fixed time 48 h after ECP. Pregnancy was diagnosed by ultrasonography at 30 d and by palpation at 44 and 58 d after AI. Plasma progesterone was measured in 4 samples per cow collected on days of 1) second PGF(2alpha), 2) GnRH, 3) third PGF(2alpha), and 4) 48 h after third PGF(2alpha). Cows were classified as cyclic or anovulatory based on progesterone concentrations in samples 1 and 2. Similarly, cows were classified according to progesterone concentrations in samples 2, 3, and 4 (H = >or=1 ng/mL; L = <1 ng/mL), resulting in 8 combinations (LLL, LHL, LLH, LHH, HHH, HHL, HLH, and HLL). Conception rates and pregnancy rates were higher for cows in the timed AI group than in the estrus-detected group at 30, 44, and 58 d (e.g., at 58 d, pregnancy rates were 42.2% for multiparous cows or 34.4% for primiparous cows in the group receiving ECP and timed AI compared with only 20.8 or 18.8% for respective parity subgroups for the treatment group inseminated only at detected estrus). Pregnancy losses were 11.5% from 30 to 58 d and did not differ between treatments. Cyclic cows within both treatments had higher estrous responses, conception rates, and pregnancy rates. Cows that responded to presynchronization and to luteolysis (HHL) had the highest conception and pregnancy rates, followed by cows classified as LHL. Use of 1 mg of ECP to induce ovulation as part of a synchrony regimen improved reproduction at first postpartum insemination in dairy cows.
2697	Macrophage activation syndrome: characteristic findings on liver biopsy illustrating the key role of activated, IFN-gamma-producing lymphocytes and IL-6- and TNF-alpha-producing macrophages.	15466922	Macrophage activation syndrome (MAS) is a rare and potentially fatal disorder, thought to result from uncontrolled activation and proliferation of T cells and excessive activation of macrophages. The term MAS designates a clinicopathologic entity that occurs in different hemophagocytic syndromes (HSs). Primary hemophagocytic lymphohistiocytosis (HLH) is recognized to have an immunogenetic basis, but in the secondary HS (also referred to as secondary HLH), the cause is unknown. The pathogenesis of the accelerated disease phase typical of MAS remains incompletely understood. This report describes the immunohistochemical findings on liver tissues from 5 children, each of whom presented with MAS in the context of a different type of HS. The data provide direct evidence for the involvement of activated CD8(+) lymphocytes through the production of interferon-gamma and of macrophages through hemophagocytosis and production of interleukin 6 and tumor necrosis factor-alpha, and underscore the view that MAS in different HSs share a common effector pathway.
2698	Hemophagocytic lymphohistiocytosis (HLH).	1546612612728123	NaN
2699	Expression of a recombinant bifunctional protein from a chimera of human lutropin receptor and human chorionic gonadotropin beta-subunit.	15380943	Human lutropin (hLH) and human chorionic gonadotropin (hCG) are structurally and functionally similar and play important roles in reproduction via a common gonadal receptor (LH-R). However, hormone specific hCG-beta subunit contains 24 additional amino acid carboxyl terminal peptide (CTP), which produce specific antibodies to hCG-beta with little cross-reaction with LH. A chimeric protein containing both hLH-R and hCG-beta would provide a unique bifunctional antigen for immunocontraception. In this study is described the synthesis of a chimeric DNA construct of full-length of hLH-receptor and hCG-beta and its expression in Sf9 cells to produce a bifunctional protein. Recombinant protein was recognized by antibodies to LH-R as well as anti-hCG-beta in Western Blots, thus indicating the preservation of immunological epitopes for both hLH-R and hCG-beta in the chimera. Specific ligand binding of recombinant hLH-R component was demonstrated by the displacement of bound labeled hCG at increasing concentrations of unlabeled hCG, indicating that, the presence of hCG-beta component of the chimera did not interfere with the binding of hCG to LH-R. hCG-beta was also present in the recombinant chimeric protein as shown by a specific hCG-beta chemiluminescence assay. Treatment of transfected Sf9 cells with hCG induced dose-dependent increase in the stimulation of intracellular cAMP production, which showed that the ligand binding had functional activity. These results demonstrate that the chimeric DNA construct of hLHR-hCG-beta expressed a bifunctional protein containing both hLH-R and hCG-beta activities, which could provide a unique potential antigen for immunocontraception in vertebrates.
2700	Inhibitory helix-loop-helix transcription factors Id1/Id3 promote bone formation in vivo.	15368360	Bone formation is under the control of a set of transcription factors. Ids are inhibitory helix-loop-helix (HLH) transcription factors and expression of Id genes in osteoblasts is under the control of calciotropic agents such as BMP and vitamin D. However, the function of Ids during bone formation in vivo has not yet been elucidated. We, therefore, examined the role of Id1 and Id3 in the regulation of bone metabolism in vivo. Using wild type and Id1/Id3 heterozygous knockout mice, we analyzed calvarial bone formation in the suture by X-ray picture, proliferation, and mineralization activities of primary calvarial osteoblasts by MTT assay and alizarin red staining and onthotopic in vivo bone formation by BMP injection onto calvaria and micro CT analysis. The width of calvarial sutures was reduced by more than 50% in Id1/Id3 heterozygous knock out mice. Analyses on the cellular basis for the mechanism underlying the defects in the mutant mice revealed suppression of proliferation and mineralization in osteoblasts derived from Id1/Id3 heterozygous knock out mice. Furthermore, Id1/Id3 heterozygous knock out mice suppressed BMP-induced bone formation in vivo. These results indicated that Id1 and Id3 are positive factors to promote bone formation in vivo.
2701	LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element.	15363889	A growing family of helix-loop-helix PAS (HLH-PAS) transcription factors has emerged recently. These proteins form heterodimers and are involved in adaptation to environmental or physiologic stresses as well as in embryonic development. We describe the cloning and characterization of a novel HLH-PAS protein termed LE-PAS (limbic-enriched PAS) in mouse. LE-PAS consists of 802 amino acids and has a predicted molecular mass of 87.4 kDa. In adult mice, it is expressed exclusively in brain. Northern blot and in situ hybridization analysis indicate that LE-PAS is expressed in the limbic system and olfactory bulb. By in vitro overexpression in COS-7, cells we found that LE-PAS is a nuclear protein. Reporter gene analysis indicated that LE-PAS transactivates the CNS midline enhancer (CME) motif but not the xenobiotic response element in an Arnt-dependent fashion and without prior activation of LE-PAS protein. Our observation suggests that LE-PAS shares a similar mode of function with HLH-PAS proteins such as single minded or trachealess indicating that LE-PAS also has constitutive or developmental functions which may be critical for regulating the transcriptional control of limbic patterning and function.
2702	Identifying DNA-binding proteins using structural motifs and the electrostatic potential.	15356290	Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix-turn-helix (HTH), helix-hairpin-helix (HhH) and helix-loop-helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif.
2703	Id family of transcription factors and vascular lesion formation.	15345510	Vascular smooth muscle cell (VSMC) modulation to a de-differentiated phenotype and proliferation are key components of vascular lesion formation. Understanding how these processes are regulated is essential to understanding the progression of vascular diseases such as atherosclerosis and in-stent restenosis. The Id family of helix-loop-helix (HLH) transcription factors has emerged as important regulators of cellular growth and differentiation. Recent published findings have implicated the Id proteins as important regulators of growth and phenotypic modulation in VSMC and in the vascular response to injury. In this review, we summarize what is known regarding how the Id proteins function to control cellular growth and differentiation and their role in vascular lesion formation.
2704	Juvenile myelomonocytic leukemia presenting with features of hemophagocytic lymphohistiocytosis in association with neurofibromatosis and juvenile xanthogranulomas.	15342987	An association exists among neurofibromatosis 1 (NF1), juvenile xanthogranulomas (JXGs), and juvenile myelomonocytic leukemia (JMML). The authors describe a patient with the triple association of JXG, NF1, and JMML initially presenting with features of hemophagocytic lymphohistiocytosis (HLH). An 18-month old boy had multiple cutaneous and gastrointestinal JXG and NF1. At 3 years of age he developed anemia, thrombocytopenia, and hepatosplenomegaly. A bone marrow biopsy revealed features of HLH. Despite chemotherapy, he went on to develop JMML, which proved fatal.
2705	Increased IL-16 levels in hemophagocytic lymphohistiocytosis.	15342983	Serum levels of interleukin-16 (IL-16) were measured to investigate its role in the pathophysiology of hemophagocytic lymphohistiocytosis (HLH). Serum IL-16 levels in patients with acute HLH were significantly higher than those in healthy controls and patients with infectious mononucleosis. They returned to normal levels in the convalescent phase of the disease. In contrast to serum interferon-gamma (IFN-gamma) levels, serum IL-16 levels showed a gradual decrease over the course of the disease. Serum IL-16 levels showed a significant positive correlation with serum levels of soluble IL-2 receptor, IFN-gamma, and interleukin-18, body temperature, and serum lactic dehydrogenase (LDH) levels. An increase in IL-16 mRNA expression was detected in the liver of an HLH patient. These results suggest that IL-16 plays an important role in the pathophysiology of HLH by TH1 cell recruitment and activation at organs with inflammation.
2706	Validation of an ultrasensitive and specific immunofluorometric assay for mouse follicle-stimulating hormone.	15342359	Sensitive and specific measurement of FSH is critical to research in reproductive biology, and the increasing availability of transgenic mouse models has created a need for a robust, sensitive, and specific mouse (m) FSH assay. The present study evaluated a time-resolved immunofluorometric assay (IFMA) for mFSH using monoclonal antibody to human (h) FSHbeta as a capture antibody and a biotinylated polyclonal antibody to rat alpha subunit as a detection probe, with signaling amplified by europium-labeled streptavidin. The mFSH IFMA lowered the detection limit 34-fold (5 vs. 170 pg/sample) compared with standard mFSH RIA. The mFSH IFMA demonstrated parallelism of response to dilutions of castrated mouse serum and rat FSH but no cross-reactivity with hFSH and mLH or hLH, whereas the RIA demonstrated nonparallel cross-reactivity with hFSH. The IFMA has a wide analytical range, with a good precision profile for within- and between-assay reproducibility. Because the IFMA is a sandwich-type assay with strict dimer-specificity by design, the lower readings and recovery obtained were compared with the RIA when both assays used a pituitary-purified mFSH assay standard that contained isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mFSH IFMA demonstrated the expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mFSH in gonadotropin-deficient mice. Furthermore, the IFMA measured mFSH with fidelity in both intact and orchidectomized male mice without any interference from transgenic hFSH. The greatly enhanced sensitivity, specificity, and technical convenience of this mFSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.
2707	The NMR solution structure of a mutant of the Max b/HLH/LZ free of DNA: insights into the specific and reversible DNA binding mechanism of dimeric transcription factors.	15342239	Basic region-helix1-loop-helix2-leucine zipper (b/H(1)LH(2)/LZ) transcription factors bind specific DNA sequence in their target gene promoters as dimers. Max, a b/H(1)LH(2)/LZ transcription factor, is the obligate heterodimeric partner of the related b/H(1)LH(2)/LZ proteins of the Myc and Mad families. These heterodimers specifically bind E-box DNA sequence (CACGTG) to activate (e.g. c-Myc/Max) and repress (e.g. Mad1/Max) transcription. Max can also homodimerize and bind E-box sequences in c-Myc target gene promoters. While the X-ray structure of the Max b/H(1)LH(2)/LZ/DNA complex and that of others have been reported, the precise sequence of events leading to the reversible and specific binding of these important transcription factors is still largely unknown. In order to provide insights into the DNA binding mechanism, we have solved the NMR solution structure of a covalently homodimerized version of a Max b/H(1)LH(2)/LZ protein with two stabilizing mutations in the LZ, and characterized its backbone dynamics from (15)N spin-relaxation measurements in the absence of DNA. Apart from minor differences in the pitch of the LZ, possibly resulting from the mutations in the construct, we observe that the packing of the helices in the H(1)LH(2) domain is almost identical to that of the two crystal structures, indicating that no important conformational change in these helices occurs upon DNA binding. Conversely to the crystal structures of the DNA complexes, the first 14 residues of the basic region are found to be mostly unfolded while the loop is observed to be flexible. This indicates that these domains undergo conformational changes upon DNA binding. On the other hand, we find the last four residues of the basic region form a persistent helical turn contiguous to H(1). In addition, we provide evidence of the existence of internal motions in the backbone of H(1) that are of larger amplitude and longer time-scale (nanoseconds) than the ones in the H(2) and LZ domain. Most interestingly, we note that conformers in the ensemble of calculated structures have highly conserved basic residues (located in the persistent helical turn of the basic region and in the loop) known to be important for specific binding in a conformation that matches that of the DNA-bound state. These partially prefolded conformers can directly fit into the major groove of DNA and as such are proposed to lie on the pathway leading to the reversible and specific DNA binding. In these conformers, the conserved basic side-chains form a cluster that elevates the local electrostatic potential and could provide the necessary driving force for the generation of the internal motions localized in the H(1) and therefore link structural determinants with the DNA binding function. Overall, our results suggests that the Max homodimeric b/H(1)LH(2)/LZ can rapidly and preferentially bind DNA sequence through transient and partially prefolded states and subsequently, adopt the fully helical bound state in a DNA-assisted mechanism or induced-fit.
2708	Tor signaling and nutrient-based signals converge on Mks1p phosphorylation to regulate expression of Rtg1.Rtg3p-dependent target genes.	15326168	The heterodimeric bZip/HLH transcription factors Rtg1p and Rtg3p regulate the expression of a concise set of metabolic genes (termed RTG target genes) required for de novo biosynthesis of glutamate and glutamine. Several components have now been identified that control both the intracellular localization as well as activity of the Rtg1p.Rtg3p complex, yet the precise upstream regulatory signals involved remain unclear. For example, it has been proposed that Rtg1p.Rtg3p activity is repressed by glutamate, acting through the mitochondrial retrograde response pathway or, alternatively, by glutamine, acting through the Tor kinase pathway. Here we demonstrate that RTG target gene regulation is remarkably complex, with glutamate and glutamine as well as ammonia collaborating as potentially distinct signals to regulate RTG target gene expression. We show that both Tor and these nutrient-based signals converge on Mks1p, the immediate upstream inhibitor of Rtg1p.Rtg3p, and that a direct correlation exists between the degree of Mks1p phosphorylation and the extent of RTG target gene repression. Finally, we find that Tor- and glutamine-mediated RTG-target gene repression can be experimentally uncoupled, indicating that glutamine and Tor act, at least in part, independently to inhibit this pathway.
2709	TEL/AML1 shows dominant-negative effects over TEL as well as AML1.	15325275	The TEL/AML1 chimeric gene is generated by the t(12;21) translocation in pre-B cell acute lymphoblastic leukemia. TEL/AML1 consists of the helix-loop-helix (HLH) dimerization domain from TEL and almost the entire of AML1, but loses the ETS DNA-binding domain from TEL. Dominant-negative effects of TEL/AML1 over wild-type-AML1 are believed to trigger the development of this type of leukemia. However, it could also be possible that TEL/AML1 affects wild-type-TEL's molecular and tumor suppressive functions through the HLH domain. To test this possibility, we first confirmed that TEL/AML1 associates with wild-type-TEL. TEL/AML1 neither bound to the ETS-binding consensus site nor repressed transcription through it. Regardless, this prevented wild-type-TEL-induced transcriptional repression. Moreover, TEL/AML1 concomitantly inhibited wild-type-TEL-induced growth suppression and wild-type-AML1-mediated transforming activity in NIH3T3 cells. All these data indicate that TEL/AML1 exerts dominant-interfering effects on both AML1 and TEL, and that expression of TEL/AML1 could result in inactivation of TEL's tumor suppressive functions in t(12;21)-carrying leukemia.
2710	Decoding hematopoietic specificity in the helix-loop-helix domain of the transcription factor SCL/Tal-1.	15314159	The helix-loop-helix (HLH) domain is employed by many transcription factors that control cell fate choice in multiple developmental settings. Previously, we demonstrated that the HLH domain of the class II basic HLH (bHLH) protein SCL/Tal-1 is critical for hematopoietic specification. We have now identified residues in this domain that are essential for restoring hematopoietic development to SCL-/- embryonic stem cells and sufficient to convert a muscle-specific HLH domain to one able to rescue hematopoiesis. Most of these critical residues are distributed in the loop of SCL, with one in helix 2. This is in contrast to the case for MyoD, the prototype of class II bHLH proteins, where the loop seems to serve mainly as a linker between the two helices. Among the identified residues, some promote heterodimerization with the bHLH partners of SCL (E12/E47), while others, unimportant for this property, are still crucial for the biological function of SCL. Importantly, the residue in helix 2 specifically promotes interaction with a known partner of SCL, the LIM-only protein LMO2, a finding that strengthens genetic evidence that these proteins interact. Our data highlight the functional complexity of bHLH proteins, provide mechanistic insight into SCL function, and strongly support the existence of an active SCL/LMO2-containing multiprotein complex in early hematopoietic cells.
2711	Multiple roles for the E/Daughterless ortholog HLH-2 during C. elegans gonadogenesis.	15282161	HLH-2 is the Caenorhabditis elegans ortholog of the Drosophila Daughterless and mammalian E basic helix-loop-helix (bHLH) transcriptional activators that function during diverse events during animal development. HLH-2 has been implicated in cell fate specification in different neural lineages and in the LIN-12/Notch-mediated anchor cell (AC)/ventral uterine precursor cell (VU) decision in the somatic gonad. Here, we show that hlh-2 plays several distinct roles during somatic gonadogenesis. Our analysis suggests that hlh-2 is required to endow specific somatic gonadal cells with the competence to undergo the AC/VU decision, as well as functioning in the AC/VU decision per se; this novel "proAC" role appears to be analogous to the proneural role of Drosophila Daughterless. In addition to its two distinct roles in the specification of the AC, hlh-2 is also required for correct differentiation and function of the AC. hlh-2 also acts at an independent point in the gonadal lineage both to specify distal tip cells (DTCs) and in DTC differentiation and function.
2712	Id-1 expression and cell survival.	15258459	The Id (inhibitor of differentiation or DNA binding) helix-loop-helix (HLH) proteins are a group of dominant negative regulators of basic HLH transcriptional factors which promote cell differentiation. Recent evidence has revealed that Id proteins, especially Id-1, are also able to promote cell proliferation and cell cycle progression through inactivation of tumour suppressor and activation of growth promoting pathways in mammalian cells. In addition, upregulation of Id-1 has been found in many types of human cancer and its expression levels are also associated with advanced tumour stage. Furthermore, ectopic expression of Id-1 in human cancer cells is able to induce cell proliferation under sub-optimal conditions and protect the cells against apoptosis. These lines of evidence strongly indicate Id-1 as a positive regulator of cell growth and its expression may be a key factor required for tumour cell proliferation. This review will discuss recent evidence on the role of Id-1 in cell proliferation and survival, and its significance in malignant transformation. In addition, we will highlight the recent development in the understanding of the molecular mechanisms responsible for the action of Id-1 in promoting cell survival and tumourigenesis. Finally, the therapeutic implications through inactivation of Id-1 in the treatment of human cancer will also be addressed.
2713	Demyelinating peripheral neuropathy associated with hemophagocytic lymphohistiocytosis. An immuno-electron microscopic study.	15243760	We report the case of an 11-year-old male who developed subacute diffuse polyradiculoneuropathy, associated with digestive symptoms and Epstein-Barr virus infection. Parental consanguinity was present. The laboratory findings including bone marrow smear were consistent with hemophagocytic lymphohistiocytosis (HLH). Electrophysiological study of peripheral nerves revealed an intense and diffuse demyelinating process. The histological nerve lesions were severe and purely demyelinating. Most axons were intact. There was a diffuse infiltration of the nerve parenchyma by mononuclear cells. Immuno-electron microscopic study evidenced entry of macrophages into Schwann cell cytoplasm with dissociation of myelin sheaths. This boy died several months after the onset of the neuropathic symptoms. HLH is a rare genetic or acquired disorder in childhood characterized by abnormal immune activation, which induces an uncontrolled inflammatory response with sustained hyperactivation of T lymphocytes and macrophages. Only very rare cases of peripheral nerve involvement have been described in HLH. This is the first case showing that peripheral nerves, as other viscera, may be destroyed by the macrophagic infiltration, which characterizes HLH.
2714	Hemophagocytic lymphohistiocytosis presenting with nonimmune hydrops fetalis.	15224121	Hydrops fetalis is a condition of diverse etiologies. An association between hemophagocytic lymphohistiocytosis (HLH) and hydrops fetalis has not been reported in the English literature. We describe an intrauterine case of HLH in an infant who presented with hydrops fetalis at 32 weeks gestation. We suggest that HLH should be considered in the differential diagnosis of nonimmune hydrops fetalis, especially in the presence of cytopenias.
2715	STRA13 interacts with STAT3 and modulates transcription of STAT3-dependent targets.	15223310	STRA13 is a pVHL-dependent bHLH transcription factor up-regulated on the mRNA level in multiple cancer cell lines and implicated recently in the regulation of immune cell homeostasis and autoimmunity. In searching for STRA13-interacting proteins with oncogenic potential by the yeast two-hybrid screening, we identified STAT3 beta as a STRA13-binding partner. We showed that STRA13 binds predominantly to phosphorylated (active) STAT3 alpha and beta isoforms via its HLH and C-terminal regions. We also found that STRA13 was able to activate transcription from STAT-dependent cis-elements. Expression of endogenous STRA13 was shown to be cytokine-inducible, consistent with STRA13 involvement in STAT-dependent transcription regulation. We demonstrated that the STAT3-regulated promoter of the pro-apoptotic Fas gene was activated upon STRA13 over-expression and that co-expression of STRA13 with STAT3 beta or STAT3 alpha modulated the transcriptional outcome. Forced expression of STRA13 induced apoptosis, in agreement with the STRA13 activation effect on the Fas promoter. Simultaneous expression of STRA13 and STAT3 beta resulted in alleviation of the STRA13 pro-apoptotic effect. Thus, for the first time, we identify STRA13 as a STAT3 partner and provide a consistent line of evidence for STRA13 involvement into regulation of apoptosis via the STAT pathways.
2716	Identification of C. elegans sensory ray genes using whole-genome expression profiling.	15183729	The three cells that comprise each C. elegans sensory ray (two sensory neurons and a structural cell) descend from a single neuroblast precursor cell. The atonal ortholog lin-32 and the E/daughterless ortholog hlh-2 act to confer neural competence during ray development, but additional regulatory factors that control specific aspects of cell fate are largely unknown. Here, we use full-genome DNA microarrays to compare gene expression profiles in adult males of two mutant strains to identify new components of the regulatory network that controls ray development and function. This approach identified a large set of candidate ray genes. Using reporter genes, we confirmed ray expression for 13 of these, including a beta-tubulin, a TWK-family channel, a putative chemoreceptor and four novel genes (the cwp genes) with a potential role in sensory signaling through the C. elegans polycystins lov-1 and pkd-2. Additionally, we have found several ray-expressed transcription factors, including the Zn-finger factor egl-46 and the bHLH gene hlh-10. The expression of many of these genes requires lin-32 function, though this requirement may not reflect direct activation by lin-32. Our strategy provides a complementary foundation for modeling the genetic network that controls the development of a simple sensory organ.
2717	Mechanism of transcriptional repression by TEL/RUNX1 fusion protein.	15179033	Human chromosomal translocation t(12;21)(p12;q22) is one of the most frequent rearrangement in human leukemia, and produces the TEL/RUNX1 fusion protein. The TEL/RUNX1 fusion protein creates a transcriptional repressor that interferes in dominant fashion with RUNX1-dependent transactivation. Here, we demonstrate that the repressor activity of TEL/ RUNX1 differs from that of TEL, even though both TEL and TEL/RUNX1 interact with the nuclear hormone co-repressor (N-CoR) and histone deacetylase (mSin3A) in vivo. Co-immunoprecipitation experiments demonstrated that TEL/RUNX1 forms homodimers in vivo, and heterodimerizes with the TEL when the two proteins are expressed together. These interactions require the HLH (helix-loop-helix) region of TEL. Immunoprecipitation and immunofluorescence analysis showed that p300 interacts with TEL/RUNX1 and is sequestered in the cytoplasm by it. These results suggest that the p300-TEL/RUNX1 complex and heterodimerization of TEL/RUNX1 with TEL may be responsible for the ability of TEL/RUNX1 to inhibit RUNX1-mediated transactivation. It appears that loss of TEL function activates a pathway that cooperates with TEL/RUNX1 and sequesters coactivator(s) into nonfunctional complex in the cytoplasm thus inhibiting transcription of target genes.
2718	Neurogenic phenotypes induced by RNA interference with bHLH genes of the Enhancer of split complex of Drosophila melanogaster.	15170696	The Enhancer of split gene complex [E(spl)-C] of Drosophila melanogaster harbors seven highly related genes encoding transcriptional regulators with a basic helix-loop-helix (bHLH) domain. They are activated by the Notch signaling pathway in order to inhibit proneural gene activity, for example, during neurogenesis in the developing embryo. The E(spl) proteins are at least partly redundant, despite some remarkable differences in their expression patterns. We attempted to address the degree of redundancy by means of RNA interference. We find a quantitative correlation between the degree of a neurogenic phenotype and the number of genes affected. Surprisingly, interference with m3 results in a high rate of mortality which cannot be reproduced by genetic mutation. Most likely, m3 dsRNA interferes with unrelated genes involved in other aspects of embryonic development.
2719	Effects of recombinant LH supplementation in women undergoing assisted reproduction with GnRH agonist down-regulation and stimulation with recombinant FSH: an opening study.	15169576	Circulating endogenous concentrations of LH are reduced in women undergoing down-regulation with gonadotrophin-releasing hormone agonists (GnRHa) and ovarian stimulation with recombinant human FSH (r-hFSH). The effect of recombinant human LH (r-hLH) supplementation on ovarian response and pregnancy outcome was evaluated in a prospective randomized study (sealed envelopes) including 231 cycles. Normogonadotrophic women were stimulated with either r-hFSH or a combination of r-hFSH and r-hLH in a ratio of 2:1. LH supplementation was started from day 8 of the cycle. Blood samples for oestradiol, LH and androstendione were prospectively collected on days 1, 8 and on the day of aspiration and analysed retrospectively. Overall, the two groups did not differ with respect to pregnancy rate. In contrast, women aged > or =35 years responded to exogenous LH supplementation with significantly increased implantation rates and significantly reduced total FSH consumption as compared with the non-supplemented group. In addition, the implantation rate for a subgroup of patients with the highest endogenous LH concentrations (i.e. > or =1.99 IU/l) on day 8 was significantly increased by LH supplementation. Exogenous LH supplementation from day 8 has no detrimental effect on ovarian response and pregnancy outcome. On the contrary supplementation with r-hLH seems to benefit treatment outcome for women above 35 years of age and for the subgroup of women exhibiting LH concentrations above 1.99 IU/l on stimulation day 8.
2720	Near fatal cerebellar swelling in familial hemophagocytic lymphohistiocytosis.	15165642	We describe a 3 year-old male who presented with fever and cerebellar dysfunction after varicella. He developed transient hepatosplenomegaly, cytopenias, and progressive central nervous system involvement (coma, status epilepticus, and hydrocephalus). Despite normal initial cranial computed tomographic scan, diffuse swelling of the cerebellum with downward tonsillar herniation ensued. Diagnosis of hemophagocytic lymphohistiocytosis was difficult and delayed because of the relapsing course, and complete diagnostic criteria were not fulfilled at initial presentation. Central nervous system disease preceded the typical clinical picture of this disease; it dominated the clinical course and caused life-threatening complications and sequelae. The patient improved after treatment, according to the hemophagocytic lymphohistiocytosis protocol (HLH-94) of the Histiocyte Society, with dexamethasone, etoposide, and cyclosporine and unrelated cord blood stem cell transplantation. A mutation in the perforin gene confirmed the diagnosis of familial hemophagocytic lymphohistiocytosis.
2721	Identification of cross-reactive and restricted epitopes localized on human chorionic gonadotropin beta-subunit by monoclonal antibodies.	15165483	Human chorionic gonadotropin (hCG) belongs to the family of glycoprotein hormones. All members of the family are composed of an identical alpha subunit and structurally related beta subunit which confers biological specificity. Specific quantification and functional analysis of hCG require the use of monoclonal antibodies recognizing different epitopes of hCGbeta. This study describes the production and characterization of monoclonal antibodies (MAbs) to hCGbeta with no cross-reactivity to other glycoprotein hormones. Spleen cells from Balb/c mice immunized with hCG were fused with mouse SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of highly purified and recombinant glycoprotein hormones, their subunits and peptides representing the C-terminal end of hCGbeta (hCGbeta-CTP) by ELISA and immunoblotting. The affinity constant (K(aff)) was also determined by ELISA. Three murine hybridomas designated G5M1, B12M2 and F4M3 were obtained that secrete MAbs specific for hCGbeta. The G5M1 MAb reacts only with hCGbeta, hCGbeta-CTP and intact hCG with no detectable cross-reaction with hCGalpha or any of the other glycoprotein hormones. The specificity of B12M2 MAb is very similar to G5M1, but it does not react with hCGbeta-CTP. The F4M3 MAb also has similar specificity to G5M1 and B12M2, but it strongly cross-reacts with hLH. The affinity constant (Kaff) of G5M1, B12M2 and F4M3 was found to be 4.28 x 10(9), 5.2 x 10(8), and 1.97 x 10(9) M(-1), respectively. Our results indicate that G5M1 and B12M2 MAbs are specific for hCG and recognize epitopes restricted to hCGbeta, but F4M3 recognizes a common epitope expressed both on hCGbeta and hLHbeta.
2722	Prednisone reduces muscle degeneration in dystrophin-deficient Caenorhabditis elegans.	1514533715351430	Duchenne muscular dystrophy is a degenerative muscular disease caused by mutations in the dystrophin gene. There is no curative treatment against Duchenne muscular dystrophy. In several countries, the steroid prednisone (or analogs) is prescribed as a palliative treatment. In the model animal Caenorhabditis elegans, mutations of the dys-1 dystrophin-like gene lead to a muscular degenerative phenotype when they are associated with a mild MyoD mutation. This cheap and fast-growing model of dystrophinopathy may be used to screen for molecules able to slow muscle degeneration. In a blind screen of approximately 100 compounds covering a wide spectrum of targets, we found that prednisone is beneficial to the C. elegans dystrophin-deficient muscles. Prednisone reduces by 40% the number of degenerating cells in this animal. This result is a proof-of-principle for the use of C. elegans as a tool in the search for molecules active against the effects of dystrophin-deficiency. Moreover, since C. elegans is not susceptible to inflammation, this suggests that prednisone exerts a direct effect on muscle survival.
2723	JAB1 enhances HAND2 transcriptional activity by regulating HAND2 DNA binding.	15139020	HAND2 (also known as dHAND) is a basic helix-loop-helix (bHLH) transcription factor essential for development of the heart, limbs, and neural crest-derived lineages. HAND2 expression is observed in a number of tissues derived from the neural crest, including components of the peripheral nervous system, where it has been shown to regulate sympathetic nervous system development. Here we show that HAND2 is expressed in both the sympathetic and the parasympathetic divisions of the autonomic nervous system (ANS). How HAND2 functions during development of these neuronal lineages is uncertain. An important mechanism involved in HAND2's function is its interactions with other proteins. To understand better the molecular interactions regulating HAND2 during ANS development, we employed a yeast two-hybrid screen to identify HAND2-interacting proteins. One protein identified in this screen, Jun activation domain-binding protein (JAB1), is involved in numerous cell processes, including regulation of transcription and protein turnover. We show that JAB1 binds directly to the HLH domain of HAND2 and increases HAND2 transcription-stimulating activity. However, JAB1 does not contain a transcriptional activation domain, nor does it recruit an activation domain to HAND2. Our data indicate that JAB1 augments HAND2 transcriptional activity by enhancing HAND2 DNA binding. We further show that enhanced HAND2 DNA binding is mediated through the HLH domain and not through the DNA binding domain. These results show that JAB1 regulates the transcriptional activity of HAND2 in a unique manner that may account, in part, for the apparent ability of this bHLH factor to regulate gene expression through numerous mechanisms.
2724	Notch signaling patterns Drosophila mesodermal segments by regulating the bHLH transcription factor twist.	15128668	One of the first steps in embryonic mesodermal differentiation is allocation of cells to particular tissue fates. In Drosophila, this process of mesodermal subdivision requires regulation of the bHLH transcription factor Twist. During subdivision, Twist expression is modulated into stripes of low and high levels within each mesodermal segment. High Twist levels direct cells to the body wall muscle fate, whereas low levels are permissive for gut muscle and fat body fate. We show that Su(H)-mediated Notch signaling represses Twist expression during subdivision and thus plays a critical role in patterning mesodermal segments. Our work demonstrates that Notch acts as a transcriptional switch on mesodermal target genes, and it suggests that Notch/Su(H) directly regulates twist, as well as indirectly regulating twist by activating proteins that repress Twist. We propose that Notch signaling targets two distinct 'Repressors of twist' - the proteins encoded by the Enhancer of split complex [E(spl)C] and the HLH gene extra machrochaetae (emc). Hence, the patterning of Drosophila mesodermal segments relies on Notch signaling changing the activities of a network of bHLH transcriptional regulators, which, in turn, control mesodermal cell fate. Since this same cassette of Notch, Su(H) and bHLH regulators is active during vertebrate mesodermal segmentation and/or subdivision, our work suggests a conserved mechanism for Notch in early mesodermal patterning.
2725	A single measurement is inadequate to estimate enterolactone levels in danish postmenopausal women due to large intraindividual variation.	15113970	Single measurements of enterolactone (ENL) used in epidemiologic studies are influenced by intraindividual variation. The objective of this controlled study was to investigate short-term intraindividual variations in serum and urine ENL. Based on these variations, the number of samples required to describe the basal ENL level was estimated. Healthy Danish postmenopausal women (n = 6) aged 54-67 y completed 3 study periods of 24 h within 2 mo. Blood samples were collected at 0, 4, 6, 8, 12, and 24 h and 24-h urine samples were collected. A low-lignan, standardized diet of 3 meals was served. ENL was measured by time-resolved fluoroimmunoassay. Intraindividual and interindividual variations were estimated using a mixed model with repeated measurements. Significant and systematic intraindividual within-day variations (CV) of 31% were observed in serum. Intraindividual day-to-day variations were 56% and overall intraindividual variation of samples collected at random times and on different days was estimated to be 64%. Describing this overall variation required 7 blood samples when estimated with a precision of 50% and 95% confidence. Day-to-day variations in 24-h urine samples were 49%. Large within-day and day-to-day variations suggest that a single measurement of ENL is inadequate to estimate the basal ENL level.
2726	Outcomes of unrelated cord blood transplantation in pediatric recipients.	15107815	We report results of unrelated cord blood transplants (UCBT) in 29 pediatric recipients in one center and the risk factors associated with survival. Median age: 9 years (0.5-20); diagnosis: ALL (9), AML (4), CML (1), HD (3), HLH (1), NHL (3), NBL (2); B-thal (1), FA (1), FEL (1), Krabbe (1), WAS (1), SAA (1); median follow-up: 11 months; conditioning: total body irradiation (TBI)-ablative (14), chemotherapy-ablative (6) and reduced intensity chemotherapy (9); GVHD prophylaxis: MMF/FK506 (18), cyclosporin A (CsA)+steroids+/-MMF (7) or CsA+methotrexate (MTX) (4); median total nucleated cells (TNC): 3.8 x 10(7)/kg (1.1-11); median CD34+: 2.3 x 10(5)/kg (0.2-9.9); and HLA match: 2 (6/6), 5 (5/6), 22 (4/6). Neutrophil engraftment by cumulative incidence curves 63% (median 28 (95% confidence interval (CI) 18-32)). Probability of >/=grade II acute graft-versus-host disease (aGVHD) by day +60 27%, >/=grade III aGVHD 20% and chronic graft-versus-host disease 3%. Estimated 1-year overall survival (OS) 46% (95% CI 30-71) and standard risk 60% (95% CI 29-100%). Variables associated with improved survival by multivariate analysis include non-TBI-ablative conditioning (P=0.024), CD34+/kg (P=0.038) and gender (P=0.048). These results suggest that CD34/kg cell dose and non-TBI-ablative conditioning may be important variables influencing OS following UCBT in pediatric recipients. Given the small number of patients, these results should be viewed cautiously.
2727	Cell cycle-dependent expression of a hairy and Enhancer of split (hes) homolog during cleavage and segmentation in leech embryos.	15081366	We have cloned genes related to hairy and Enhancer of split (hes) from glossiphoniid leeches, Helobdella robusta and Theromyzon rude. In leech, segments arise sequentially in anteroposterior progression from a posterior growth zone that consists of five bilaterally paired embryonic stem cells called teloblasts. Each teloblast gives rise to segmental founder cells (primary blast cells) that contribute iterated sets of definitive progeny in each segment. Thus, in leech, the "segmentation clock," is closely identified with the cell cycle clock of the teloblasts. We have characterized normal expression patterns of mRNA and protein for the H. robusta hes-class gene (Hro-hes). Semiquantitative RT-PCR revealed that Hro-hes mRNA levels peak while the teloblasts are actively producing primary blast cells. RT-PCR, in situ hybridization and immunostaining revealed that Hro-hes is expressed as early as the first zygotic mitosis and throughout early development. Hro-hes is expressed in macromeres, pro-teloblasts, teloblasts and primary blast cells. HRO-HES protein is localized in the nuclei of cells expressing HRO-HES during interphase; nuclear HRO-HES is reduced during mitosis. In contrast, Hro-hes is transcribed during mitosis and its transcripts are associated with mitotic apparatus (MA). Thus, Hro-hes transcription cycles in antiphase to the nuclear localization of HRO-HES protein. These results indicate that Hro-hes expression, and thus possibly its biological activity, is linked to the cell cycle.
2728	An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder.	15069016	Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder with familial and acquired forms. The familial form is associated with mutations in the perforin gene and both forms are associated with severe defects in lymphocyte cytotoxic function. We examined perforin-deficient mice as a model of HLH in order to gain insight into this poorly understood disorder. While these mice do not spontaneously develop HLH-like symptoms, we found that they manifest all of the features of HLH after infection with lymphocytic choriomeningitic virus (LCMV). Following LCMV infection, perforin-deficient mice develop fever, splenomegaly, pancytopenia, hypertriglyceridemia, hypofibrinogenemia, and elevation of multiple serum cytokine levels, and hemophagocytosis is evident in many tissues. Investigation into how this phenotype develops has revealed that CD8+ T cells, but not natural killer (NK) cells, are necessary for the development of this disorder. Cytokine neutralization studies have revealed that interferon gamma (IFNgamma) is uniquely essential as well. Finally, the excessive amount of IFNgamma seen in affected mice appears to be driven by increased antigen presentation to CD8+ T cells. These studies provide insight into the pathophysiology of HLH, and provide new targets for specific therapeutic intervention in this fatal disorder.
2729	Mitf and Tfe3: members of a b-HLH-ZIP transcription factor family essential for osteoclast development and function.	15050900	The Microphthalmia-associated transcription factor (Mitf) is required for the proper development of several cell lineages including osteoclasts, melanocytes, retinal pigment epithelial cells, mast cells and natural killer cells. Mutations in Mitf in multiple organisms result in osteopetrosis due to defective osteoclast development. Mitf is a member of the basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) transcription factor subfamily named MiT, which also includes Tfe3. Genetic evidence indicates that Mitf and Tfe3 carry out essential functions in osteoclast development. Mitf has been shown to reside downstream of the macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) signaling pathways that are critical for osteoclast proliferation, differentiation and function. Mitf and Tfe3 have been shown to regulate the expression of several target genes necessary for bone degradation by mature osteoclasts. Here, we review the bone and osteoclast phenotypes of animals with mutations in Mitf and Tfe3, Mitf's interaction partners and signaling pathways, and known target genes which, together with others yet to be identified, likely represent key effectors of bone resorption.
2730	Basic helix-loop-helix proteins bind to TrkB and p21(Cip1) promoters linking differentiation and cell cycle arrest in neuroblastoma cells.	15024057	Differentiation of precursor into specialized cells involves an increasing restriction in proliferative capacity, culminating in cell cycle exit. In this report we used a human neuroblastoma cell line to study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Exposure to retinoic acid (RA), a differentiation agent in many cell types, causes an upregulation of neurotrophin receptor TrkB and the cyclin kinase inhibitor p21(Cip1) at a transcriptional level. Full transcriptional activation of these two genes requires canonical E-box sequences found in the TrkB and p21(Cip1) promoters. As reported for other E-box-regulated promoters, ectopic expression of E47 and E12 basic helix-loop-helix (bHLH) proteins enhances RA-dependent expression of TrkB and p21(Cip1), whereas the inhibitory HLH Id2 exerts opposite effects. In addition, ectopic expression of E47 and NeuroD, a neuronal bHLH protein, is able to activate TrkB transcription in the absence of RA. More importantly, we show that E47 and NeuroD proteins bind the TrkB and p21(Cip1) promoter sequences in vivo. Since they establish a direct transcriptional link between a cell cycle inhibitor, p21(Cip1), and a neurotrophic receptor, TrkB, bHLH proteins would play an important role in coordinating key events of cell cycle arrest and neuronal differentiation.
2731	Longitudinal follow-up of patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	15003893	Although immunochemotherapy has been reported to be an effective initial treatment for patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH), the long-term outcome of these patients remains unknown. The main purpose of this study was to determine the outcome of the EBV-HLH patients treated between 1992 and 2001.During this period, a total of 78 EBV-HLH patients were consecutively registered in 3 separate studies. The rates of initial response, reactivation, and survival as well as causes of death were analyzed. The outcome of the patients who received hematopoietic stem cell transplantation was also studied.With a median follow-up of 43 months, clinical reactivation was noted in 13 patients (19.4%) and a total of 12 patients needed hematopoietic stem cell transplantation, of whom 9 are alive and well. There had been 19 deaths: early deaths were due to hemorrhages and infections (n=11), while late deaths were related to late reactivation (n=4), transplant-associated causes (n=3) and secondary leukemia (n=1). Overall, after a median follow-up of 43 months, 59 (75.6%) of the 78 patients are alive and well.The majority of successfully treated EBV-HLH patients have a good outcome and remain disease-free.
2732	Drosophila CK2 regulates eye morphogenesis via phosphorylation of E(spl)M8.	15003630	The Notch effector E(spl)M8 is phosphorylated at Ser159 by CK2, a highly conserved Ser/Thr protein kinase. We have used the Gal4-UAS system to assess the role of M8 phosphorylation during bristle and eye morphogenesis by employing a non-phosphorylatable variant (M8SA) or one predicted to mimic the 'constitutively' phosphorylated protein (M8SD). We find that phosphorylation of M8 does not appear to be critical during bristle morphogenesis. In contrast, only M8SD elicits a severe 'reduced eye' phenotype when it is expressed in the morphogenetic furrow of the eye disc. M8SD elicits neural hypoplasia in eye discs, elicits loss of phase-shifted Atonal-positive cells, i.e. the 'founding' R8 photoreceptors, and consequently leads to apoptosis. The ommatidial phenotype of M8SD is similar to that in Nspl/Y; E(spl)D/+ flies. E(spl)D, an allele of m8, encodes a truncated protein known as M8*, which, unlike wild type M8, displays exacerbated antagonism of Atonal via direct protein-protein interactions. In line with this, we find that the M8SD-Atonal interaction appears indistinguishable from that of M8*-Atonal, whereas interaction of M8 or M8SA appears marginal, at best. These results raise the possibility that phosphorylation of M8 (at Ser159) might be required for its ability to mediate 'lateral inhibition' within proneural clusters in the developing retina. This is the first identification of a dominant allele encoding a phosphorylation-site variant of an E(spl) protein. Our studies uncover a novel functional domain that is conserved amongst a subset of E(spl)/Hes repressors in Drosophila and mammals, and suggests a potential role for CK2 during retinal patterning.
2733	Distinct domains within Mash1 and Math1 are required for function in neuronal differentiation versus neuronal cell-type specification.	14993186	Many members of the basic helix-loop-helix (bHLH) family of transcription factors play pivotal roles in the development of a variety of tissues and organisms. We identify activities for the neural bHLH proteins Mash1 and Math1 in inducing neuronal differentiation, and in inducing the formation of distinct dorsal interneuron subtypes in the chick neural tube. Although both factors induce neuronal differentiation, each factor has a distinct activity in the type of dorsal interneuron that forms, with overexpression of Math1 increasing dI1 interneurons, and Mash1 increasing dI3 interneurons. Math1 and Mash1 function as transcriptional activators for both of these functions. Furthermore, we define discrete domains within the bHLH motif that are required for these different activities in neural development. Helix 1 of the Mash1 HLH domain is necessary for Mash1 to be able to promote neuronal differentiation, and is sufficient to confer this activity to the non-neural bHLH factor MyoD. In contrast, helix 2 of Math1, and both helix 1 and 2 of Mash1, are the domains required for the neuronal specification activities of these factors. The requirement for distinct domains within the HLH motif of Mash1 and Math1 for driving neuronal differentiation and cell-type specification probably reflects the importance of unique protein-protein interactions involved in these functions.
2734	Hemophagocytic lymphohistiocytosis in children: abdominal US findings within 1 week of presentation.	14990835	To evaluate the ultrasonographic (US) findings at presentation in a series of children who received a diagnosis of hemophagocytic lymphohistiocytosis (HLH) at a single institution.All available results of imaging studies of children who received a diagnosis of HLH between January 1985 and June 2000 were retrieved. For nine patients, abdominal US images obtained within 1 week of presentation to the hospital were reviewed retrospectively by two radiologists who were aware of the diagnosis and of the original interpretation of each study. US images were examined for evidence of splenomegaly, hepatomegaly, ascites, gallbladder wall thickening, increased periportal echogenicity, lymphadenopathy, pleural effusion, and nephromegaly. Any other abnormalities were also recorded. Differences in interpretation were resolved by consensus. The patients ranged in age from 2 months to 4(1/2) years. The male-to-female ratio was 5:4.Findings at presentation included splenomegaly (in eight of the nine children), hepatomegaly (in seven children), ascites (in six children), gallbladder wall thickening (in six children), increased periportal echogenicity (in three children), lymphadenopathy (in three children), and pleural effusion (in two children). Miscellaneous findings in individual patients included coarse hepatic echotexture with a single 9-mm hypoechoic focus in the liver, multiple hypoechoic foci in the spleen, nephromegaly, gallstone, increased renal cortical echogenicity, and mural thickening of the duodenum.In the appropriate clinical setting, the differential diagnosis of a combination of hepatosplenomegaly, ascites, gallbladder wall thickening, increased periportal echogenicity, lymphadenopathy, and/or pleural effusion should include HLH.
2735	Randomized trial to compare the effect of recombinant human FSH (follitropin alfa) with or without recombinant human LH in women undergoing assisted reproduction treatment.	14989794	Women undergoing intracytoplasmic sperm injection (ICSI) for male factor infertility were randomly assigned to receive ovarian stimulation in a long agonist protocol with a combination of recombinant human FSH (r-hFSH; Gonal-F) and recombinant human LH (r-hLH; Luveris) (n = 212) starting on day 6 of FSH stimulation until human chorionic gonadotrophin (HCG) at a daily fixed dose of 150 IU r-hLH, or with r-hFSH alone (n = 219). There was no significant difference in the number of metaphase II oocytes retrieved (10.3 versus 10.4) in patients treated with r-hFSH and r-hLH versus r-hFSH alone; however, more embryos were transferred in the LH-supplemented group (2.9 versus 2.8, P = 0.037). Overall, the implantation rates were 22.9 versus 27.0% in patients treated with r-hFSH and r-hLH versus with r-hFSH alone respectively (NS). The respective numbers of MII oocytes retrieved in patients <35 or >or=35 years were 11 versus 8.3 (P = 0.010) for patients treated with r-hFSH alone, and 10.7 versus 9.3 (NS) for those given supplemental r-hLH (150 IU) from day 6. Implantation rates in patients <35 years treated with r-hFSH were higher (30.7%) than those receiving r-hFSH and r-hLH, (23.5%) (P = 0.068). In patients >or=35 years, the implantation rates were 21.7% for those patients supplemented with 150 IU r-hLH from day 6 of stimulation versus 15.7% when treated with FSH alone (NS). Younger patients therefore do not seem to benefit from an LH-supplemented ovarian stimulation protocol, but women >or=35 years undergoing assisted reproduction may benefit from using r-hLH in addition to r-hFSH.
2736	Determination of binding constant of transcription factor myc-max/max-max and E-box DNA: the effect of inhibitors on the binding.	14980448	The truncated myc and max proteins, only containing basic regions and helix-loop-helix/zipper (b/HLH/Zip) regions were over-expressed in E. coli and used for the determination of the binding constant and of the inhibitory mechanism on myc-max (or max-max)-DNA complex formation. The association kinetic constants (k(1) and k(-1)) of truncated max-max or myc-max dimer and DNA were determined as k(1)=(1.7+/-0.6)x10(5) M(-1) s(-1), k(-1)=(3.4+/-1.2)x10(-2) s(-1) for max-max and DNA or k(1)=(2.1+/-0.7)x10(5) M(-1) s(-1), k(-1)=(3.2+/-1.4)x10(-2) s(-1) for myc-max and DNA. The equilibrium binding constant (K(1)) was determined using these kinetic parameters [K(XXD)=(7.8+/-2.6)x10(6) M(-1) for max-max and DNA or K(XYD)=(6.9+/-2.2)x10(6) M(-1) for myc-max and DNA]. The binding constants of myc-max or max-max dimer formation were K(XX)=(2.6+/-0.9)x10(5) M(-1) or K(XY)=(1.3+/-0.4)x10(4) M(-1), respectively. When truncated proteins were used, the max-max dimer formation was easier than the myc-max dimer formation, contrary to the physiologically determined case. This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions. The truncated myc and max proteins, which were expressed in E. coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc-max-DNA complex formation. A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA. The dissociation constant of max-max dimer and the inhibitor was 9 microM, when investigated using in vitro expressed b/HLH/Zip dimer proteins. The curcuminoid 004 showed an inhibitory effect on the binding of myc-max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over-expressed human stomach cancer cell line SNU16.
2737	Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris.	14965762	The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.
2738	SCL assembles a multifactorial complex that determines glycophorin A expression.	14749362	SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.
2739	Prevalence of congenital cardiovascular malformations among relatives of infants with hypoplastic left heart, coarctation of the aorta, and d-transposition of the great arteries.	14708093	Cardiovascular malformations (CVM) are the most common birth defects and carry significant and lifelong personal and societal costs. Research into genetic and environmental risk factors is therefore critical in identifying clues to causation and prevention. The purpose of this study was to investigate patterns of familial aggregation in CVM, specifically among infants with left-sided obstructive heart defects. We ascertained families of probands with hypoplastic left heart (HLH: N = 38), coarctation of the aorta (CoA: N = 46), and d-transposition of the great arteries (dTGA: N = 22). First degree relatives had clinical examinations and echocardiograms; all other relatives had detailed reviews of medical records. A total of 2,694 relatives were included in the study: 379 1st degree, 986 2nd degree, and 1,329 3rd degree. Mean nuclear family size and sibship size were similar among the groups. CVM were detected more frequently in 1st degree relatives of probands with HLH (19.3%) or CoA (9.4%) than among dTGA families (2.7%). The proportions of affected 2nd degree relatives were similar across groups (</=1%). In 3rd degree relatives, CVM was detected in 1.8% of the HLH families compared to 1.2% in CoA and 0.4% in dTGA families. The predominant types of CVM among relatives of HLH and CoA probands were left-sided obstructive lesions, in 72% (21 of 29) and 67% (25 of 37) of the affected relatives, respectively. Familial aggregation of these types of CVM is therefore confirmed in this study, potentially facilitating the search for specific genetic and other risk factors in recurrent CVM.
2740	SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative.	14702347	The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.
2741	Post-transcriptional regulation of the E/Daughterless ortholog HLH-2, negative feedback, and birth order bias during the AC/VU decision in C. elegans.	14701877	The anchor cell/ventral uterine precursor cell (AC/VU) decision in Caenorhabditis elegans is a canonical example of lin-12/Notch-mediated lateral specification. Two initially equivalent cells interact via the receptor LIN-12 and its ligand LAG-2, so that one becomes the AC and the other a VU. During this interaction, feedback loops amplify a small difference in lin-12 activity, limiting lin-12 transcription to the presumptive VU and lag-2 transcription to the presumptive AC. Here, we find that hlh-2 appears to be required for the VU fate and directly activates lag-2 transcription in the presumptive AC. HLH-2 appears to accumulate selectively in the presumptive AC prior to differential transcription of lin-12 or lag-2, and is therefore the earliest detectable difference between the two cells undergoing the AC/VU decision. The restricted accumulation of HLH-2 to the presumptive AC reflects post-transcriptional down-regulation of HLH-2 in the presumptive VU. Our observations suggest that hlh-2 is regulated as part of the negative feedback that down-regulates lag-2 transcription in the presumptive VU. Finally, we show that the AC/VU decision in an individual hermaphrodite is biased by the relative birth order of the two cells, so that the first-born cell is more likely to become the VU. We propose models to suggest how birth order, HLH-2 accumulation, and transcription of lag-2 may be linked during the AC/VU decision.
2742	Quantum states far from the energy eigenstates of any local Hamiltonian.	14683281	What quantum states are possible energy eigenstates of a many-body Hamiltonian? Suppose the Hamiltonian is nontrivial, i.e., not a multiple of the identity, and L local, in the sense of containing interaction terms involving at most L bodies, for some fixed L. We construct quantum states psi which are "far away" from all the eigenstates E of any nontrivial L-local Hamiltonian, in the sense that ||psi-E|| is greater than some constant lower bound, independent of the form of the Hamiltonian.
2743	Senseless acts as a binary switch during sensory organ precursor selection.	14665671	During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Here, we present evidence that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. We show that Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express low levels of Sens, it acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express high levels of Sens, it acts as a transcriptional activator and synergizes with proneural proteins. We therefore propose that Sens acts as a binary switch that is fundamental to SOP selection.
2744	The 31-kDa caspase-generated cleavage product of p130cas functions as a transcriptional repressor of E2A in apoptotic cells.	14660614	In response to integrin receptor binding to the extracellular matrix, the multidomain docking protein p130(cas) (Crk-associated substrate) activates various signaling cascades modulating such cellular processes as proliferation, migration, and apoptosis. During apoptosis, caspase-mediated cleavage of p130(cas) generated a C-terminal 31-kDa fragment (31-kDa) and promoted morphological changes characteristic of apoptosis, including loss of focal adhesions, cell rounding, and nuclear condensation and fragmentation. By contrast, a p130(cas) D748E mutant, which was unable to produce 31-kDa, attenuated the disassembly of focal adhesions at the bottom of the cell. 31-kDa contains a helix-loop-helix (HLH) domain that shows greater sequence homology with Id proteins than with basic HLH proteins, which enabled heterodimerization with E2A. Once coupled to E2A, 31-kDa was translocated to the cell nucleus, where it inhibited E2A-mediated p21(Waf1/Cip1) transcription. Moreover, overexpression of 31-kDa led to cell death that could be inhibited by treatment with the caspase inhibitor ZVAD-fluoromethyl ketone or by ectopic expression of E2A or p21(Waf1/Cip1). These data suggest that during etoposide-induced apoptosis, 31-kDa promotes caspase-mediated cell death by inhibiting E2A-mediated activation of p21(Waf1/Cip1) transcription.
2745	A cell-specific enhancer that specifies lin-3 expression in the C. elegans anchor cell for vulval development.	14660442	During C. elegans vulval development, the anchor cell (AC) in the somatic gonad expresses lin-3, activating the EGF receptor signaling pathway in vulval precursor cells (VPCs) and thereby inducing and patterning VPCs. Previous studies with lin-3 mutants and transgene expression have revealed that the level of LIN-3 in the AC must be precisely regulated for proper vulval development. To understand how lin-3 expression is achieved in the AC, we identified a 59 bp lin-3 enhancer sufficient to activate lin-3 transcription solely in the AC. The enhancer contains two E-box elements, and one FTZ-F1 nuclear hormone receptor (NHR) binding site that is mutated in a vulvaless mutant, lin-3(e1417). Mutagenesis studies show that both E-boxes and the NHR binding site are necessary to express lin-3 in the AC. In vitro DNA-binding studies and in vivo functional assays indicate that distinct trans-acting factors, including the E-protein/Daughterless homolog HLH-2 and unidentified nuclear hormone receptor(s), are necessary for lin-3 transcription in the AC and thus are involved in vulval development.
2746	Helix-loop-helix proteins in mammary gland development and breast cancer.	14635797	The basic helix-loop-helix (bHLH) family of transcription factors functions in the coordinated regulation of gene expression, cell lineage commitment, and cell differentiation in most mammalian tissues. Helix-loop-helix Id (Inhibitor of DNA binding) proteins are distinct from bHLH transcription factors in that they lack the basic domain necessary for DNA binding. Id proteins thus function as dominant negative regulators of bHLH transcription factors. The inhibition of bHLH factor activity by forced constitutive expression of Id proteins is closely associated with the inhibition of differentiation in a number of different cell types, including mammary epithelial cells. Moreover, recent literature suggests important roles of HLH proteins in many normal and transformed tissues, including mammary gland. Therefore, future directions for prognosis or therapeutic treatments of breast cancer may be able to exploit bHLH and Id genes as useful molecular targets. The purpose of this review is to summarize the evidence implicating HLH proteins in the regulation of normal and transformed mammary epithelial cell phenotypes.
2747	Id4 regulates mammary epithelial cell growth and differentiation and is overexpressed in rat mammary gland carcinomas.	14633621	Id4 belongs to a family of helix-loop-helix (HLH) proteins that impact cellular growth and differentiation via regulation of basic HLH transcription factors. Herein the rat Id4 gene was cloned (GenBank Accession No. AF468681). The expression of rat Id4 was examined in rat mammary gland tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in the human diet. By real-time polymerase chain reaction analysis, relative expression of Id4 mRNA in carcinomas, adenomas, and normal tissue was 27, 6, and 1, respectively. Immunohistochemical analysis indicated statistically elevated nuclear expression for Id4 protein in carcinomas in comparison to adenomas and normal mammary gland. In carcinomas, Id4 nuclear expression was positively correlated with proliferation, invasiveness, and tumor weight (Fisher Exact Test or Spearman Correlation, P < 0.05). The consequence of enforced expression of Id4 on mammary epithelial cell proliferation, differentiation, and growth in soft agar was examined in HC11 cells, a well-characterized model for studying various aspects of mammary epithelial cell biology. After transient and stable transfection of HC11 cells, Id4 overexpression increased cell proliferation and inhibited lactogenic hormone-mediated differentiation as revealed by inhibition of beta-casein promoter activity and beta-casein expression. In addition, enforced expression of Id4 in HC11 cells induced a statistically significant increase in colony growth in soft agar. The results implicate Id4 in rat mammary gland carcinogenesis and suggest that Id4 may contribute to carcinogenesis by inhibiting mammary epithelial cell differentiation and stimulating mammary epithelial cell growth.
2748	Abnormal cell surface antigen expression in individuals with variant CD45 splicing and histiocytosis.	14630980	Hemophagocytic lymphohistiocytosis (HLH) and Langerhans cell histiocytosis (LCH) are members of a group of rare heterogenous disorders, the histiocytoses, characterized by uncontrolled accumulation of pleomorphic infiltrates of leukocytes. The etiology of these diseases is mainly unknown. CD45 is a hemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signaling in lymphocytes and different patterns of CD45 splicing are associated with distinct functions. Recently a polymorphism (C77G) in exon 4 of CD45 causing abnormal CD45 splicing and a point mutation affecting CD45 dimerization were implicated in multiple sclerosis in humans and lymphoproliferation and autoimmunity in mice respectively. Here we show that two patients with HLH exhibited abnormal CD45 splicing caused by the C77G variant allele, while a further 21 HLH patients have normal CD45. We have also examined 62 LCH patients and found three to have the C77G mutation. Peripheral blood thymus-derived (T) CD8(+) cells from normal individuals carrying the C77G mutation show a significant decrease in the proportion of cells expressing L-selectin and increased frequency of cells with LFA-1(hi) expression. It remains to be established whether C77G is a contributing factor in these histiocytic disorders.
2749	HLH-14 is a C. elegans achaete-scute protein that promotes neurogenesis through asymmetric cell division.	14627726	Achaete-Scute basic helix-loop-helix (bHLH) proteins promote neurogenesis during metazoan development. In this study, we characterize a C. elegans Achaete-Scute homolog, HLH-14. We find that a number of neuroblasts express HLH-14 in the C. elegans embryo, including the PVQ/HSN/PHB neuroblast, a cell that generates the PVQ interneuron, the HSN motoneuron and the PHB sensory neuron. hlh-14 mutants lack all three of these neurons. The fact that HLH-14 promotes all three classes of neuron indicates that C. elegans proneural bHLH factors may act less specifically than their fly and mammalian homologs. Furthermore, neural loss in hlh-14 mutants results from a defect in an asymmetric cell division: the PVQ/HSN/PHB neuroblast inappropriately assumes characteristics of its sister cell, the hyp7/T blast cell. We argue that bHLH proteins, which control various aspects of metazoan development, can control cell fate choices in C. elegans by regulating asymmetric cell divisions. Finally, a reduction in the function of hlh-2, which encodes the C. elegans E/Daughterless bHLH homolog, results in similar neuron loss as hlh-14 mutants and enhances the effects of partially reducing hlh-14 function. We propose that HLH-14 and HLH-2 act together to specify neuroblast lineages and promote neuronal fate.
2750	Morpholino-based gene knockdown screen of novel genes with developmental function in Ciona intestinalis.	14627717	In the present study, we conducted an extensive analysis to identify novel genes with developmental function among Ciona intestinalis genes discovered by cDNA projects. Translation of a total of 200 genes expressed during embryogenesis was suppressed by using specific morpholino antisense oligonucleotides. Suppression of the translation of any of 40 genes (one-fifth of the genes tested) was thereby shown to cause specific embryonic defects. Most of these genes have counterpart(s) in mouse and human, suggesting that the present approach will be useful for identifying candidate genes essential for the development of vertebrates. Suppression of translation of 14 of these 40 genes resulted in the 'disorganized body plan' phenotype characterized by gross morphological abnormalities caused by early defects in embryogenesis. These genes encode zinc-finger, transmembrane or Pbx homeodomain proteins. The morphological features of larvae of this phenotypic class varied according to the gene suppressed, suggesting that a distinct developmental event such as tissue specification or cell cycle progression was affected in each type of larva. Suppression of the remaining 26 genes resulted in the 'abnormal tail' phenotype. Some of these genes encode proteins with known functional structures such as Zn-finger and HLH motifs. Twelve genes among them are especially interesting, because their suppression produced defects in the nervous system, as demonstrated by the loss of the sensory pigment cells or palps of the adhesive organ in the knockdown larvae. These results suggest that screening for developmental genes by the reverse genetic approach in Ciona intestinalis embryos is effective for identifying novel genes with developmental functions required for the development of chordates.
2751	Candidiasis-associated hemophagocytic lymphohistiocytosis in a patient infected with human immunodeficiency virus.	14614689	We describe a patient with acquired immunodeficiency syndrome who developed candidiasis-associated hemophagocytic lymphohistiocytosis (HLH), and we review the previously reported cases of this unusual clinical syndrome in patients infected with human immunodeficiency virus (HIV). HLH appears to follow a fulminant course in HIV-infected patients, which warrants an aggressive diagnostic and therapeutic approach. HIV itself may play a role in the pathogenesis of HLH, which is usually associated with opportunistic infections or malignancies. Therapy is usually directed at supportive care and treatment of the underlying disorders, although initiation of antiretroviral therapy may improve the eventual outcome in some cases.
2752	Patient with trisomy 9p and a hypoplastic left heart with a tricentric chromosome 9.	14608650	We present a patient with a hypoplastic left heart (HLH), dislocations of the hips and knees, and minor dysmorphic features, who had an abnormal karyotype that resulted in trisomy for 9p and a portion of 9q: 46,((, dic(or tri?)(9)(9pter --> 9q34::9q21 --> 9pter).ish(WCP9++).ish(D9Z5X4 +/+++). The derivative chromosome consisted of an additional copy of the proximal q arm and p arm attached to 9qter in an inverted fashion. Fluorescence in situ hybridization (FISH) using a chromosome 9 beta-satellite probe revealed three signals on the abnormal chromosome 9, suggesting the presence of 3 pericentromeric regions on the der(9). The 9q subtelomere was present on both the normal and derivative chromosome 9, suggesting that very little material, if any, is deleted.
2753	Controlled ovulation of the dominant follicle: a critical role for LH in the late follicular phase of the menstrual cycle.	14585870	A method was sought to control ovulation of the dominant follicle and to test the importance of LH during the late follicular phase of the menstrual cycle. Menstrual cycles of rhesus monkeys were monitored, and treatment initiated at the late follicular phase (after dominant follicle selection, before ovulation).The 2-day treatment consisted of GnRH antagonist plus either r-hFSH and r-hLH (1:1 or 2:1 dose ratio) or r-hFSH alone. In addition, half of the females received an ovulatory bolus of hCG.When treatment was initiated at estradiol levels >120 pg/ml, neither the endogenous LH surge, ovulation nor luteal function were controlled. However, when treatment was initiated at estradiol levels 80-120 pg/ml using either 1:1 or 2:1 dose ratios of FSH:LH, the LH surge was prevented, and ovulation occurred following hCG treatment. FSH-only treatment also prevented the LH surge, but follicle development appeared abnormal, and hCG failed to stimulate ovulation.Control over the naturally dominant follicle is possible during the late follicular phase using an abbreviated GnRH antagonist, FSH+LH protocol. This method offers a model to investigate periovulatory events and their regulation by gonadotrophins/local factors during the natural menstrual cycle in primates.
2754	Clinical and genetic studies of familial hemophagocytic lymphohistiocytosis in Oman: need for early treatment.	14578030	Hemophagocytic lymphohistiocytosis (HLH) embraces the frequently indistinguishable conditions of familial hemophagocytic lymphohistiocytosis (FHL) and virus-associated hemophagocytic syndrome (VAHS). Without therapy FHL is invariably fatal, but successful therapy, including chemotherapy and immunotherapy followed by bone marrow transplantation (BMT), has been presented. To clarify the outcome of HLH in a developing country, with regard to clinical, laboratory, and genetic features, a nationwide study on all patients diagnosed with HLH in Oman during the 5-year period 1997-2001 was performed. In 5 patients and their families, mutational analysis was made. Thirteen patients with HLH were identified, 5 of whom had clinical manifestations of central nervous system involvement at presentation. In none of the patients could an infectious cause be identified. Ten children were referred late in the disease course, and the concern about starting chemotherapy before exclusion of an acute viral infection resulted in delayed treatment in some patients. Two children were started early on the HLH-94-therapy followed by successful BMT in one child. In the successfully transplanted child, the response to intrathecal hydrocortisone appeared to be better than standard therapy with intrathecal methotrexate. Finally, a novel missense mutation in the perforin gene was identified in 2 patients and their family members, causing a transition of proline to threonine at codon 89. Early diagnosis and treatment is important to improve outcome. Intrathecal corticosteroids may be considered, in addition to intrathecal methotrexate, in certain patients. Since the novel perforin mutation has been reported in only 2 patients from Oman, and since similar polymorphism in the sequencing data of the members of their families has been identified, a founder effect is possible in this population.
2755	Low molecular weight inhibitors of Myc-Max interaction and function.	13679853	c-Myc is helix-loop-helix-leucine zipper (HLH-ZIP) oncoprotein that is frequently deregulated in human cancers. In order to bind DNA, regulate target gene expression, and function in a biological context, c-Myc must dimerize with another HLH-ZIP protein, Max. A large number of c-Myc target genes have been identified, and many of the encoded proteins are transforming. Such functional redundancy, however, complicates therapeutic strategies aimed at inhibiting any single target gene product. Given this consideration, we have instead attempted to identify ways by which c-Myc itself could be effectively disabled. We have used a yeast two-hybrid approach to identify low-molecular-weight compounds that inhibit c-Myc-Max association. All of the compounds prevented transactivation by c-Myc-Max heterodimers, inhibited cell cycle progression, and prevented the in vitro growth of fibroblasts in a c-Myc-dependent manner. Several of the compounds also inhibited tumor growth in vivo. These results show that the yeast two-hybrid screen is useful for identifying compounds that can be exploited in mammalian cells. More specifically, they provide a means by which structural analogs, based upon these first-generation Myc-Max inhibitors, can be developed to enhance antitumor efficacy.
2756	Id4 is deregulated by a t(6;14)(p22;q32) chromosomal translocation in a B-cell lineage acute lymphoblastic leukemia.	12969807	Chromosome translocations resulting in gene overexpression are commonly associated with lymphoid neoplasia. Enhancer elements of the immunoglobulin or T-cell receptor (TCR) loci are abnormally located in the vicinity of the entire coding sequences of genes which exert an influence on the normal maturation and differentiation program of lymphoid cells.A patient who presented with a B-cell lineage acute lymphoblastic leukemia had a t(6;14)(p22;q32). Cytogenetic and molecular findings confirmed the involvement of IgH. Molecular cloning of the breakpoint revealed that this was located near the coding sequence of the Id4 gene, a helix-loop-helix (HLH) inhibitor protein. Alu-repeated sequences at the 6p22 end flanked a short stretch of 10 bases shared by the 6p22 and 14q32 ends, suggesting that a deletion or a looping-Alu mediated mispairing mechanism may lead to this chromosome translocation.Northern blot and real-time polymerase chain reaction analyses showed that the Id4 mRNA was abnormally overexpressed in this case. Only the two smaller Id4 mRNA products were detected (1.6 and 1.1 kb). Immunohistochemical analysis of Id4 protein was also assayed in a series of hematologic malignancies. Marked overexpression was found in two cases of T-cell prolymphocytic leukemias and in four B-cell lineage acute lymphoblastic leukemia including one case with the t(8;14) and another case with a p53 mutation.The Id4 gene may behave as an oncogene in some human leukemias, perhaps through its capacity to sequester specific B-cell transcription factors. A genetic recombination between Alu-repeated sequences may not be the exclusive mechanism of generating pathogenic chromosomal translocations.
2757	Effect of hyper- and microgravity on collagen post-translational controls of MC3T3-E1 osteoblasts.	12968680	We attempted to study the effects of microgravity (by clinostat) and hypergravity (using centrifugation) on collagen metabolism using murine MC3T3-E1 osteoblasts, especially focusing on collagen cross-link formation. We found that altered gravitational load affected the post-translational modification of collagen, particularly the collagen maturation pathway, through altered expression of enzymes involved in cross-link formation.Gravitational loading plays important roles in the stimulation of differentiated osteoblast function and in the maintenance of skeletal tissues, whereas microgravity seems to result in osteopenia caused by impaired osteoblast differentiation. The aim of our study was to clarify the effects of altered gravitational environments on collagen metabolism, particularly the relationship between post-translational collagen quality and enzymes involved in cross-link formation, using murine osteoblastic MC3T3-E1 cells.Cells were cultured under vector-averaged microgravity (1 x 10(-3) g) using a clinostat or under conventional centrifugation techniques to generate hypergravity (20 g and 40 g) for 72 h. We then examined the expression patterns of lysyl oxidase and the two lysyl hydroxylase isoforms telopeptidyl lysyl hydroxylase (TLH; procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 2 [PLOD2]) and helical lysyl hydroxylase (HLH; [PLOD1]) by quantitative real time polymerase chain reaction (PCR) analysis. Quantitative analysis of reducible immature (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature (pyridinoline and deoxypyridinoline) cross-links, and maturation rate analysis of immature to mature cross-links by conventional metabolic labeling using tritium lysine were also performed.Hypergravity upregulated both TLH mRNA expression and enzyme activity compared with stationary cultures, whereas microgravity stimulated both HLH mRNA expression and enzyme activity. These results were consistent with increased relative occupancy rates of telopeptidyl hydroxylysine-derived cross-links and helical hydroxylysine-derived forms observed under hypergravity and microgravity, respectively. Hypergravity stimulated not only lysyl oxidase mRNA expression but also increased enzyme activity and the sum of immature and mature cross-links. Furthermore, the conversion rate of immature cross-links to mature compounds was markedly increased under hypergravity but decreased under microgravity.Altered gravitational loading may affect the post-translational modification of collagen through altered expression of enzymes involved in cross-link formation. These observations may be important in elucidating the mechanisms of osteopenia during space flight.
2758	Id proteins negatively regulate basic helix-loop-helix transcription factor function by disrupting subnuclear compartmentalization.	12952978	Id helix-loop-helix (HLH) proteins act as global regulators of metazoan cell fate, cell growth, and differentiation. They heterodimerize with and inhibit the DNA-binding function of members of the basic helix-loop-helix (bHLH) family of transcription factors. Using real time fluorescence microscopy techniques in single living cells, we show here that nuclear pools of chromatin-associated bHLH transcription factor are freely exchangeable and in constant flux. The existence of a dynamic equilibrium between DNA-bound and free bHLH protein is also directly demonstrable in vitro. By contrast, Id protein is not associated with any subcellular, macromolecular structures and displays a more highly mobile, diffuse nuclear-cytoplasmic distribution. When co-expressed with antagonist Id protein, the chromatin-associated sublocalization of bHLH protein is abolished, and there is an accompanying 100-fold increase in its nuclear mobility to a level expected for freely diffusible Id-bHLH heterodimer. These results suggest that nuclear Id protein acts by sequestering pools of transiently diffusing bHLH protein to prevent reassociation with chromatin domains. Such a mechanism would explain how Id proteins are able to overcome the large DNA-binding free energy of bHLH proteins that is necessary to accomplish their inhibitory effect.
2759	Muscle regulatory factor MRF4 activates differentiation in rhabdomyosarcoma RD cells through a positive-acting C-terminal protein domain.	12944914	Rhabdomyosarcoma (RMS) has deregulated proliferation and is blocked in the differentiation program despite Myf-5, MyoD and myogenin expression. Here we show that ectopic expression of MRF4, which is not subject to an autoregulatory pathway but regulated by the other MRFs protein family, induces growth arrest and terminal differentiation in RD cells. Deletion mapping identified a positive-acting C-terminal domain in MRF4 as the mediator of transcriptional activity, revealing a conserved motif with helix III in MyoD previously found to initiate expression of endogenous skeletal muscle genes. By using chimeric MyoD/MRF4 proteins, we observe that the C-terminal motif of MRF4 rescues MyoD activity in RD cells. Moreover, comparative induction of muscle-specific genes following activation of MyoD, through the expression of a constitutively activated MKK6 either in the absence or presence of MRF4, shows that MyoD and MRF4 can differently regulate muscle genes expression. Together, these results demonstrate that the MRF4 C-terminus functions as specification as well as activation domain in tumor cells. They provide a basis to identify gene products necessary for b-HLH-mediated differentiation versus tumor progression.
2760	Heterodimers of bHLH-PAS protein fragments derived from AhR, AhRR, and Arnt prepared by co-expression in Escherichia coli: characterization of their DNA binding activity and preparation of a DNA complex.	12944374	AhR (aryl hydrocarbon receptor), AhRR (AhR repressor), and Arnt (AhR nuclear translocator) are members of the bHLH (basic-helix-loop-helix)-PAS (Per-AhR/Arnt-Sim homology sequence) transcription factor superfamily. They associate with each other to form heterodimers, AhR/Arnt or AhRR/Arnt, and bind the XRE (xenobiotic responsive element) sequences in the promoter regions of the target genes to regulate their expression. Their basic regions and HLH motifs mediate DNA binding activity and protein dimerization, respectively. The PAS domain includes two incomplete repeats, PAS-A and PAS-B, and is considered to determine the specificity on protein dimerization. However, the three-dimensional structures of PAS folds reported so far are all monomeric, therefore, little is known about the structural basis of protein interaction through PAS domains. In the present study, we prepared heterodimeric bHLH-PAS domains derived from AhR and Arnt, and AhRR and Arnt by co-expressing each pair in E. coli, and showed that the heterodimers formed exhibited full DNA-binding activity, which was not apparently affected by deletion of the highly basic amino acid cluster most N-terminal as to the HLH region of AhR or AhRR. Methylation of the two CpG sites in the XRE core sequence reduced the binding affinity to heterodimeric proteins, with 5-methylcytosine in the AhR recognition site exhibiting a greater inhibitory effect than that in the Arnt recognition site.
2761	Increased serum levels of interferon-gamma-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphohistiocytosis.	12930373	We measured serum interferon-gamma-inducible protein 10 (IP-10) and monokine induced by gamma interferon (MIG) levels to investigate the role of these molecules in the pathophysiology of haemophagocytic lymphohistiocytosis (HLH). Serum IP-10 and MIG levels were significantly increased in patients with active HLH compared with those of healthy controls. Serum MIG levels decreased gradually during the course of disease in a patient who recovered without therapy. On the other hand, rapid reduction of MIG and IP-10 levels was observed after chemotherapy in a patient with severe HLH. IP-10 and MIG mRNA expression was enhanced in liver and spleen, and IP-10 mRNA expression was enhanced in bone marrow in the patients, suggesting activated macrophages that infiltrated in these organs as one of the main producers of these cytokines. Serum IP-10 and MIG levels showed a significant correlation with serum IFN-gamma levels. In addition, these chemokines had a significant correlation with fever and serum LDH levels, which are clinical indicators of disease activity of HLH. These results suggest that IP-10 and MIG which are produced by activated macrophages by the stimulation of IFN-gamma, play an important role in the pathophysiology of HLH, by recruitment of activated Th1 cells into the tissues or organs.
2762	Cha, a basic helix-loop-helix transcription factor involved in the regulation of upstream stimulatory factor activity.	12923186	We report here the characterization of Cha, a transcription factor of the basic helix-loop-helix (bHLH) family. The basic region of Cha shares DNA-interacting amino acids with members of class C bHLH transcription factors. In addition, the HLH region of Cha presents a Myc-type dimerization domain signature required for heterodimer formation between members of this class. Cha protein and mRNA were ubiquitously expressed in many human tissues. Electrophoretic mobility shift assays showed that Cha and upstream stimulatory factor (USF)-1 formed a complex that specifically bound to E-box DNA elements. Moreover, pull-down and co-immunoprecipitation experiments showed an interaction between Cha and USF-1. Cha did not bind to E-box DNA elements and required USF-1 for protein-DNA complex formation. Moreover, Cha inhibited USF-1-stimulated transcription of CD2 (a USF-1-dependent gene) and E-box promoter reporter plasmids. Chromatin immunoprecipitation assays showed that Cha occupied the CD2 promoter in resting, but not in mitogen-stimulated, T cells. Finally, Cha mRNA and protein expression were high in resting T cells and absent in mitogen-activated T cells and inversely correlated with CD2 expression. Contrarily, overexpression of Cha in T cells significantly reduced CD2 expression. In summary, our results indicated that Cha is a new bHLH transcription factor that negatively regulates USF-dependent transcription.
2763	Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation.	12917338	The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.
2764	Atypical hemophagocytic lymphohistiocytosis following bacterial tonsillitis in acute lymphoblastic leukemia.	12916881	We report a rare case of a cute lymphoblasticleukemia (ALL) who developed dyspnea, neurological disturbance with illusions, pancytopenia, phagocytosis and coagulation disturbances following bacterial tonsillitis. The values of soluble interleukin-2 receptor (sIL-2R), IL-6 and IL-8 were also elevated. Her clinicolaboratory findings were similar to hemophagocytic lymphohistiocytosis (HLH), which is a cytokine disease induced by activated T cells and macrophages. Atypical HLH following bacterial tonsillitis should be kept in mind in leukemia patients.
2765	Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.	12907712	DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.
2766	Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis in Wiskott-Aldrich syndrome.	12892170	A patient with Wiskott-Aldrich syndrome who developed Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis (EBV-HLH) is described in this study. At 4 mo of age the patient developed fever associated with bicytopenia and splenomegaly. Analysis of a bone marrow specimen revealed extensive haemophagocytosis, and in situ hybridization for EBV of the bone marrow specimen using an EBV-encoded RNA probe was positive. Diagnosis of EBV-HLH was established and immunotherapy with HLH-94 protocol was started. HLH has been described in patients with other well-defined primary immunodeficiencies such as X-linked lymphoproliferative syndrome, Chediak-Higashi syndrome and Griscelli disease. Also, HLH was reported recently in severe combined immunodeficiency and DiGeorge syndrome.The possibility of an underlying primary immunodeficiency should be considered in paediatric patients who present with HLH during infancy.
2767	The Snail-like CES-1 protein of C. elegans can block the expression of the BH3-only cell-death activator gene egl-1 by antagonizing the function of bHLH proteins.	12874127	The NSM cells of the nematode Caenorhabditis elegans differentiate into serotonergic neurons, while their sisters, the NSM sister cells, undergo programmed cell death during embryogenesis. The programmed death of the NSM sister cells is dependent on the cell-death activator EGL-1, a BH3-only protein required for programmed cell death in C. elegans, and can be prevented by a gain-of-function (gf) mutation in the cell-death specification gene ces-1, which encodes a Snail-like DNA-binding protein. Here, we show that the genes hlh-2 and hlh-3, which encode a Daughterless-like and an Achaete-scute-like bHLH protein, respectively, are required to kill the NSM sister cells. A heterodimer composed of HLH-2 and HLH-3, HLH-2/HLH-3, binds to Snail-binding sites/E-boxes in a cis-regulatory region of the egl-1 locus in vitro that is required for the death of the NSM sister cells in vivo. Hence, we propose that HLH-2/HLH-3 is a direct, cell-type specific activator of egl-1 transcription. Furthermore, the Snail-like CES-1 protein can block the death of the NSM sister cells by acting through the same Snail-binding sites/E-boxes in the egl-1 locus. In ces-1(gf) animals, CES-1 might therefore prevent the death of the NSM sister cells by successfully competing with HLH-2/HLH-3 for binding to the egl-1 locus.
2768	Opposite contribution of two ligand-selective determinants in the N-terminal hormone-binding exodomain of human gonadotropin receptors.	12869592	The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the human LH/chorionic gonadotropin receptor (hLH-R) harbor molecular determinants that allow the mutually exclusive binding of human FSH (hFSH) and human LH (hLH)/human chorionic gonadotropin (hCG) when these hormones are present in physiological concentrations. Previously, we have shown that the beta-strands of hLH-R leucine-rich repeats 3 and 6 can confer full hCG/hLH responsiveness and binding when simultaneously introduced into a hFSH-R background without affecting the receptor's responsiveness to hFSH. In the present study, we have determined the nature of contribution of each of these two beta-strands in conferring hCG/hLH responsiveness to this mutant hFSH-R. Human LH-R beta-strand 3 appeared to function as a positive hCG/hLH determinant by increasing the hCG/hLH responsiveness of the hFSH-R. In contrast, mutagenesis of hFSH-R beta-strand 6, rather than the introduction of its corresponding hLH-R beta-strand, appeared to allow the interaction of hCG/hLH with the hFSH-R. Hence, hFSH-R beta-strand 6 functions as a negative determinant and, as such, restrains binding of hCG/hLH to the hFSH-R. Detailed mutagenic analysis revealed that the ability of the hFSH-R to interact with hCG/hLH depends primarily on the identity of two amino acids (Asn104, a positive LH-R determinant, and Lys179 a negative FSH-R determinant) that are situated on the C-terminal ends of beta-strands 3 and 6, respectively.
2769	Control of erythroid cell production via caspase-mediated cleavage of transcription factor SCL/Tal-1.	12867998	SCL/Tal-1 is a helix-loop-helix (HLH) transcription factor required for blood cell development, whose abnormal expression is responsible for induction of T-cell acute lymphoblastic leukemia. We show here that SCL/Tal-1 is a key target of caspases in developing erythroblasts. SCL/Tal-1 degradation occurred rapidly after caspase activation and preceded the cleavage of the major erythroid transcription factor GATA-1. Expression of a caspase-resistant SCL/Tal-1 in erythroid progenitors was able to prevent amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis induced by growth factor deprivation or death receptor triggering. The potent proerythropoietic activity of uncleavable SCL/Tal-1 was clearly evident in the absence of erythropoietin, a condition that did not allow survival of normal erythroid cells or expansion of erythroblasts expressing caspase-resistant GATA-1. In the absence of erythropoietin, cells expressing caspase-resistant SCL/Tal-1 maintain high levels of Bcl-X(L), which inhibits amplification of the caspase cascade and mediates protection from apoptosis. Thus, SCL/TAL-1 is a survival factor for erythroid cells, whereas caspase-mediated cleavage of SCL/Tal-1 results in amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis.
2770	The Ala45Thr polymorphism of BETA2/NeuroD1 gene and susceptibility to type 2 diabetes mellitus in a Polish population.	12861411	The BETA2/NeuroD1 gene product is a transcription factor, a member of a helix-loop-helix (HLH) family that is specifically expressed in the endocrine pancreas. HLH and homeobox proteins are involved in the development and function of pancreatic islets cells. Mice homozygous for a targeted disruption of BETA2/NeuroD1 showed abnormal pancreatic islet morphogenesis and developed overt diabetes. Mutations in the NeuroD/BETA2 gene were linked to the development of type 2 diabetes (T2DM). The aims of the study were to determine the allele and genotype frequency of Ala45Thr polymorphism of BETA2/NeuroD1 in a Polish population and to examine the role of this amino acid variant in the genetic susceptibility to T2DM. We included 394 individuals into this study: 223 T2DM patients with the age at diagnosis above 35 years and 171 controls without a family history of T2DM. The fragment of the gene, corresponding to the Ala45Thr amino acid variant, was amplified by polymerase chain reaction. Alleles and genotypes were determined based on electrophoresis of the specific restriction enzyme EcoI57 DNA digestion products. Differences in distribution between the groups were examined by chi(2) test. The frequencies of the Ala and Thr alleles in T2DM patients (62% and 37.9%) were similar to those in the controls (65.5% and 34.5%; p=0.32). Similarly, there was no difference between the groups when we analyzed the genotype distribution (p=0.24). The stratification analysis based on family history of T2DM, obesity, and age of diagnosis did not show any difference between the groups. In conclusion, the frequency of Ala45Thr polymorphism in this studied Polish population is similar to its frequency in other Caucasians. We did not find evidence that the Ala45Thr polymorphism of BETA2/NeuroD1 played a role in the risk of T2DM in the examined Polish population.
2771	Dynamic gene expression during the onset of myoblast differentiation in vitro.	12837262	Skeletal myogenesis is a well-studied differentiation process. However, despite the identification and functional characterization of the myogenic basic HLH transcription factors, molecular details are still lacking. With the advent of microarray technology, it has become possible to look at changes in gene expression profiles in a biological process on an unprecedented scale. In this study, we applied this technology to profile gene expression during the in vitro differentiation of an established myoblast cell line, C2C12. We report over 1500 genes whose expression is altered when these cells differentiate, including 624, or about 40% of the total number of genes, with unknown functions. This analysis reveals the existence of 12 groups of coordinately regulated genes that are expressed in temporal waves of gene expression prior to the transcriptional induction of myogenin. Among these are multiple families of transcription factors that are important for the process of myogenesis. In addition, the induction of the Notch signaling pathway suggests that previously unappreciated intercellular signaling occurs during myogenic differentiation. These results provide a molecular description of the skeletal myogenesis up to the activation of myogenin.
2772	Treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) in young adults: a report from the HLH study center.	12825212	Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH), also known as EBV-associated hemophagocytic syndrome, develops mostly in children and young adults and may be fatal. Early etoposide treatment has been confirmed to be effective in children. However, it is unclear whether the same treatment is useful in adults.To assess whether etoposide is effective in treating young adult cases, we retrospectively studied the therapeutic measures taken and outcomes in 20 young adult cases of EBV-HLH. Eleven cases were registered in our HLH study center in Kyoto and nine derived from the literature. The patients were between 17 and 33 years old and eight were males. The influence of gender, cell lineage (T- or natural killer-), EBV serology pattern, jaundice and treatment on the outcome was assessed.Patients receiving etoposide within four weeks after diagnosis had a good prognosis as five of the seven patients survived compared to one of 13 not treated with etoposide or treated late (chi-square test for survival, P = 0.0095). The Kaplan-Meier analysis showed the 2.5-year survival of 85.7 +/- 13.2% in the early etoposide-treated patients, compared to 10.3 +/- 9.4% in the remaining patients (log-rank test, P = 0.0141). Thus, early etoposide treatment is effective in treating EBV-HLH in young adults as well as in children.
2773	Role for Id-1 in immunobiology of normal keratinocytes and in basal cell carcinoma.	12823438	It has been established that Id proteins can block the basic helix-loop-helix (HLH) transcription factors, thereby impacting the onset of senescence in keratinocytes, as well as influencing tumorigenesis involving squamous cell carcinomas. However, the ability of Id-1 to influence the immunologic response of epithelial cells to cytokines implicated in cutaneous oncology such as gamma interferon (IFN-gamma) has not been determined. Using a whole population of human keratinocytes infected with a retrovirus to induce over-expression of Id-1, the influence on early differentiation of rapidly proliferating keratinocytes was assessed, as was the response to IFN-gamma. While induction of involucrin, a marker of early differentiation, was not altered in Id-1 overexpressing keratinocytes, the IFN-gamma mediated increase in intercellular adhesion molecule-1 (ICAM-1) and HLA-DR was reduced. No change in constitutive or inducible levels of MHC class I antigen, CD95 (Fas antigen) or LFA-3 (CD58) was observed in this system. Immunostaining and Western blot analysis revealed over-expression of Id-1 in basal cell carcinomas (BCCs). These tumors not only strongly and diffusely expressed Id-1, but were also characterized by reduced ICAM-1 and HLA-DR expression. Thus, dysregulated Id-1 may not only contribute to delaying the senescence program in keratinocytes, it may also contribute to the escape of the relatively undifferentiated tumor cells in BCC from immune surveillance.
2774	The role of class I HLH genes in neural development--have they been overlooked?	12815726	Helix-loop-helix (HLH) genes encode for transcription factors affecting a whole variety of developmental programs, including neurogenesis. At least seven functional classes (denoted I to VII) of HLH genes exist, (1) with subclass members exhibiting homo- and heterodimerisation for proper DNA binding and transcriptional regulation of downstream target genes. In the developing nervous system, members of class II, V and VI have been most extensively studied concerning their roles in neural programming. In contrast, the function of class I proteins (such as E12 and E47) is poorly defined and the orthodox view relegates them to general dimerisation duties that are necessary for the activity of the other classes. However, closer scrutiny of the spatiotemporal expression patterns of class I factors, combined with recent biochemical evidence, would suggest that class I proteins possess specific functions during early neural differentiation. This essay supports this possibility, in addition to putting forward the hypothesis that, outside their general dimerisation activity, class I genes have independent roles in regulating neurogenesis.
2775	Id proteins are overexpressed in human oral squamous cell carcinomas.	12787042	The helix-loop-helix (HLH) proteins Id-1, Id-2 and Id-3 have been demonstrated to inhibit the activity of transcription factors and play an important role in regulating cell growth and tissue-specific differentiation.To elucidate the involvement of Id in human oral squamous cell carcinoma (OSCC), we examined 83 surgical specimens and eight normal gingival mucosae for the expression of Id proteins by immunohistochemistry; in addition, some specimens of the OSCC and the normal gingivae were also examined for the expression of Id-1 mRNA by in situ hybridization (ISH), while Western blots were performed on six of the tumours and on cell lysates of five OSCC cell lines. We also explored the correlation between Id expressions and cellular proliferation indicating Ki-67 or clinical parameters.We discovered a higher and more frequent expression of Id-1 protein (27.7%) in human OSCC compared to that of Id-2 and Id-3 proteins (6.0 and 7.2%, respectively). Western blot analysis detected Id-1 protein in four of six tumour samples and in all cell lines. ISH demonstrated strong cytoplasmic localization of Id-1 mRNA in tumour samples at significantly higher levels compared to those of normal gingival mucosae.Id-1 protein expression significantly correlates with lymph node status, indicating that increased Id expression may relate to the metastatic behaviour in human OSCC.
2776	Primary hemophagocytic lymphohistiocytosis in Turkish children.	12775534	Nineteen children with hemophagocytic lymphohistiocytosis (HLH) were studied in the Department of Pediatric Hematology, Hacettepe University. Patients were divided into two groups. Group 1: Thirteen patients were classified as having a genetic etiology (7 familial, 6 presumed familial) on the basis of an affected sibling and consanguinity. There was a history of consanguineous marriage in 13 of the families. Seven of them had a history of a sibling with HLH. Group 2: Six patients were diagnosed with sporadic HLH. The age at presentation for familial patients was 0.7-84 months (mean 21.9 +/- 24.9 months), and for sporadic cases it was 2.5-48 months (mean 22.7 +/- 19.8 months). The clinical and laboratory data of these two groups were similar at diagnosis. Thirteen cases were diagnosed premortem by bone marrow aspiration. Splenic biopsy was performed in 2 patients. Four patients were diagnosed by postmortem examination. Elevated LDH levels were found in all patients tested. No significant differences for clinical and laboratory data were found between the two groups.
2777	Novel genes regulated by gonadotropins in granulosa cells: new perspectives on their physiological functions.	12770742	Follicular stimulating hormone (FSH) is a key hormone secreted from the pituitary, which controls the development of the follicle-enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, yet the entire spectrum of genes which are regulated by FSH are not fully characterized. We have established rat and human FSH responsive granulosa cell lines, which express FSH receptors at 20-times higher rates compared to primary cells. Since the lines are monoclonal, they are expected to have a homogeneous composition of RNA among the entire cell population, which increases the probability of yielding a distinct view of genes modulated by FSH eliminating the possibility of other cell types contamination. Using Affymetrix DNA microarrays to uncover novel FSH-regulated genes, we discovered genes not reported earlier to be regulated by FSH. These include genes coding for (1) proteases; (2) growth factors and cytokines; (3) proteins involved in intercellular communication and connection with the nervous system; (4) protein phosphatases and kinases; (5) anti oxidants and anti-toxicants; (6) G-coupled proteins. These findings can deepen our understanding in the mechanism of FSH action in stimulation of the development of the ovarian follicular cells, in the modulation of ovarian intracellular and intercellular communication and in the process of selection of the dominant follicle. When human granulosa cells, obtained from in vitro fertilization patients were exposed to either hLH- or hFSH stimulation and mRNAs of these cells were analyzed by DNA microarrays, novel genes, similar to those found modulated by FSH in FSH responsive cell lines, were discovered in the human primary cells. This suggests that the immortalized cell systems established in our laboratory could serve as a useful system expanding the spectrum of authentic genes modulated by gonadotropin stimulation in normal ovarian function and in ovarian malfunction.
2778	Imaging characteristics of hemophagocytic lymphohistiocytosis.	1276825517576549	Hemophagocytic lymphohistiocytosis (HLH) is a nonmalignant disorder of immune regulation, with overproduction of cytokines and diminished immune surveillance. Symptoms are nonspecific and may affect multiple organs, including the central nervous system. Neuroimaging findings have been described in case reports and small series; body imaging findings have not been described extensively. OBJECTIVE. To summarize findings of the most frequently performed imaging studies of the brain, chest and abdomen in patients with HLH.Retrospective review of chest radiographs and CT, abdominal ultrasound and CT, brain CT and MRI, skeletal surveys, and autopsy data.Twenty-five patients were diagnosed and treated for HLH at our institution over an 11-year period; 15 patients (60%) died. Common chest radiograph findings included alveolar-interstitial opacities with pleural effusions, often with rapid evolution and resolution. Hepatosplenomegaly, gallbladder wall thickening, hyperechoic kidneys and ascites were common abdominal findings, which resolved after therapy in some cases. Brain-imaging studies revealed nonspecific periventricular white-matter abnormalities, brain-volume loss and enlargement of extra-axial fluid spaces. Three infant cases, one with intracranial hemorrhage, one with multiple pathologic rib fractures and one with diaphyseal periosteal reaction involving multiple long bones on skeletal survey, raised suspicion of child abuse at presentation. Abuse was not substantiated in any case.Clinicians and radiologists should be aware of the radiographic manifestations of HLH, which are nonspecific and overlap with infectious, inflammatory and neoplastic disorders. Findings in the chest (similar to acute respiratory distress syndrome) and abdomen may progress rapidly and then regress with institution of appropriate anti-HLH therapy. CNS findings may be progressive. In some infants, initial imaging findings may mimic nonaccidental trauma.
2779	Rapid LightCycler assay for identification of the perforin codon 374 Trp --> stop mutation in patients and families with hemophagocytic lymphohistiocytosis (HLH).	12764739	Recently, point mutations in the Perforin gene on chromosome 10q21 have been described to be the cause of hemophagocytic lymphohistiocytosis (HLH) in a subset of patients. Small deletions, missense, or nonsense mutations were found in both coding exons of the gene. One mutation was found apparently independently in different families of Turkish origin. This Trp374stop mutation is located within a cystein-rich epidermal growth factor (EGF) precursor type domain in exon 3 of the Perforin gene.To detect this mutation, we developed a rapid "fluorescence resonance energy transfer" (FRET) assay on the LightCycler based on the different melting behavior of mutated and wild-type sequences.In seven out of 20 analyzed patients with defined HLH history, this Trp374stop mutation was detectable within the different genotypes. Additionally, one out of 90 alleles tested in 45 healthy Turkish controls had this mutation.We present a rapid and reliable test for the most common Perforin (Trp374stop) mutation in HLH suitable for genetic testing and prenatal diagnosis. The mutation was detected in a large proportion of Turkish patients. Furthermore, a rapid and precise diagnosis of HLH will shorten the initiation of therapy in this subset of patients.
2780	TFIIB and subunits of the SAGA complex are involved in transcriptional activation of phospholipid biosynthetic genes by the regulatory protein Ino2 in the yeast Saccharomyces cerevisiae.	12753200	In the yeast Saccharomyces cerevisiae, genes involved in phospholipid biosynthesis are activated by ICRE (inositol/choline-responsive element) up-stream motifs and the corresponding heterodimeric binding factor, Ino2 + Ino4. Both Ino2 and Ino4 contain basic helix-loop-helix (bHLH) domains required for ICRE binding, whereas transcriptional activation is mediated exclusively by Ino2. In this work, we describe a molecular analysis of functional minimal domains responsible for specific DNA recognition and transcriptional activation (TAD1 and TAD2). We also define the importance of individual amino acids within the more important activation domain TAD1. Random mutagenesis at five amino acid positions showed the importance of acidic as well as hydrophobic residues within this minimal TAD. We also investigated the contribution of known general transcription factors and co-activators for Ino2-dependent gene activation. Although an ada5 single mutant and a gal11 paf1 double mutant were severely affected, a partial reduction in activation was found for gcn5 and srb2. Ino2 interacts physically with the basal transcription factor Sua7 (TFIIB of yeast). Interestingly, interaction is mediated by the HLH dimerization domain of Ino2 and by two non-overlapping domains within Sua7. Thus, Sua7 may compete with Ino4 for binding to the Ino2 activator, creating the possibility of positive and negative influence of Sua7 on ICRE-dependent gene expression.
2781	Comparison of recombinant human luteinising hormone (r-hLH) and human menopausal gonadotropin (hMG) in assisted reproductive technology.	12740151	Follicle-stimulating hormone (FSH) and luteinising hormone (LH) act in concert in the stimulation of folliculogenesis and ovulation. However, high levels of LH promote follicular atresia and early miscarriage, and this has led to the concept of a 'therapeutic window' of LH for successful conception in assisted reproductive technology (ART) and ovulation induction. Until now, urinary-derived human menopausal gonadotropin (hMG) has been the only available source of exogenous LH activity. hMG preparations contain highly variable levels of LH, and are often augmented with human chorionic gonadotropin (hCG), which mimics LH activity. Accumulation of hCG bioactivity, however, may have detrimental effects on follicular development and oocyte quality. Recombinant human LH (r-hLH) (Luveris) is the only pure source of LH activity. r-hLH is well characterised and production is tightly controlled, resulting in a highly consistent product. Clinical studies in hypogonadotropic hypogonadal women have demonstrated the efficacy of r-hLH, 75 IU/day, together with r-hFSH, 150 IU/day, in promoting optimal follicular development, oestrogen secretion and endometrial thickness. r-hLH therefore provides the clinician with the opportunity for precise and consistent dosing within the therapeutic window for patients requiring exogenous LH, without the risk of LH overexposure that is associated with hCG.
2782	Hemophagocytic lymphohistiocytosis masquerading as child abuse: presentation of three cases and review of central nervous system findings in hemophagocytic lymphohistiocytosis.	127281231546612618004555	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease resulting from abnormal proliferation of histiocytes in tissues and organs. Although the disease generally presents with systemic symptoms such as pancytopenia, coagulopathy, and organomegaly, HLH may also present with central nervous system (CNS) manifestations. CNS events can range from irritability to encephalopathy and coma. Retinal and intracranial hemorrhages are among the neuropathologic findings in these children. Patients who present with CNS findings may have symptoms that mimic those of inflicted injury. These children are at risk, therefore, for misdiagnosis as victims of child abuse. Such an error causes not only unnecessary additional trauma to the family but also, more important, a delay in initiating effective therapy. We present 3 cases of children with HLH who initially came to medical attention with neurologic findings, all suspected to be victims of child abuse. Subsequent laboratory evaluations, however, were consistent with the diagnosis of HLH. No additional evidence of child abuse was obtained, and the charges eventually were dropped. Two of the 3 children died from their disease shortly after presentation; the third is surviving with no evidence of HLH several months after allogeneic bone marrow transplantation. Although the diagnosis of child abuse certainly is all too common, clinicians need to be diligent and informed to avoid assigning this label erroneously. Several laboratory findings of HLH may alert physicians to the possibility of this diagnosis. The timely diagnosis of and institution of therapy for HLH may reduce ultimate morbidity and mortality.
2783	Acute inflammatory demyelinating polyradiculoneuropathy associated with perforin-deficient familial haemophagocytic lymphohistiocytosis.	12725560	This study reports the first paediatric case of acute inflammatory demyelinating polyradiculoneuropathy (AIDP) associated with a fatal haemophagocytic lymphohistiocytosis (HLH). The patient developed progressive weakness of the lower limbs in the context of a picture of infectious mononucleosis and Epstein-Barr virus (EBV) infection. After an apparent improvement, a fulminant hepatic failure and pancytopenia ensued, leading to death. Molecular genetic studies documented a compound heterozygosity for two mutations in the perforin (PRF1) gene as the background defect for a familial haemophagocytic lymphohistiocytosis (FHL).In this patient EBV infection triggered both AIDP and FHL. The latter condition was due to PRF1 deficiency. Two novel mutations in the PRF1 gene were concomitantly present in the patient. The first caused an amino acid change, while the second introduced a stop codon in the sequence which resulted in a truncated protein.
2784	Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan.	12716377	Perforin gene (PRF1) mutations appear to occur in about 30% of patients with haemophagocytic lymphohistiocytosis (HLH). We tested perforin expression and gene mutations in 14 HLH patients and six patients with Epstein-Barr virus-associated HLH (EBV-HLH) in Japan. Five of the 14 HLH patients had perforin abnormalities. The presence of PRF1 genetic abnormality correlated well with the lack of perforin expression as determined by flow cytometry. Sequencing showed that four patients had a compound heterozygous mutation while the fifth patient had a homozygous mutation. Three of the mutations we detected were novel. In contrast, none of the six EBV-HLH patients showed perforin abnormalities. Our data, combined with the PRF1 mutations in three previously reported Japanese patients, suggest that the 1090-1091delCT and 207delC mutations of the perforin gene are frequently present in Japanese HLH patients (62.5% and 37.5% respectively). Examination of the geographical origins of the ancestors in the perforin-mutant HLH patients revealed that they mostly came from the Western part of Japan, suggesting that the present-day cases may largely derive from a common ancestor.
2785	Epstein-Barr virus-associated T-/natural killer cell lymphoproliferative diseases.	12704589	Epstein-Barr virus (EBV) is a ubiquitous virus that infects the majority of the world population by adulthood. The major target for infection is the B lymphocyte, and acute infection causes vigorous EBV-specific killer T-cell responses exemplified clinically by acute infectious mononucleosis (IM). EBV infection usually persists latently life-long without eliciting any clinical symptoms. Rarely, active EBV infection is prolonged, with abnormal expansions of EBV-infected T or NK cells, conditions collectively defined here as EBV-associated T/NK lymphoproliferative diseases. Hemophagocytic lymphohistiocytosis (HLH), chronic active EBV infection (CAEBV), NK lymphoma/leukemia, and T-cell lymphoma are entities included in this category. Hypersensitivity to mosquito bite (HMB) represents a unique syndrome characterized by expansion of EBV-infected NK cells in the peripheral circulation and within the inflammatory skin lesions induced by mosquito bites. Target cell specificity, defects in host immune responses, and strain differences of EBV may account for ectopic EBV infections and for the unique clinical presentations characteristic of each illness.
2786	BIR-1, a Caenorhabditis elegans homologue of Survivin, regulates transcription and development.	12682297	bir-1, a Caenorhabditis elegans inhibitor-of-apoptosis gene homologous to Survivin is organized in an operon with the transcription cofactor C. elegans SKIP (skp-1). Because genes arranged in operons are frequently linked functionally, we have asked whether BIR-1 also functions in transcription. bir-1 inhibition resulted in multiple developmental defects that overlapped with C. elegans SKIP loss-of-function phenotypes: retention of eggs, dumpy, movement defects, and lethality. bir-1 RNA-mediated interference decreased expression of several gfp transgenes and the endogenous genes dpy-7 and hlh-1. Immunoblot analysis revealed decreased phosphoacetylated histones in bir-1 RNA-mediated interference-treated worms. In a heterologous transfection system, BIR-1 augments thyroid hormone-regulated transcription and has an additive effect with SKIP. These results show that BIR-1 functions in the regulation of transcription and development.
2787	Engraftment and dissemination of T lymphocytes from primary haemophagocytic lymphohistiocytosis in scid mice.	12694259	Although primary haemophagocytic lymphohistiocytosis (HLH) is a genetic disorder of T lymphocytes, it remains unclear why T lymphocytes of primary HLH patients preferentially infiltrate the central nervous system and peripheral blood, in addition to the reticuloendothelial systems. We engrafted Herpesvirus saimiri (HVS)-immortalized T-lymphocyte lines established from primary HLH patients into severe combined immunodeficient (scid) mice and examined their capacity to infiltrate mouse organs. A diffuse infiltration of human T lymphocytes was detected in each organ of scid mice treated with 1 x 10(6) T lymphocytes from all four primary HLH patients assessed, whereas no infiltration of T lymphocytes from healthy individuals was observed in any organ. The infiltration of T lymphocytes was mainly observed in the lung, brain and peripheral blood, in association with haemophagocytosis. These cells were positive for HLA-DR, CD3 and either CD8 or CD4, but negative for CD68. Certain markers of proliferation and apoptotic activities were highly positive in these cells. There was no difference between the infiltration pattern of T lymphocytes of primary HLH patients with a perforin deficiency and those without. By Southern blot analysis, T lymphocytes infiltrating mouse organs were observed to be polyclonal. These findings suggest that our murine model implementing HVS-immortalized human T lymphocytes is suitable to clarify the pathogenesis of primary HLH.
2788	Id proteins in epithelial cells.	12681293	Id helix-loop-helix (Id HLH) proteins are negative regulators of basic HLH transcription factors. They are expressed during embryonic development and are important for the regulation of cell phenotypes in adults. They participate in the molecular networks controlling cell growth, differentiation, and carcinogenesis, through specific basic HLH and non-basic HLH protein interactions. Recent in vitro and in vivo data implicate Id HLH as important orchestrating proteins of homeostasis in glandular and protective epithelia. In particular, Id proteins have been reported to be involved in cell behavior in epidermis, respiratory system, digestive tract, pancreas, liver, thyroid, urinary system, prostate, testis, endometrium, cervix, ovary, and mammary gland. The purpose of this review is to summarize the evidence implicating Id proteins in the regulation of mammalian epithelial cell phenotypes.
2789	Smooth muscle alpha-actin gene requires two E-boxes for proper expression in vivo and is a target of class I basic helix-loop-helix proteins.	12663487	Changes in the differentiated state of smooth muscle cells (SMCs) play a key role in vascular diseases, yet the mechanisms controlling SMC differentiation are still largely undefined. We addressed the role of basic helix-loop-helix (bHLH) proteins in SMC differentiation by first determining the role of two E-box (CAnnTG) motifs, binding sites for bHLH proteins, in the transcriptional regulation of the SMC differentiation marker gene, smooth muscle alpha-actin (SM alpha-actin), in vivo. Mutation of one or both E-boxes significantly reduced the expression of a -2560- to 2784-bp SM alpha-actin promoter/LacZ reporter gene in vivo in transgenic mice. We then determined the potential role of class I bHLH proteins, E12, E47, HEB, and E2-2, in SM alpha-actin regulation. In cotransfection experiments, E12, HEB, and E2-2 activated the SM alpha-actin promoter. Activation by HEB and E2-2 was synergistic with serum response factor. Additionally, the dominant-negative/inhibitory HLH proteins, Id2, Id3, and Twist, inhibited both the E12 and serum response factor-induced activations of the SM alpha-actin promoter. Finally, we demonstrated that E2A proteins (E12/E47) specifically bound the E-box-containing region of the SM alpha-actin promoter in vivo in the context of intact chromatin in SMCs. Taken together, these results provide the first evidence of E-box-dependent regulation of a SMC differentiation marker gene in vivo in transgenic mice. Moreover, they demonstrate a potential role for class I bHLH factors and their inhibitors, Id and Twist, in SM alpha-actin regulation and suggest that these factors may play an important role in control of SMC differentiation and phenotypic modulation.
2790	A rare syndrome in the differential diagnosis of hepatosplenomegaly and pancytopenia: report of identical twins with Griscelli disease.	12648328	White, identical twin boys aged 3 months were referred to our centre with persisting fever, mouth ulcers, hepatosplenomegaly, pancytopenia and failure to thrive. The parents were first cousins and there was a history of a sibling with similar manifestations who had died. The infants had silvery-grey hair and pigment clumps on the hair shafts, and skin biopsy showed accumulation of melanocytes on melanosomes. Bone marrow revealed hypercellularity and haemophagocytosis. HLH-94 chemotherapy (initial therapy with daily dexamethasone and etoposide, maintenance with dexamethasone pulses, etoposide and cyclosporin A) was started. Though partial haematological remission was achieved, one of the boys died on the 34th day following aspiration pneumonia. No pathogen could be identified. The second boy responded to therapy but had a haematological relapse and died 68 days after first being admitted. Genetic study revealed a 5 bp deletion in the RAB27A gene (510 del AAGCC in exon 5). Transient haematological remission can be achieved with chemotherapy but allogeneic bone marrow transplantation is the only curative therapy in Griscelli disease, as in other familial haemophagocytic syndromes. Identification of the mutation also provides an opportunity for prenatal diagnosis.
2791	Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency.	12626416	The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species. Whereas the structure found for the N-glycans of the gonadotrophins was in agreement with the complex type model, the structure for an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoelectrospray ion trap mass spectrometry. Using these two glycan mapping methods, the high level of batch-to-batch consistency achieved for the glycosylation of the three recombinant gonadotrophins in commercial production has been shown. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins, which is a prerequisite to the delivering of a guaranteed dose of drug from vial to vial, and in turn to ensuring the clinical efficacy of the product.
2792	A clinician's view of spinal cord injury.	12619085	The primary cause of spinal cord injury (SCI) is automobile collisions, followed by violence, falls, and injuries in sporting events. The patient is most frequently a young male. Regardless of cause and age, SCI is a potentially catastrophic injury. The unique anatomical relationship of the spinal cord, being enclosed in the dural sac within the bony vertebral column, make it venerable to a wide range of traumatic insults. SCI is classified as complete or incomplete with several subclasses arranged under each of these respective headings. The probability of recovery to a functional state is usually better for patients with incomplete injuries. Treatment for SCI involves initially immobilizing the injured vertebral column, medications to prevent secondary injury, and potential surgery to release pressure on the spinal cord and restore stability to the vertebral column. Postsurgical care is directed toward prevention and treatment of secondary complications of SCI such as respiratory failure, deep venous thrombosis, and decubitus ulcers. Advances in these areas are providing patients with a greater probability of recovery, a longer life, and a better quality of life. Research in the clinical and basic sciences is opening new avenues of hope for the spinal cord injury patient.
2793	Novel perforin mutation in a patient with hemophagocytic lymphohistiocytosis and CD45 abnormal splicing.	12599189	Hemophagocytic lymphohistiocytosis (HLH) composes a group of rare heterogenous disorders characterized by uncontrolled accumulation and infiltration of activated T lymphocytes and macrophages. Cytotoxic T and natural killer cell activity is significantly reduced or absent in these patients. Mutations in the important mediator of lymphocyte cytotoxicity perforin were identified in a number of HLH individuals. Here we report a novel missense mutation thr435met in the conserved Ca(2+) binding domain of perforin in a patient with HLH. Prediction of the 3-dimensional structure of the thr435met perforin mutant using comparative molecular modeling indicates that the protein's ability to bind Ca(2+), and therefore its cytolytic function, would be strongly compromised. In addition, this patient exhibited abnormal CD45 splicing caused by a C77G mutation in the gene encoding CD45 (PTPRC). Our findings suggest a combined role for perforin mutation and abnormal CD45 splicing as significant contributory factors in the pathogenesis of HLH.
2794	Ligand selectivity of gonadotropin receptors. Role of the beta-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor.	12598521	The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their approximately 350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeat-containing subdomain (LRR subdomain) flanked by N- and C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel beta-strands. Receptors in which individual beta-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R beta-strands 3 and 6. A mutant receptor in which the hFSH-R beta-strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most beta-strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.
2795	Improving the thermodynamic stability of the leucine zipper of max increases the stability of its b-HLH-LZ:E-box complex.	12595267	Max is a member of the b-HLH-LZ (basic region-helix1-loop-helix2-leucine zipper) family of eukaryotic transcription factors. It is the obligate partner of the related b-HLH-LZ proteins, c-Myc and Mad1, with which it forms heterodimers on target DNA. While c-Myc and Mad1 require Max for DNA-binding, Max itself can form a homodimer that recognizes E-box DNA sequences (CACGTG) in gene promoters that are targeted by c-Myc. Evidence suggests that this mode of binding by Max may repress c-Myc transcriptional activity, and this may have applications in the control of the aberrant activity of c-Myc during certain oncogenic transformations. To enhance this repressive potential of Max, we sought to stabilize Max homodimers. We have designed a double mutant (N78V/H81L) located in the coiled-coil interface of the leucine zipper domain and we demonstrate that these mutations do indeed increase the stability of the protein. The mutations also improve the stability of the complex with cognate DNA. Thermal denaturations monitored by circular dichroism reveal two transitions that are due to intermediate folding states for both the wild-type and mutant proteins; this is supported by detailed thermodynamic analyses. A formalism to characterize the temperature-dependence of the unfolding, including the effect of intermediates, is presented.
2796	STAT5-induced Id-1 transcription involves recruitment of HDAC1 and deacetylation of C/EBPbeta.	12574125	Transcriptional activation is associated commonly with recruitment of histone acetylases, while repression involves histone deacetylases (HDACs). Here, we provide evidence to suggest that STAT5 activates gene expression by recruiting HDAC. The interleukin-3 (IL-3)-dependent expression of the Id-1 gene, encoding a helix-loop-helix (HLH) transcriptional inhibitor, is activated by both C/EBPbeta and STAT5 transcription factors bound to its pro-B-cell enhancer (PBE), but is inhibited by HDAC inhibitors in Ba/F3 cells. STAT5 interacts with HDAC1 in the PBE region, resulting in deacetylation of histones, as well as C/EBPbeta, whose acetylation diminishes its DNA-binding activity. Consistently, expression of an acetylation-resistant mutant of C/EBPbeta results in IL-3-independent expression of the Id-1 gene. Thus, we propose a novel mechanism by which STAT5 mediates the deacetylation of C/EBPbeta, allowing transcriptional activation.
2797	Mitotic G2-arrest is required for neural cell fate determination in Drosophila.	12559497	In the wing discs of Drosophila, the mecanosensory precursor cells are singled out from clusters of cells blocked at the G2 phase of the cell cycle. This mitotic quiescence and the selection of the precursors are under strict spatio-temporal control. We forced G2 cells to enter mitosis by overexpression of string, the Drosophila homologue of the cdc25 gene. Premature entrance in the cell cycle is associated to a loss of precursor cells. Precursors are lost consecutively to a transcriptional down-regulation of the determinant proneural achaete/scute genes. This down-regulation results from an over-activation of the Enhancer of Split genes, known as effectors of the Notch signalling pathway. We conclude that exit from the cell cycle is required for proper neural cell fate determination.
2798	Successful engraftment and stable full donor chimerism after myeloablation with thiotepa, fludarabine, and melphalan and CD34-selected peripheral allogeneic stem cell transplantation in hemophagocytic lymphohistiocytosis.	12555220	Allogeneic hematopoietic stem cell transplantation (HSCT) represents the only curative option for primary hemophagocytic lymphohistiocytosis (HLH), a rare disease of infants and young children, characterized by recurrent fever, hepatosplenomegaly, and cytopenia. We report a case of successful engraftment and stable full-donor chimerism in a patient with HLH who underwent peripheral allogeneic CD34-selected HSCT. The donor was his 1-antigen-HLA-mismatched grandmother. After a conditioning regimen based on the combination of thiotepa, fludarabine, melphalan, and rabbit antilymphocyte serum, the patient received a megadose of 26.3 x 10(6)/kg of CD34(+) peripheral blood cells. Neutrophil (>0.5 x 10(9)/L) and platelet (>50 x 10(9)/L) engraftment was observed on days +16 and +12, respectively, and the patient was discharged home on day +24. No acute or chronic GVHD was observed. Infectious complications were the main causes of re-hospitalization in the first year after transplantation, but no significant morbidity was observed thereafter. Thirty-two months after HSCT, the patient is alive and well, still in complete clinical remission of his underlying disease with a durable engraftment, normal NK activity and full donor chimerism. This case suggests that a fludarabine-based conditioning regimen and CD34-selected peripheral allogeneic HSCT may be a feasible option in case of unavailability of a fully HLA-matched related or unrelated donor.
2799	Identification of differentially expressed genes in classical and atypical multidrug-resistant gastric carcinoma cells.	12530067	The phenomenon of multidrug resistance (MDR) in human cancers is the major cause of failure of chemotherapy. To better understand the molecular events associated with the development of different types of MDR, a classical MDR P-glycoprotein expressing gastric carcinoma cell line and an atypical MDR P-glycoprotein-negative variant were analyzed by cDNA array hybridization. Of 588 cDNAs spotted on the array, a total of 9 genes showed differences in mRNA expressions. An enhanced expression level of 3 genes could be detected in both different MDR models (HLH, IR21, CCT5, Tx P-1), 4 genes were overexpressed solely in classical MDR cells (Hsp27, Rcl, aldehyde dehydrogenase 1, vimentin), whereas the expression of one gene was increased in atypical MDR cells (ProTa). Furthermore, the mRNA expressions of 3 genes were down-regulated in atypical MDR cells (Hsp27, vimentin, JNK2), whereas a decrease in mRNA expression in classical MDR cells could be determined for none of them. These differences were confirmed by a semiquantitative RT-PCR approach. Further characterization of these factors may provide more insight into the biology and development of different types of MDR in human gastric carcinomas. Moreover, these genes may be potential candidate factors for the diagnostics and/or prognosis of clinical drug resistance.
2800	Inhibition of BETA2/NeuroD by Id2.	12526101	Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.
2801	Natural TWIST protein variants in a panel of eleven non-human primates: possible implications of TWIST gene-tree for primate species tree.	12523351	The twist gene is implied in head morphogenesis, as human patients heterozygous at TWIST and heterozygous M-twist mutant mice present similar cranial-facial abnormalities. M-twist and TWIST are respectively unique genes, coding for a B-HLH transcription factor. We identified twist coding sequences from 11 species representing 7 families of primates, report their conservation and genus-specific amino acid substitutions, and present a tentative gene-tree of these sequences. Amino acid changes result in natural Twist variants, which might contribute to generating distinct head morphologies in species. These data suggest twist as a molecular marker, which could be used to refine controversial classification.
2802	Apoptosis. Life and death decisions.	1252223912522243	NaN
2803	Molecular recognition in helix-loop-helix and helix-loop-helix-leucine zipper domains. Design of repertoires and selection of high affinity ligands for natural proteins.	12514181	Helix-loop-helix (HLH) and helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that mediate selective pairing among members of a large transcription factor family involved in cell fate determination. To investigate the molecular rules underlying recognition specificity and to isolate molecules interfering with cell proliferation and differentiation control, we assembled two molecular repertoires obtained by directed randomization of the binding surface in these two domains. For this strategy we selected the Heb HLH and Max Zip regions as molecular scaffolds for the randomization process and displayed the two resulting molecular repertoires on lambda phage capsids. By affinity selection, many domains were isolated that bound to the proteins Mad, Rox, MyoD, and Id2 with different levels of affinity. Although several residues along an extended surface within each domain appeared to contribute to dimerization, some key residues critically involved in molecular recognition could be identified. Furthermore, a number of charged residues appeared to act as switch points facilitating partner exchange. By successfully selecting ligands for four of four HLH or HLHZip proteins, we have shown that the repertoires assembled are rather general and possibly contain elements that bind with sufficient affinity to any natural HLH or HLHZip molecule. Thus they represent a valuable source of ligands that could be used as reagents for molecular dissection of functional regulatory pathways.
2804	Neuronal injury affects expression of helix-loop-helix transcription factors.	12499834	Helix-loop-helix transcription (HLH) factors regulate several stages of neuronal development including differentiation of individual populations of neurons and neurite growth. Here we demonstrate that axonal injury of corticospinal and dorsal root ganglion neurons induces changes in the expression of several HLH transcription factors that function as negative regulators of neurogenesis and neurite outgrowth. However, the expression of HLH transcription factors that stimulate neurogenesis is not affected by the axonal injury. Expression of HES, SHARP and Id family members is suppressed shortly after axonal injury and expression returns to normal levels after 14 days. We hypothesize that down-regulation of these HLH transcription factors is required for initiation of regenerative response to axonal injury.
2805	Id-1 expression defines a subset of vimentin/S-100beta-positive, GFAP-negative astrocytes in the adult rat pineal gland.	12495223	Id proteins are dominant negative members of the helix-loop-helix (HLH) transcription factor family which are involved in the differentation of many cell types, including glia. We have recently identified the adult rat pineal gland as a major site of Id-1 and Id-3 expression. In the present study, double fluorescence immunocytochemical analysis was used to examine the co-localization of Id-1 and Id-3 with both neuronal (synaptophysin, betaIII-tubulin) and astrocytic markers (GFAP, vimentin, S-100beta) in the rat pineal. In addition to localizing Id-1 and Id-3 protein to the melatonin-producing pinealocytes, we have also made the novel observation that Id-1, but not Id-3, is highly expressed in a population of vimentin-positive/S-100beta-positive/GFAP-negative astrocytes. Surprisingly, Id-1 was primarily cytoplasmic in these cells, and expression extended throughout the cellular processes. The pineal has been recognized previously as a unique region of the central nervous system in which a vimentin-positive/GFAP-negative glial phenotype is maintained in adult mammals. The exclusion of Id-1 from GFAP-positive cells, and expression in a population of vimentin-positive pineal astrocytes is evidence of a role for Id-1 in the adult stabilization of one form of astrocyte. These results identify the rat pineal gland as a model system for the functional analysis of Id-1.
2806	[Ovarian stimulation: is exogenous LH necessary in all patients?].	12476696	FSH and LH play an essential but different role in the growth of ovarian follicles during the cycle. In stimulation protocols, good follicular development is obtained in most patients treated with FSH alone whereas the role of LH is more complex and controversial. Clinical and pre-clinical studies have shown that optimal follicular development is obtain if (i) exposure to endogenous and/or exogenous LH is sufficient ("threshold" concept) and (ii) exposure to LH is not excessive ("ceiling" concept). The recombinant luteinizing hormone (r-hLH, Luveris) is the only available stand-alone preparation of LH. Its characteristics are a high specific activity, the absence of undesirable proteins and an excellent batch to batch consistency. Luveris is indicated in association with FSH for stimulating follicular development in LH and FSH deficient women (defined by an endogenous LH level < 1.2 UI/l). In this subgroup of patients, the therapeutic benefit of exogenous LH at a daily dose of 75 IU is only observed when endogenous serum LH is below than 1.2 IU/l: LH threshold concept. In ART, the combination of exogenous LH at a daily dose from 75 to 150 IU and recombinant FSH improved the ovarian stimulation results only in a minority of patients (5 to 17%). On the opposite, studies conducted in OMS I and II patients showed that high doses of exogenous LH lead to atresia of secondary follicles. So, a daily dose of exogenous LH greater than 225 IU had a deleterious effect on follicular growth: LH ceiling concept.
2807	Cell type specific infection of Epstein-Barr virus (EBV) in EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection.	12467968	While Epstein-Barr virus (EBV) tropism in B cells and nasopharygeal epithelial cells in the normal host has been demonstrated, recently the role of its infection into non-B cell populations has been suggested to play a pivotal role in the pathogenesis of several EBV-related hematological as well as non-hematological diseases. Ectopic EBV infection in T cells or natural killer (NK) cells has been reported in EBV-associated hematological diseases, such as acute fulminant EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV). Recent advances in the analysis of EBV infection in lymphocyte subpopulations have clarified the differential virus-cell interaction within these EBV-related disorders. EBV infection was predominantly found in CD8(+) T-cells from EBV-HLH, and in CD4(+) T-cells or NK cells from CAEBV, while the majority of EBV infected cells were found in B cells from acute infectious mononucleosis (IM). Different virus-cell interactions between acute EBV-HLH and CAEBV have indicated different pathogenic mechanisms against EBV infection between the two EBV-associated diseases, accounting for the difference in clinical manifestations between the two diseases.
2808	Clinical features and treatment strategies of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	12467966	Epstein-Barr virus (EBV) is the major triggering factor producing hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH). In this review, diagnostic problems, clinical and histopathological features, and treatment strategies of EBV-HLH have been described. In patients with EBV-HLH, the EBV-infected T cells or natural killer (NK) cells are mostly mono- or oligo-clonally proliferating, where hypercytokinemia plays a major role and causes hemophagocytosis, cellular damage and dysfunction of various organs. Although the majority of EBV-HLH cases develop in apparently immunocompetent children and adolescents, it also occurs in association with infectious mononucleosis, chronic active EBV infection, familial HLH, X-linked lymphoproliferative disease, lymphoproliferative disease like peripheral T-cell lymphoma and NK cell leukemia. In terms of treatment, special therapeutic measures are required to control the cytokine storm generated by EBV and to suppress proliferating EBV-genome-containing cells, because the clinical courses are often fulminant and result in a poor outcome.
2809	Immunological aspects of Epstein-Barr virus infection.	12467961	Epstein-Barr virus (EBV) is a member of ubiquitous gamma herpes viruses, which primarily induces acute infectious mononucleosis (IM) or subclinical infection in susceptible subjects. The host reactions account for the clinical manifestation of IM. This virus also contributes to the development of lymphoid or epithelial malignancies. The outgrowth of EBV-infected B-cells is first controlled by interferon (IFN)-gamma and natural killer (NK) cells, and later by EBV-specific cytotoxic T-lymphocytes (CTL). To overcome the host responses and establish the persistent infection, EBV conducts the protean strategies of immune evasion. Several EBV genes modulate apoptotic signals and cytokine balances to persist B-cell infection without insulting the host. Uncontrolled lymphoproliferation occurs as EBV(+) B-cell lymphoproliferative disease (LPD)/lymphoma in AIDS, posttransplant, or primary immunodeficiency diseases (PID). On the other hand, EBV(+) T/NK cells are involved in EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) or chronic active EBV infection (CAEBV) in children having no underlying immunodeficiencies, and at times lead to the clonal evolution of T/NK-cell LPD/lymphomas. Recent advance in molecular techniques has enabled us to analyze the clonality of EBV-infected lymphocytes and to quantify the gene expression of EBV and cytokines. Dominant autocrine loop of T helper (Th) 2 and Th1 may exert in EBV(+) B-LPD and T-LPD, respectively. Intensive studies on the immunological interface between effector components and EBV(+) target cells will provide more information on clarifying the pathogenesis of EBV-associated lymphoid malignancies, as well as on exploiting the therapeutic and preventive strategies for the formidable EBV-associated disease in childhood.
2810	Administration of insulin-like growth factor I to rhesus monkeys does not augment gonadotropin-stimulated ovarian steroidogenesis.	12466377	Although it is well established that IGF-I is able to amplify the actions of FSH and LH on ovarian cells in vitro, little information is available regarding the effects of IGF-I on ovarian function in vivo. To address this question, rhesus monkeys whose spontaneous gonadotropin secretion was interrupted with a GnRH antagonist received continuous iv infusions of saline, IGF-I (240 microg/kg.d), or IGF-I (240 microg/kg.d) plus human GH (hGH) (200 microg/kg.d) 7 d before and continuing throughout a 15-d iv infusion of hFSH and hLH during which serum LH concentrations were maintained at 7-10 mIU/ml and FSH concentrations were incrementally increased every 3 d from 7.5 to 17.5 mIU/ml. Serum estradiol concentrations in saline-treated control animals did not differ (P > 0.05) from animals treated with IGF-I + hGH. In contrast, serum estradiol levels in IGF-I-treated animals were significantly less (P < 0.05) than those of control or IGF-I + hGH-treated animals. Serum androstenedione levels did not differ among the three treatment groups. Analysis of follicular fluids on the final day of gonadotropin infusion indicated that intrafollicular IGF-I concentrations paralleled serum IGF-I concentrations in all treatment groups. Measurement of the ratio of IGF-I to IGF-binding protein-3 in follicular fluids indicated that there was not a disproportionate increase in I-binding protein-3 in animals infused with either IGF-I alone or IGF-I + hGH. Concentrations of GH in follicular fluids of IGF-I treated animals were less than control animals suggesting that the diminished responsiveness of ovaries to FSH in the IGF-I treatment group may have been due to reduced GH.
2811	Two modes of recruitment of E(spl) repressors onto target genes.	12466194	The decision of ectodermal cells to adopt the sensory organ precursor fate in Drosophila is controlled by two classes of basic-helix-loop-helix transcription factors: the proneural Ac and Sc activators promote neural fate, whereas the E(spl) repressors suppress it. We show here that E(spl) proteins m7 and mgamma are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mgamma correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. Irrespective of whether E(spl) are recruited via direct DNA binding or interaction with proneural proteins, the co-repressor Groucho is always needed for target gene repression.
2812	Steroid receptor coactivator SRC-1 exhibits high expression in steroid-sensitive brain areas regulating reproductive behaviors in the quail brain.	12457041	The steroid receptor coactivator SRC-1 modulates ligand-dependent transactivation of several nuclear receptors, including the receptors for sex steroid hormones. Reducing the expression of SRC-1 by injection of specific antisense oligonucleotides markedly inhibits the effects of estrogens of the sexual differentiation of brain and behavior in rats and inhibits the activation of female sexual behavior in adult female rats. SRC-1 thus appears to be involved in both the development and activation of sexual behavior. In the Japanese quail brain, we amplified by RT-PCR a 3,411-bp fragment extending from the HLH domain to the activating domain-2 of the protein. The quail SRC-1 is closely related to the mammalian (m) SRC-1 and contains a high proportion of GC nucleotides (62.5%). Its amino acid sequence presents 70% identity with mammalian SRC-1 and contains the three conserved LXXLL boxes involved in the interaction with nuclear receptors. In both males and females, RT-PCR demonstrates a similarly high level of expression in the telencephalon, diencephalon, optic lobes, brain stem, spinal cord, pituitary, liver, kidney, adrenal gland, heart, lung, gonads and gonoducts. Males express significantly higher levels of SRC-1 in the preoptic area-hypothalamus than females. In both sexes, lower levels of expression are observed in the cerebellum and muscles. In situ hybridization utilizing a mixture of four digoxigenin-labeled oligonucleotides confirms at the cellular level the widespread distribution of SRC-1 mRNA in the brain and a particularly dense expression in steroid-sensitive areas that play a key role in the control of male sexual behavior. These data confirm the presence and describe for the first time the SRC-1 distribution in the brain of an avian species. They confirm its broad, nearly ubiquitous, distribution in the entire body including the brain as could be expected for a coactivator that regulates to the action of many nuclear receptors. However this distribution is heterogeneous in the brain and sexually differentiated in at least some areas. The very dense expression of SRC-1 in limbic and mesencephalic nuclei that are associated with the control of male sexual behavior is consistent with the notion that this coactivator plays a significant role in the activation of this behavior.
2813	Xath5 regulates neurogenesis in the Xenopus olfactory placode.	12454929	Helix-loop-helix (HLH) genes function as important regulators of neurogenesis in both the peripheral and central nervous systems. The olfactory system is an ideal tissue in which to study the role of these genes in regulating the acquisition of neuronal cell fate, particularly that of the olfactory receptor neuron (ORN). Here we describe the expression of several basic HLH (bHLH) and repeat HLH (rHLH) factors during olfactory placode development in Xenopus laevis. Our work reveals that a combination of both bHLH and rHLH genes are sequentially expressed within the nascent olfactory placode during normal development. Moreover, overexpression of the bHLH factor, Xenopus atonal homologue 5 (Xath5), promotes olfactory neural fate independent of cellular proliferation within a restricted domain at the anterior of the embryo. Collectively, our data argue that HLH genes are expressed in a cascade during olfactory placode development and that the activity of an atonal homologue, Xath5, can promote ORN fate but only in the appropriate developmental context.
2814	Notch activation during endothelial cell network formation in vitro targets the basic HLH transcription factor HESR-1 and downregulates VEGFR-2/KDR expression.	12453432	Angiogenesis is essential for normal homeostasis, wound healing, and tumor growth and involves a switch in endothelial cell (EC) phenotype from quiescence to migration, proliferation and network formation, and back to quiescence. The notch signaling pathway is critically involved in cell fate decisions during development, and mice deficient in several notch/notch ligand genes have vascular phenotypes. Here we show that notch signaling is activated during EC capillary-like network formation in vitro and that EC express transcripts for notch 1, notch 4, the notch ligand delta 4, and the putative notch processing enzymes ADAM-10 and presenilin. Expression of dominant negative notch blocks network formation; however, constitutively active notch (NICD) does not induce morphologic changes. Furthermore, both EC network formation and expression of activated notch 1 or notch 4 induce expression of the bHLH transcription factor HESR-1 and downregulate the known HESR-1 target VEGFR-2 (KDR). Notch-mediated reduction in VEGFR-2 expression results in decreased EC proliferation in response to VEGF but not bFGF. These data suggest that HESR-1 may be involved in the phenotypic changes that characterize the progression from EC proliferation and migration to network formation and quiescence.
2815	[Hemophagocytic lymphohistiocytosis as a manifestation of visceral leishmaniasis].	12422569	A 7-year-old previously healthy Czech boy was admitted due to fever, hepatosplenomegaly and pancytopenia. Aspiration of bone marrow revealed no signs of hemoblastosis (nor hemophagocytosis). He was treated with antibiotics and virostatics without effect. Progression of hepatosplenomegaly and pancytopenia induced suspicion of hemophagocytic lymphohistiocytosis (HLH). Five weeks later, bone marrow hemophagocytosis of erythrocytes, nuclear elements and platelets was detected. He was given corticoids and intravenous immunoglobulins and transferred to our haematology department. Laboratory findings of mild pancytopenia, hypofibrinogenaemia, hyperlipidaemia and elevated levels of ferritin, LDH and immunoglobulins were compatible to the diagnosis of HLH. Immunologic evaluation revealed T-lymphocyte activation. Appropriate immunosuppressive treatment with Dexamethasone, etoposide and Cyclosporine A was launched, followed by transient subside of fever and improvement of peripheral blood count, but not regression of hepatosplenomegaly. Four weeks later, relapse of fever and deterioration of blood count led to intensification of immunosuppression. However, no effect was evident. Moreover, hypertrophic cardiomyopathy with ventricular arrhythmia occurred. Treatment with antilymphocytic globulin for resistant course of HLH was planned. Before that, a fifth bone marrow aspiration was performed. Surprisingly, many Leishmania amastigotes were observed within marrow macrophages. Leishmania infection was confirmed by positive serology. Immunosuppressive treatment was withdrawn and changed for causal treatment with liposomal Amphotericin B. Positive clinical effect with subside of fever was evident in ten days, splenomegaly gradually resolved during three weeks, restoration of normal blood count lasted six weeks. No relapses of HLH nor leishmaniasis occurred. In control bone marrow aspirate performed three months later, the parasites were not detected. Ten months after the event, the patient is in complete remission of HLH with normal immunologic parameters. Most probably, he contracted visceral leishmaniasis during a visit of a Neapol area in Italy 3 months before the onset of the disease.
2816	The Caenorhabditis elegans presenilin sel-12 is required for mesodermal patterning and muscle function.	12413907	Mutations in presenilin genes impair Notch signalling and, in humans, have been implicated in the development of familial Alzheimer's disease. We show here that a reduction of the activity of the Caenorhabditis elegans presenilin sel-12 results in a late defect during sex muscle development. The morphological abnormalities and functional deficits in the sex muscles contribute to the egg-laying defects seen in sel-12 hermaphrodites and to the severely reduced mating efficiency of sel-12 males. Both defects can be rescued by expressing sel-12 from the hlh-8 promoter that is active during the development of the sex muscle-specific M lineage, but not by expressing sel-12 from late muscle-specific promoters. Both weak and strong sel-12 mutations cause defects in the sex muscles that resemble the defects we found in lin-12 hypomorphic alleles, suggesting a previously uncharacterised LIN-12 signalling event late in postembryonic mesoderm development. Together with a previous study indicating a role of lin-12 and sel-12 during the specification of the pi cell lineage required for proper vulva-uterine connection, our data suggest that the failure of sel-12 animals to lay eggs properly is caused by defects in at least two independent signalling events in different tissues during development.
2817	Unsaturated fatty acids bind Myc-Max transcription factor and inhibit Myc-Max-DNA complex formation.	12406560	Oncoprotein Myc, hetero-dimerized with Max through a b/HLH/Zip region, is a transcription factor that governs important cellular processes such as cell cycle entry, proliferation and differentiation. We found that linoleic acid, isolated from Pollen Typhae, and other unsaturated fatty acids have strong inhibitory effects on the binding of Myc-Max heterodimer to an E-box DNA site (CA(C/T)GTG). The interaction of a fatty acid with a protein dimer, not with DNA, is assumed to block the entire Myc-Max-DNA complex formation. Unsaturated fatty acids also showed cytotoxicity against a SNU16 human stomach cancer cell line and conjugated linoleic acid suppressed mRNA expression of several myc-target genes; ornithine decarboxylase, p53, cdc25a in the SNU16 cells.
2818	Down-regulation of Delta by proteolytic processing.	12403816	Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, we examined the biological activity of purified soluble Delta forms and conclude that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner.
2819	Secondary hemophagocytic lymphohistiocytosis in children: an analysis of etiology and outcome.	12403229	Fifty-two pediatric patients were diagnosed with secondary hemophagocytic lymphohistiocytosis (HLH) at the Department of Pediatrics, Siriraj Hospital between 1989 and 1998. Of these, 15 were infection-associated (IAHS), 25 were malignancy-associated (MAHS) and 12 were idiopathic HLH. Causative organisms for IAHS were Salmonella (3), Staphylococcus (2), enterobactor (2), dengue virus (3), malaria (2) and one each of Ebstein Barr virus (EBV), Serratia marcesens and Penicillium maneffei. Unlike those reported in adults and in the Western literature, 47 of 52 children in the present series were immunocompetent hosts. In addition, the proportion of MAHS was higher than expected (48.1%). Twenty-two of 25 MAHS presented with hemophagocytic syndrome and were subsequently found to have malignant diseases. Sixty per cent of MAHS (15 cases) were associated with non-Hodgkin's lymphoma (NHL), mainly T-cell. Other malignancies included acute leukemias (7) MDS (1), Langerhans cell histiocytosis (1) and histiocytic sarcoma (1). Treatment approaches were specific therapy for individuals with known causes. Supportive treatment with blood components transfusions, steroid, intravenous immunoglobulins (IVIG), and chemotherapeutic agents, mainly vinblastine and etoposides, were used in indicated cases. Of the 52 cases, 15 (28.8%) had a fatal outcome during the acute phase, and other 4 died of their subsequent malignant diseases. There was a statistically significant association between poorer prognosis and patients' age < 3 years (p= 0.004) or MAHS (p=0.005).Secondary HLH is not uncommon in Thai children who are immunocompetent. Malignancies, particulary NHL, are highly suspicious especially for cases not responsive to conventional therapy. Poor prognostic factors are age less than 3 years and MAHS.
2820	Adverse outcomes in primary hemophagocytic lymphohistiocytosis.	12368693	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition characterized by abnormal proliferation of macrophages. Although the mortality rate in children diagnosed with primary HLH is high, little has been described about the nature of adverse events. This review evaluates unfavorable events in children with primary HLH to suggest methods of improving outcomes.Charts of patients who met diagnostic criteria for primary HLH at the Hospital for Sick Children between January 1985 and June 2000 were retrospectively reviewed. The primary outcome measure was an adverse event, defined as death, the subsequent diagnosis of malignancy, or developmental delay.Twenty children were diagnosed with primary HLH. The median age at diagnosis was 6.5 months (range 1-78 months). Nineteen children received chemotherapy and two underwent matched sibling donor bone marrow transplantation. Of the 20 children, 12 (60%) died. These deaths were attributed to progressive HLH in 4 cases and invasive infection in 8 cases. These infections consisted of disseminated cytomegalovirus infection (n = 1), sepsis (n = 1), and invasive fungal infections (n = 6). Eight children survived. Two were subsequently diagnosed with malignancy. Two others were found to have significant developmental delay.The overall mortality rate was 60% in our series of 20 children with primary HLH; 50% of deaths were directly attributable to invasive fungal infection. Developmental delay and the diagnosis of malignancy are important events in this cohort.
2821	Haemophagocytic lymphohistiocytosis: proposal of a diagnostic algorithm based on perforin expression.	12358924	Haemophagocytic lymphohistiocytosis (HLH) is a rare, fatal disorder of early infancy. Mutations of the PRF1 gene have been identified in a subset of patients. However, the distinction between the different genetically determined and environmental subtypes of the disease remains a major issue to be solved. This may result in delayed or inappropriate application of bone marrow transplantation (BMT). We propose an algorithm that uses a combination of three rapid laboratory tests, i.e. perforin expression by peripheral lymphocytes, assessment of the behaviour of the 2B4 lymphocyte receptor and natural killer (NK) cell activity, to identify the different subgroups of HLH. In 19 patients diagnosed according to current criteria, we tested perforin expression, 2B4 receptor function and NK cell activity. PRF1 mutations were found in all seven patients showing absent perforin expression. In one male with abnormal behaviour of the 2B4 receptor, SH2D1A mutation confirmed the diagnosis of X-linked lymphoproliferative disease. Four patients with normal NK cell activity had evidence of associated infections. Of the seven with impaired NK cell activity, two had a probable genetically determined subtype of HLH and five appeared as sporadic, infection-associated cases. Improving the diagnostic approach may restrict the use of BMT, the only recognized curative treatment, to HLH patients with a documented poor prognosis while patients with milder disorders may be treated less intensively. Our flow chart could also lead to better selection of patients for specific gene analysis.
2822	Hemophagocytic lymphohistiocytosis is associated with deficiencies of cellular cytolysis but normal expression of transcripts relevant to killer-cell-induced apoptosis.	12351400	In 65 patients with hemophagocytic lymphohistiocytosis (HLH), we found an as yet undescribed heterogeneity of defects in cellular cytotoxicity when assay conditions were modified by the incubation time, the presence of mitogen, or interleukin-2 (IL-2). The standard 4-hour natural killer (NK) test against K562 targets was negative in all patients. In patients deficient in type 1 (n = 21), type 2 (n = 5), and type 4 (n = 8) HLH, negative NK function could be reconstituted by mitogen, by IL-2, or by prolongation of the incubation time (16 hours), respectively. Most patients (n = 31) displayed the type 3 defect, defined by a lack of any cellular cytotoxicity independent of assay variations. The characteristic hypercytokinemia also concerned counterregulatory cytokines, such as proinflammatory interferon-gamma (IFN-gamma), simultaneously elevated with suppressive IL-10 in 38% of types 1-, 2-, and 4-deficient patients and in 71% of type 3-deficient patients. Elevated IFN-gamma alone correlated with high liver enzymes, but sCD95-ligand and sCD25 did not-though these markers were expected to indicate the extent of histiocytic organ infiltration. Outcome analysis revealed more deaths in patients with type 3 deficiency (P =.017). Molecular defects were associated with homozygously mutated perforin only in 4 patients, but other type 3 patients expressed normal transcripts of effector molecules for target-cell apoptosis, including perforin and granzyme family members, as demonstrated by RNase protection analysis. Thus, target-cell recognition or differentiation defects are likely to explain this severe phenotype in HLH. Hyperactive phagocytes combined with NK defects may imply defects on the level of the antigen-presenting cell.
2823	Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation.	12239144	Hemophagocytic lymphohistiocytosis (HLH) comprises familial (primary) hemophagocytic lymphohistiocytosis (FHL) and secondary HLH (SHLH), both clinically characterized by fever, hepatosplenomegaly, and cytopenia. FHL, an autosomal recessive disease invariably fatal when untreated, is associated with defective triggering of apoptosis and reduced cytotoxic activity, resulting in a widespread accumulation of T lymphocytes and activated macrophages. In 1994 the Histiocyte Society initiated a prospective international collaborative therapeutic study (HLH-94), aiming at improved survival. It combined chemotherapy and immunotherapy (etoposide, corticosteroids, cyclosporin A, and, in selected patients, intrathecal methotrexate), followed by bone marrow transplantation (BMT) in persistent, recurring, and/or familial disease. Between July 1, 1994, and June 30, 1998, 113 eligible patients aged no more than 15 years from 21 countries started HLH-94. All had either an affected sibling (n = 25) and/or fulfilled the Histiocyte Society diagnostic criteria. At a median follow-up of 3.1 years, the estimated 3-year probability of survival overall was 55% (95% confidence interval +/- 9%), and in the familial cases, 51% (+/- 20%). Twenty enrolled children were alive and off therapy for more than 12 months without BMT. For patients who received transplants (n = 65), died prior to BMT (n = 25), or were still on therapy (n = 3), the 3-year survival was 45% (+/- 10%). The 3-year probability of survival after BMT was 62% (+/- 12%). HLH-94 is very effective, allowing BMT in most patients. Survival of children with HLH has been greatly improved.
2824	Extracellular and intracellular regulation of oligodendrocyte development: roles of Sonic hedgehog and expression of E proteins.	12237843	Recent advances in understanding oligodendrocyte development have revealed the importance of both extra- and intracellular molecules in regulating the induction, survival, and proliferation of early oligodendrocyte progenitors. The signaling molecule Sonic hedgehog (Shh) is critical for normal development of oligodendrocytes, although the precise influences of Shh on cells of the oligodendrocyte lineage are unclear. The present study shows that Shh increased the number of oligodendrocyte precursors in both pure cultures of oligodendrocyte precursors and mixed cultures from embryonic rat spinal cord. In pure precursor cultures Shh increased cell survival. In mixed cultures, Shh increased both the survival and proliferation of oligodendrocyte precursors in a concentration dependent manner. One intracellular consequence of exposure to Shh is the activation of transcription factors in oligodendrocyte lineage cells, which are critical for oligodendrocyte development, helix-loop-helix (HLH) transcription factors, Olig1 and 2. In many cases, HLH proteins such as Olig1 and Olig2 heterodimerize with other HLH proteins, such as members of the E subfamily, which are critical regulators of cell proliferation and differentiation. Immature (A2B5(+)) and more mature (O4(+)) rat oligodendrocyte precursors in dissociated cell culture expressed Olig1 as well as E proteins, HEB and E2A. Similarly, cells bearing the morphology of oligodendrocyte precursors expressed both Olig1 and HEB or E2A. We propose that E2A and/or HEB, possibly in combination with Olig1 and 2, are critical components of oligodendrogenesis and may regulate cell survival, proliferation, and fate decisions in the oligodendrocyte lineage.
2825	The alpha-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin.	12207009	RNA polymerase II core subunit 3 (RPB3) is an a-like core subunit of RNA polymerase II (pol II). It is selectively down-regulated upon treatment with doxorubicin (dox). Due to the failure of skeletal muscle cells to differentiate when exposed to dox, we hypothesized that RPB3 is involved in muscle differentiation. To this end, we have isolated human muscle RPB3-interacting proteins by using yeast two-hybrid screening. It is of interest that an interaction between RPB3 and the myogenic transcription factor myogenin was identified. This interaction involves a specific region of RPB3 protein that is not homologous to the prokaryotic a subunit. Although RPB3 contacts the basic helix-loop-helix (HLH) region of myogenin, it does not bind other HLH myogenic factors such as MyoD, Myf5, and MRF4. Coimmunoprecipitation experiments indicate that myogenin contacts the pol II complex and that the RPB3 subunit is responsible for this interaction. We show that RPB3 expression is regulated during muscle differentiation. Exogenous expression of RPB3 slightly promotes myogenin transactivation activity and muscle differentiation, whereas the region of RPB3 that contacts myogenin, when used as a dominant negative molecule (Sud), counteracts these effects. These results indicate for the first time that the RPB3 pol II subunit is involved in the regulation of tissue-specific transcription.
2826	An EGFR/Ebi/Sno pathway promotes delta expression by inactivating Su(H)/SMRTER repression during inductive notch signaling.	12230979	The Notch and Epidermal Growth Factor Receptor (EGFR) pathways both regulate proliferation and differentiation, and the cellular response to each is often influenced by the other. Here, we describe a mechanism that links them in a sequential fashion, in the developing compound eye of Drosophila. EGFR activation induces photoreceptor (R cell) differentiation and promotes their expression of Delta. This Notch ligand then induces neighboring cells to become nonneuronal cone cells. ebi and strawberry notch (sno) regulate EGFR-dependent Delta transcription by antagonizing a repressor function of Suppressor of Hairless (Su(H)). Sno binds to Su(H), and Ebi, an F-box/WD40 protein, forms a complex with Su(H) and the corepressor SMRTER. EGFR-activated transcriptional derepression requires ebi and sno, is proteasome-dependent, and correlates with the translocation of SMRTER to the cytoplasm.
2827	Characterization of a carbohydrate response element regulating the gene for human galactose-1-phosphate uridyltransferase.	12208133	Human galactose-1-phosphate uridyltransferase (hGALT) is a central enzyme in the conserved pathway by which galactose is converted to energy, UDP-galactose and UDP-glucose. A natural mutation that deleted -119 to -116 bp (delGTCA) of the promoter decreased hGALT mRNA and enzyme activity and prompted analysis of hGALT gene regulation. Regulatory domains were identified by inspection and confirmed in a reporter system. Previous studies by others were confirmed that HepG2 cells grown in D-glucose increased hGALT enzyme activity and mRNA by 30%. We extended these observations by sequencing the promoter region and identifying a potential carbohydrate response element (ChoRE). The response to glucose rose to 190% when a plasmid construct containing a luciferase reporter and only the -165 bp region as a promoter was transfected into HepG2 and NIH:OVCAR-3. By contrast, fibroblasts transfected with the identical construct failed to respond to glucose. Within the -165 bp region there were two enhancer (E-box) motifs that encompassed the delGTCA mutation. The deletion diminished the positive regulatory response, but an additional GTCA repeat unexpectedly increased the response. Using this postulated ChoRE as a probe in electrophoretic mobility shift assays, multiple nuclear proteins bound and one was identified as a member of the basic/helix-loop-helix/leucine zipper enhancer-binding (b/HLH/LZ) family. Increased binding of proteins correlated with increased hGALT expression when the spacing between E-box motifs was enlarged but the carbohydrate response was dampened. When the 3(')E-box was mutated, b/HLH/LZ binding and gene expression were abolished. We conclude that the hGALT promoter region contains a ChoRE in which the spacing between and the sequence of its E-box motifs are critical. One nuclear protein of the b/HLH/LZ family is necessary, but not sufficient for the carbohydrate response.
2828	Cloning and characterization of GRIPE, a novel interacting partner of the transcription factor E12 in developing mouse forebrain.	12200424	The helix-loop-helix (HLH) family of transcription factors are key contributors to a wide array of developmental processes, including neurogenesis and hematopoiesis. These factors are thought to exert their regulatory influences by binding to cognate promoter-DNA sequences as dimers. Although studies in mice have convincingly demonstrated that neurogenic HLH proteins such as NeuroD are intimately involved in neuronal fate determination, the role of the ubiquitously expressed HLH protein, E12, in mammalian neurogenesis remains ambiguous. To address this, a yeast two-hybrid interaction screen was employed to identify dimerization partners to E12. Screening of an embryonic day 11.5 forebrain library resulted in the cloning of GRIPE, a novel GAP-related interacting protein to E12. GRIPE binds to the HLH region of E12 and may require E12 for nuclear import. Furthermore, GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidentally detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were recapitulated through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein dimerization of which with E12 has important consequences for cells undergoing neuronal differentiation.
2829	[Unrelated donor stem cell transplantation in children: low toxicity using a GvHD-prophylaxis regimen with CSA, MTX, metronidazole,iv-immunoglobulin and ATG].	12165903	Unrelated donor (UD) hematopoietic stem cell transplantation (HSCT) is accepted as a therapy for leukaemic diseases and varying inborn diseases if a suitable related donor cannot be found. The goal of immunosuppressive therapy with UD-HSCT is an effective prevention of graft-versus-host-disease (GvHD) on one hand. On the other hand an optimal balance with immunocompetence of the transplanted bone marrow is desirable in order to prevent graft failure, infection and, in the case of leukaemic diseases, potentially control the underlying disease.Between 1992 and 2000 49 patients aged 11 months to 16.7 years received an UD-HSCT in Hamburg. Underlying diseases were leukaemia or MDS in 35, of these ALL in 21, hemophagocytic lymphohistiocytosis (HLH) in 9, immunodeficiency or inborn error of metabolism in 5 patients. GvHD-prophylaxis consisted of a combination of Cyclosporin A (CSA), methotrexate (MTX), metronidazole, IgM-enriched iv-immunoglobulin (ivIg) (Pentaglobin(R)) or ivIgG and anti-thymocyte-globulin (ATG). Within the same time span 10 patients with ALL received a matched related donor HSCT (MRD-HSCT). GvHD-prophylaxis in these patients was done without ATG in 8 of 10 cases. UD-HSCT were analyzed for survival, relapse and toxicity. Probability of survival of the patients with ALL after UD-HSCT was compared with results of MRD-HSCT in children with ALL.The Kaplan-Meier estimates of three year overall-survival (OS) were 74 % for all patients. Probability of disease-free survival (DFS) at three years was 62 % for leukaemia/MDS-patients and 100 % for the HLH-patients. Acute GvHD (aGvHD) grades II or III occurred in 51 % of patients. Chronic GvHD (cGvHD) occurred in 22 % of patients. There were 5 cases of treatment-related mortality (TRM). Probability of DFS for patients with ALL at three years was 65 % after UD-HSCT and 30 % in the patients after MRD-HSCT.UD-HSCT in children is an effective and safe therapy. A GvHD-prophylaxis regimen combining the standard immunosuppressive agents CSA and MTX with ivIg, metronidazole and serotherapy using ATG may result in a low incidence of severe GvHD-complications and low TRM rate without increase in relapse rates.
2830	Transcriptional regulation of erythropoiesis. Fine tuning of combinatorial multi-domain elements.	12153557	Haematopoiesis, the differentiation of haematopoietic stem cells and progenitors into various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Commitment of pluripotent haematopoietic stem cells to the erythroid lineage induces erythropoiesis, the production of red blood cells. This process involves a concerted progression through an erythroid burst forming unit (BFU-E), an erythroid colony forming unit (CFU-E), proerythroblast and an erythroblast. The terminally differentiated erythrocytes, in mammals, lose their nucleus yet function several more months. A well-coordinated cohort of transcription factors regulates the formation, survival, proliferation and differentiation of multipotent progenitor into the erythroid lineage. Here, we discuss broad-spectrum factors essential for self-renewal and/or differentiation of multipotent cells as well as specific factors required for proper erythroid development. These factors may operate solely or as part of transcriptional complexes, and exert activation or repression. Sequence comparisons reveal evolutionarily conserved modular composition for these factors; X-ray crystallography demonstrates that they include multidomain elements (e.g. HLH or zinc finger motifs), consistent with their complex interactions with other proteins. Finally, transfections and genomic studies show that the timing of each factor's expression during the hematopoietic process, the cell lineages affected and the existing combination of other factors determine the erythroid cell fate.
2831	Hemophagocytic lymphohistiocytosis presenting with thrombocytopenia in the newborn.	12142793	Hemophagocytic lymphohistiocytosis (HLH) may present with thrombocytopenia during the newborn period. Three neonates (one term and two preterm) presented during the newborn period with thrombocytopenia. Transient recovery occurred in two newborns. The diagnosis of HLH was made after the recurrence of thrombocytopenia and the clinical symptoms at 5 and 7 weeks. The third infant was a premature baby diagnosed at 8 days of age after manifesting the clinical and laboratory features of HLH. All three neonates were treated with chemotherapy and responded well. After hematologic and clinical remission was achieved, the two newborns received hematopoietic stem cell transplantation from allogeneic donors. The third neonate is currently receiving chemotherapy. Persistent or recurrent thrombocytopenia of undetermined cause during the neonatal period should raise the suspicion of HLH, even though other symptoms or signs are not yet evident.
2832	Elevated sphingomyelinase and hypercytokinemia in hemophagocytic lymphohistiocytosis.	12142792	Ceramide generated from sphingomyelinase activation has been reported to play a role in cytokine-mediated events. Secretory sphingomyelinase (S-SMase), a product of the acid sphingomyelinase gene, has been found to be derived from many cell types and to exist in human serum. The purpose of the current study was to investigate the serum level of S-SMase.Three patients with hypercytokinemia caused by hemophagocytic lymphohistiocytosis (HLH) were studied. Serum samples were collected from the three patients with HLH, patients with a deficiency of acid sphingomyelinase, or type B Niemann-Pick disease, and normal controls. The S-SMase activities were determined using 14C-sphingomyelin as a substrate.The serum S-SMase activities were increased 10-to 20-fold in the sera from the three patients with HLH (P < 0.0001). The high S-SMase activity was decreased to the normal level along with the clinical improvement of HLH.Increased release of S-SMase from cells into the circulation is observed in patients with active HLH. The authors suggest that S-SMase is related to the pathophysiology of hypercytokinemia.
2833	Prenatal diagnosis of congenital heart disease in the Naples area during the years 1994-1999 -- the experience of a joint fetal-pediatric cardiology unit.	12124685	To analyse the spectrum and frequencies of observed malformations; to evaluate associated extracardiac and chromosomal anomalies and outcomes in each diagnostic category; to demonstrate the need for a multidisciplinary approach to the diagnosis of CHD in the fetus.From January 1994 to December 1999, 450 cases of CHD were detected among 4052 pregnancies at risk of fetal CHD seen at our combined unit. Confirmation of the diagnosis was not available in 50 cases, leaving 400 cases for analysis. From our computerized database, the following variables were retrieved and analysed: indication, gestational age at diagnosis, associated extracardiac anomalies, karyotype, natural history, pregnancy and feto-neonatal outcome.CHDs most commonly detected were VSD (75 cases), AVSD (40 cases) and HLH (37 cases). The aneuploidy rate was 29.3% in the 355 cases submitted for karyotyping (25.9% in the whole series), with a prevalence of trisomy 21 and 18 (48 and 30 cases, respectively). The aneuploidy rate was highest for AVSD (80%), coarctation (49%), tetralogy of Fallot and VSD (45%). Associated extracardiac anomalies were present in 29.5% of the cases (118/400). As for pregnancy outcome, there were 150 (37.5%) terminations of pregnancy, 16 (4%) intrauterine fetal deaths and 85 (21.3%) neonatal deaths. The remaining 149 neonates are alive (37.3% survival rate). The termination rate for pregnancies in which CHD was detected at a gestational age <25 weeks was 65.2%. Evolutive changes determined progressive prognostic deterioration in 21 cases (5%), consisting of semilunar valve obstructions and development of ventricular hypoplasia.The high association rate with extracardiac and chromosomal anomalies (29.3% and 25.9%) and the possible progressive prognostic deterioration require a multidisciplinary team for correct management and follow-up. Survival of fetuses with certain CHD is severely reduced, in comparison with postnatal figures, for the common association with aneuploidies.
2834	Abundant expression of Dec1/stra13/sharp2 in colon carcinoma: its antagonizing role in serum deprivation-induced apoptosis and selective inhibition of procaspase activation.	12119049	The basic helix-loop-helix (bHLH) proteins are intimately associated with developmental events such as cell differentiation and lineage commitment. The HLH domain in the bHLH motif is responsible for dimerization, whereas the basic region mediates DNA binding. Based on sequence alignment and domain analysis, differentially expressed in chondrocytes/stimulated with retinoic acid/split and hairy-related proteins (DEC/STRA/SHARPs) represent a new class of bHLH proteins. The present study describes the functional characterization of DEC1. Subtractive experiments and blotting analyses demonstrated that DEC1 was highly expressed in colon carcinomas, but not in the adjacent normal tissues. Several cell cycle blockers markedly induced DEC1 expression. Stable transfectants with a tetracycline-inducible construct demonstrated that DEC1 caused proliferation inhibition, antagonized serum deprivation-induced apoptosis and selectively inhibited the activation of procaspases. These activities were highly correlated with the abundance of tetracycline-induced DEC1. Stable transfectants expressing a mutant DEC1 (lacking the DNA-binding domain) exhibited neither proliferation inhibition nor apoptotic antagonism, which suggests that DNA binding is required for these actions. Enzymic assays and immunoblotting analyses demonstrated that induction of DEC1 by tetracycline significantly decreased the activation of procaspases 3, 7 and 9 but not procaspase 8. The selective suppression on the activation of procaspases 3, 7 and 9 over procaspase 8 suggests that DEC1-mediated anti-apoptosis is achieved by blocking apoptotic pathways initiated via the mitochondria. The results functionally distinguish DEC1 from other bHLH proteins and directly link this factor to oncogenesis.
2835	Low natural killer activity and central nervous system disease as a high-risk prognostic indicator in young patients with hemophagocytic lymphohistiocytosis.	12115393	Familial hemophagocytic lymphohistiocytosis HLH (FHL) is fatal, unless patients are rescued with hematopoietic stem cell transplantation (SCT). Although the molecular identification of FHL now is possible at least in part from perforin gene study, many cases escape detection or never are tested due to the lack of specific hallmarks, making diagnosis difficult. To the authors' knowledge, it remains to be determined whether persistently low natural killer cell (NK) activity and a high incidence of central nervous system (CNS) disease increase the probability of FHL.The authors analyzed 42 HLH patients age < 2 years, 13 of whom developed overt CNS disease and 5 of whom demonstrated persistently deficient NK activity (Group 1). The remaining 24 patients had no CNS disease and had NK activity of moderate decrease to within the normal range (Group 2).In Group 1, CNS symptoms were detected in 6 cases within 1 month and between 4.5-9 months in 6 other patients. In these cases, spotty lesions demonstrating a high T2 signal in the white matter were noted on brain magnetic resonance imaging. The survival was significantly poor for patients in Group 1 unless they were rescued with SCT, which was performed in 5 of the 13 patients with CNS disease and in all 5 patients with persistent NK activity deficiency. SCT was successful in 9 patients, with no CNS sequelae reported after the transplantation. Conversely, the prognosis of the 24 patients in Group 2 was better and only 1 patient required SCT.Very young HLH patients (age < 2 years) who are at high risk of fatal FHL with persistently deficient NK activity and/or overt CNS disease require appropriate SCT to reverse CNS disease and achieve a complete cure.
2836	Successful treatment of Griscelli syndrome with unrelated donor allogeneic hematopoietic stem cell transplantation.	12098069	Griscelli syndrome (GS) is a rare autosomal recessive disorder, characterized by pigmentary dilution of the skin and hair and in most patients by abnormal regulation of the immune system, which results in a syndrome of macrophage hyperactivation, known as hemophagocytic lymophohistiocytosis (HLH). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment available for genetically induced HLH. Few cases of successful HSCT from a compatible donor have been reported in children with GS. We describe the first patient with GS cured with an allograft from a compatible unrelated bone marrow donor. We used a novel preparative regimen consisting of busulfan, thiotepa and fludarabine. The demonstrated curative effect of HSCT from an unrelated donor in a patient with genetically determined HLH also supports the use of a systematic diagnostic approach in these patients, in order to identify those with a worse prognosis and needing an urgent allograft in a timely manner.
2837	Effect of maternal undernutrition on fetal testicular steroidogenesis during the CNS androgen-responsive period in male sheep fetuses.	12090916	The aim of this study was to determine the effects of maternal undernutrition, applied during physiologically relevant stages of development of the reproductive system, on reproductive development in male sheep fetuses. Groups of ewes (n = 11-19) were fed rations providing either 100% (high; H) or 50% (low; L) of metabolizable energy requirements for live weight maintenance during selected 'windows', bounded by days 0, 30, 50, 65 and 110 after mating. Ewes of control groups (HH (Expts 1 and 2) and HHH (Expt 3)) were fed the H ration from mating until they were killed at day 50 (Expt 1), day 65 (Expt 2) or day 110 (Expt 3) of gestation, whereas ewes of other groups were fed the L ration for the periods days 0-30 of gestation (LH and LHH), days 31-50 or days 31-65 of gestation (HL and HLH), days 65-110 of gestation (HHL), or day 0 to day 50, day 65 or day 110 of gestation (LL and LLL) when the animals were killed. At day 50 of gestation, there was no effect of nutritional treatment on mean fetal mass or fetal testicular mass, but there was increased expression of mRNA for steroidogenic acute regulatory protein (StAR) in the testes of LL animals (P < 0.05) compared with HH controls. Compared with HH animals, the mean plasma testosterone concentrations of LL fetuses tended to be higher, but this result did not reach significance. At day 65 of gestation there were no significant differences between treatments in mean fetal masses, testicular masses, mean plasma testosterone concentrations or StAR mRNA content. At day 110 of gestation, fetal masses in the LLL group were lower (P < 0.01) than those of control fetuses, although no differences in testicular size or fetal plasma testosterone concentrations were recorded. It is concluded that the effects of undernutrition on reproductive development of male sheep fetuses are dependent on the timing of the period of undernutrition.
2838	Transformation of Ba/F3 cells and Rat-1 cells by ETV6/ARG.	12080468	ETV6/ARG, a novel fusion gene composed of the ETV6 HLH oligomerization domain and most of sequences of the ARG protein tyrosine, was recently identified in human leukemia cells. The presence of the ETV6/ARG translocation raises the possibility that the resulting fusion protein functions as an oncogene. However, the transforming activity of the ETV6/ARG protein has not been determined and its contribution to leukemogenesis is therefore unknown. Here we address this question by analysing the oncogenic activity of ETV6/ARG in hematopoietic and fibroblast cells. It is demonstrated that expression of ETV6/ARG confers IL3-independent growth to Ba/F3 cells and anchorage independent growth to Rat-1 fibroblasts. It is also shown that multiple signaling molecules, including PI3K, SHC, ras-GAP and CRK-L, are tyrosine phosphorylated in Ba/F3 cells that express ETV6/ARG. Analysis of four different types of ETV6/ARG transcripts previously identified in the AML-M3 leukemia cell line HT93A suggest that ETV6 HLH domain is required for oncogenic activity. Based upon these results it is concluded that ARG can be activated as an oncogene in human malignancy and that the ETV6/ARG oncoprotein triggers some of the same signaling pathways associated with activated ABL oncogenes.
2839	Distinct patterns of downstream target activation are specified by the helix-loop-helix domain of proneural basic helix-loop-helix transcription factors.	12074558	Both gain- and loss-of-function analyses indicate that proneural basic/helix-loop-helix (bHLH) proteins direct not only general aspects of neuronal differentiation but also specific aspects of neuronal identity within neural progenitors. In order to better understand the function of this family of transcription factors, we have used hormone-inducible fusion constructs to assay temporal patterns of downstream target regulation in response to proneural bHLH overexpression. In these studies, we have compared two distantly related Xenopus proneural bHLH genes, Xash1 and XNgnr1. Our findings indicate that both Xash1 and XNgnr1 induce expression of the general neuronal differentiation marker, N-tubulin, with a similar time course in animal cap progenitor populations. In contrast, these genes each induce distinct patterns of early downstream target expression. Both genes induce expression of the HLH-containing gene, Xcoe2, at early time points, but only XNgnr1 induces early expression of the bHLH genes, Xath3 and XNeuroD. Structure:function analyses indicate that the distinct pattern of XNgnr1-induced downstream target activation is linked to the XNgnr1 HLH domain, demonstrating a novel role for this domain in mediating the differential function of individual members of the proneural bHLH gene family.
2840	Misexpression of dHAND induces ectopic digits in the developing limb bud in the absence of direct DNA binding.	12070084	Basic helix-loop-helix (bHLH) transcription factors control developmental decisions in a wide range of embryonic cell types. The HLH motif mediates homo- and heterodimerization, which juxtaposes the basic regions within the dimeric complex to form a bipartite DNA binding domain that recognizes a DNA consensus sequence known as an E-box. eHAND and dHAND (also known as HAND1 and HAND2) are closely related bHLH proteins that control cardiac, craniofacial and limb development. Within the developing limb, dHAND expression encompasses the zone of polarizing activity in the posterior region, where it has been shown to be necessary and sufficient to induce the expression of the morphogen sonic hedgehog. Misexpression of dHAND in the anterior compartment of the limb bud induces ectopic expression of sonic hedgehog, with resulting preaxial polydactyly and mirror image duplications of posterior digits. To investigate the potential transcriptional mechanisms involved in limb patterning by dHAND, we have performed a structure-function analysis of the protein in cultured cells and ectopically expressed dHAND mutant proteins in the developing limbs of transgenic mice. We show that an N-terminal transcriptional activation domain, and the bHLH region, are required for E-box-dependent transcription in vitro. Remarkably, however, digit duplication by dHAND requires neither the transcriptional activation domain nor the basic region, but only the HLH motif. eHAND has a similar limb patterning activity to dHAND in these misexpression experiments, indicating a conserved function of the HLH regions of these proteins. These findings suggest that dHAND may act via novel transcriptional mechanisms mediated by protein-protein interactions independent of direct DNA binding.
2841	Overexpression of dystrobrevin delays locomotion defects and muscle degeneration in a dystrophin-deficient Caenorhabditis elegans.	12062255	Duchenne muscular dystrophy is one of the most common neuromuscular diseases. It is caused by mutations in the dystrophin gene. Dystrobrevins are dystrophin-associated proteins potentially involved in signal transduction. The nematode Caenorhabditis elegans possesses one dystrophin-like (dys-1) and one dystrobrevin-like (dyb-1) gene. Mutations of dyb-1 and dys-1 lead to similar phenotypes, comprising hyperactivity and a tendency to hypercontract, which suggest that these proteins may participate in a common function. We show here that overexpression of the Dyb-1 protein delays the onset of the myopathy observed in the C. elegans double mutant (dys-1; hlh-1 mutations). This finding indicates that, in C. elegans, (1) the absence of dystrophin can be partly compensated for by extra doses of dystrobrevin, and (2) dystrobrevin is partly functional in absence of dystrophin.
2842	Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis.	12060139	Familial haemophagocytic lymphohistiocytosis (FHL), an inherited form of haemophagocytic lymphohistiocytosis (HLH) syndrome, is characterized by the overwhelming activation of T lymphocytes and macrophages invariably leading to death in the absence of treatment. FHL is a heterogeneous autosomal recessive disorder, with one known causative gene which codes for perforin, a cytotoxic effector protein. In this study, we have characterized the genotype and phenotype of 14 unrelated families with perforin deficiency. Four new missense mutations of the perforin gene were identified. In every case, perforin gene mutations led to undetectable intracellular perforin expression within cytotoxic cells, while some residual T-cell cytotoxic activity could be associated with certain missense mutations. Clinical and biological analyses did not differentiate between patients with nonsense or missense mutations, although age at diagnosis, which tended to be similar within members of the same family, was delayed in patients from two families belonging to the second group. In one case, consequences of perforin deficiency, diagnosed at birth, could be assessed prior to onset of clinical manifestations. No evidence for T-cell activation could be shown, suggesting that an exogenous event is required to trigger the disease manifestation. Control assessment of perforin expression and cytotoxic assays by lymphocytes from young children led to the conclusion that perforin content of natural killer cells could be a reliable diagnostic test at any age. Altogether, these data enabled a better characterization of perforin deficiency and its consequences, and defined reliable diagnostic tools.
2843	The clinical therapeutic window for luteinizing hormone in controlled ovarian stimulation.	1205772412798917	To discuss the clinical therapeutic window for LH during the follicular phase.Review of selected papers that were retrieved through a Medline search and a review of clinical trials, the results of which are in the process of publication.Women undergoing infertility treatment.Recombinant human LH (r-hLH) was administered SC as a supplement to FSH during controlled ovarian hyperstimulation.Follicular development, E2 production, and endometrial thickness.Optimal follicular maturation is the result of both FSH and LH stimulation. In patients with hypogonadotropic hypogonadism, 75 IU of r-hLH and 150 IU of FSH per day resulted in more follicles and provided sufficient E2 for optimal endometrial proliferation. Additional r-hLH (>250 IU/day), in patients with either hypogonadotropic hypogonadism or polycystic ovary disease, may precipitate a series of deleterious physiological actions leading to atresia of developing follicles. Adding r-hLH to FSH in women treated with GnRH agonist showed no benefits in terms of number of mature oocytes, fertilization, and cleavage. However, those who experience profound pituitary desensitization may benefit from adding LH to the stimulation protocol. No obvious clinical criteria have been established to define this group of patients.A "threshold" and "ceiling" level for LH (therapeutic window) is proposed, below which E2 production is not adequate and above which LH may be detrimental to follicular development.
2844	Gene abnormalities in patients with hemophagocytic lymphohistiocytosis.	12040827	Hemophagocytic lymphohistiocytosis is thought to occur as a primary (familial) form or secondary to infection or malignancy. Recently, several defects in genes important for immune functions were identified in patients with HLH. These include mutations in perforin, the gamma common chain of the receptor for interleukin-2, Slap and purine nucleoside phosphorylase. Since abnormal function of these genes is associated with a wide clinical spectrum, HLH is probably another manifestation of immune deficiency and a thorough immune evaluation should be done in all such patients.
2845	Down-regulation of Id-1 expression is associated with TGF beta 1-induced growth arrest in prostate epithelial cells.	12020803	Transforming growth factor beta1 (TGF beta 1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGF beta 1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGF beta 1 was able to suppress the expression of Id-1, a helix-loop-helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGF beta 1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGF beta 1 signaling pathway. The fact that up-regulation of p21(WAF1), one of the downstream effectors of Id-1, was observed after exposure to TGF beta 1 further indicates the involvement of Id-1 in the TGF beta 1-induced growth arrest in HPr-1 cells. However, increased expression of p27(KIP1) was also observed in the TGF beta 1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGF beta 1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGF beta 1 may be one of the upstream regulators of Id-1.
2846	Characterization of a dominant negative C. elegans Twist mutant protein with implications for human Saethre-Chotzen syndrome.	12015302	Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.
2847	Differential pulse adsorptive stripping voltammetry of osmium-modified peptides.	12009445	Complexes of osmium tetroxide with nitrogen ligands were developed and used in our laboratory as probes of the DNA structure. Here, we show that the complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) can be used for modification and electrochemical detection of proteins at neutral pH. Salmon luteinizing hormone (SLH) containing two tryptophan (Trp) residues and human luteinizing hormone (HLH) containing one Trp were modified by Os,bipy and measured by differential pulse adsorptive stripping voltammetry (DPAdSV) at a hanging mercury drop electrode (HMDE). The intensity of the DPAdSV catalytic signals corresponded to the number of Trp residues in the peptide molecule. Decreasing pH of the background electrolyte from 6.6 to 3.8 led to the increase of DPAdSV signals, suggesting that at pH 3.8, the DPAdSV detection limit might be well below 1 ng/ml. Our results suggest that Os,bipy is potentially useful for chemical modification of proteins.
2848	The SAM domain of polyhomeotic forms a helical polymer.	11992127	The polycomb group (PcG) proteins are important in the maintenance of stable repression patterns during development. Several PcG members contain a protein protein interaction module called a SAM domain (also known as SPM, PNT and HLH). Here we report the high-resolution structure of the SAM domain of polyhomeotic (Ph). Ph-SAM forms a helical polymer structure, providing a likely mechanism for the extension of PcG complexes. The structure of the polymer resembles that formed by the SAM domain of another transcriptional repressor, TEL. The formation of these polymer structures by SAM domains in two divergent repressors suggests a conserved mode of repression involving a higher order chromatin structure.
2849	Reactive hemophagocytic syndrome associated with thrombotic thrombocytopenic purpura during therapeutic plasma exchange.	11982958	Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever, cytopenia, splenomegaly, and lymphohistiocytic proliferation with hemophagocytosis. Sporadic, familial, and reactive HLH varieties exist. The latter, also termed the reactive hemophagocytic syndrome (RHS), has been associated with a variety of infectious and noninfectious etiologies. Activation of monocytes in RHS is due to stimulation by high levels of activating cytokines. RHS has not been associated previously with thrombotic thrombocytopenic purpura (TTP). TTP is a multisystem disorder characterized by consumptive thrombocytopenia, microangiopathic hemolytic anemia, neurologic symptoms, renal impairment, and fever. We report on a 33 year old male patient with a classic picture of TTP who initially responded to therapeutic plasma exchange but then became refractory to treatment and developed RHS. It is likely that a specific pathophysiology involving the activation of neutrophils during TPE is present for the development of cytokine-induced hemophagocytosis during TTP treatment. The consequent development of RHS possibly caused early TTP relapse.
2850	Elimination of luteinizing hormone cross-reactive epitopes from human chorionic gonadotropin.	11972973	The beta-chain of human chorionic gonadotropin (hCG) has been shown to have efficacy in clinical trials when used as a contraceptive vaccine. This hormone is a heterodimer, the alpha-chain being shared with the other members of the glycoprotein hormone family but the beta-chain being unique to hCG. Nevertheless, there is sequence homology between the hCG beta-chain and the beta-chain of human luteinizing hormone (hLH) which results in cross-reactive antibodies being produced following immunization with wild-type hCGbeta. To reduce or eliminate such cross-reactions we generated a number of mutants of the hCGbeta-chain. One mutant (hCGbeta(R68E)), containing an arginine to glutamic acid replacement at position 68, has been expressed as a recombinant protein in High Five insect cells. The recombinant BAChCGbeta(R68E) form of this molecule was used to immunize rabbits and the antibody response compared to the response following immunization with the recombinant wild-type protein BAChCGbeta and with the native hCGalphabeta heterodimer isolated from pregnancy urine. The mutant elicited the production of antibodies which avidly recognize native hCG. Compared to immunization with wild-type hCG, the response showed very little cross reactivity with hLH. This is demonstrated to be due to a radically altered epitope usage in the response to the mutant, which now focuses mainly upon the C-terminal region of the beta-chain.
2851	Hes6 regulates myogenic differentiation.	11959828	Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21(Cip1), and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.
2852	Intracellular calcium mobilization in response to the activation of human wild-type and chimeric gonadotropin receptors.	11956155	It is well established that LH action is mediated primarily by adenylate cyclase/cAMP. However, the role of inositol phosphate/calcium in LH signaling is less well established. We examined the effects of gonadotropins in primary culture human granulosa-lutein cells and in HEK293 cells transiently transfected with human wild-type or chimeric gonadotropin receptors. The intracellular free calcium concentration was measured using fura-2 microspectrofluorometric techniques. Human (h) LH (2-4 microg/ml) and CG (10 IU/ml) consistently evoked oscillatory calcium signals in HEK293 cells transfected with hLH receptor, whereas hFSH (2-4 microg/ml) failed to elicit any response. Conversely, both hLH and hFSH failed to elicit a calcium response from HEK293 cells transfected with hFSHR, indicating the specificity of the response to the LH receptor. Pretreatment of transfected HEK293 cells with pertussis toxin (100 ng/ml) attenuated all gonadotropin-evoked calcium mobilization. Studies with chimeric LH receptor showed that the sequence of the long extracellular portion of the receptor was not critical for stimulation of PLC activity, but maintained agonist binding specificity. The C-terminal sequence of the receptor was clearly important for the generation of the basal calcium oscillations, but the precise extent of the critical sequence has yet to be identified. Although various subdivisions of this region were capable of stimulating calcium transients, an intact carboxyl-terminal third of the receptor was required for normal and sustained intracellular calcium signaling. Our study unequivocally shows that the hLH receptor is coupled to the inositol phosphate/calcium signaling pathway via a pertussis toxin-sensitive G protein-coupled receptor.
2853	The MyoD-inducible p204 protein overcomes the inhibition of myoblast differentiation by Id proteins.	11940648	The murine p204 protein level is highest in heart and skeletal muscle. During the fusion of cultured myoblasts to myotubes, the p204 level increases due to transcription dependent on the muscle-specific MyoD protein, and p204 is phosphorylated and translocated from the nucleus to the cytoplasm. p204 overexpression accelerates myoblast fusion in differentiation medium and triggers this process even in growth medium. Here we report that p204 is required for the differentiation of C2C12 myoblasts. We propose that it enables the differentiation, at least in part, by overcoming the inhibition of the activities of the MyoD and E47 proteins by the Id proteins: Id1, Id2, and Id3. These are known to inhibit skeletal muscle differentiation by binding and blocking the activity of MyoD, E12/E47, and other myogenic basic helix-loop-helix (bHLH) proteins. Our hypothesis is based on the following findings. (i) A decrease in the p204 level in C2C12 myoblasts by antisense RNA (a) increased the level of the Id2; (b) inhibited the MyoD-, E12/E47-, and other bHLH protein-dependent accumulation of the muscle-specific myosin heavy-chain protein; and (c) inhibited the fusion of myoblasts to myotubes in differentiation medium. (ii) p204 bound to the Id proteins in vitro and in vivo. (iii) In the binding of p204 to Id2, the b segment of p204 and the HLH segment of Id2 were involved. (iv) Addition of p204 overcame the inhibition by the Id proteins of the binding of MyoD and E47 to DNA in vitro. (v) Overexpression of p204 in myoblasts (a) decreased the level of the Id proteins, even in a culture in growth medium, and (b) overcame the inhibition by the Id proteins of MyoD- and E47 dependent transcription and also overcame the inhibition by Id2 of the fusion of myoblasts to myotubes.
2854	Risk of etoposide-related acute myeloid leukemia in the treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	11939264	We studied the impact of etoposide on the prognosis of 81 patients (77 of whom were children <15 years old) with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The study group received a median cumulative dose of 1,500 mg/m2 etoposide (range, 0-14,550 mg/m2), with a median follow-up period of 44 months (range, 20-88 months) from the diagnosis. Only 1 patient, who received 3150 mg/m2 etoposide, developed therapy-related acute myeloid leukemia (t-AML), at 31 months after diagnosis. Excluding 9 patients who underwent hemopoietic stem cell transplantation during the course of treatment, the prognosis was poorer for those patients who received less than a 1,000 mg/m2 cumulative dose of etoposide. Our results indicate that the risk of etoposide-related t-AML is low. An appropriate dosage of etoposide for the treatment of EBV-HLH would be in the range of 1,000 to 3,000 mg/m2. However, even at these doses, care must be taken to prevent the rare risk of t-AML.
2855	Molecular characterization of breast cancer cell lines by expression profiling.	11935298	Gene expression patterns provide detailed insights into cellular regulation that reflect minor differences of cellular capacity not accessible by standard descriptions of the cellular phenotype or origin.To identify fundamental differences and similarities we analyzed the gene expression patterns of four breast cancer cell lines: MCF-7, SK-BR-3, T-47D, and BT-474.Although only a small subset of genes (597) is represented on the Atlas-cDNA-Array (Clontech) used, clear differences in the expression of a number of genes could be detected. For example, unique high levels of expressions were found for the HLH-protein ID-1 (MCF-7) and the receptor tyrosine kinase erbB2 (SK-BR-3 and T-47D). Most genes analyzed were expressed at comparable levels in all cell lines studied.For interpretation of the results sets of genes that show similar variation of expression among the cells were grouped together. Furthermore, our analysis allows the assignment of similarity values that lead to a relation profile of the cell lines. How these results correlate with known biological properties of the cell lines is discussed. Additionally, we demonstrate that results obtained by cDNA-Array hybridization for expression of the ErbB receptor family correlate well with competitive RT-PCR, thus confirming the reliability of the cDNA-Array analysis.
2856	Visceral leishmaniasis and haemophagocytic syndrome in an Omani child.	24019726	The paper reports the case of a previously healthy 4-year-old-girl who presented with pallor, fever and hepatosplenomegaly. Laboratory findings included pancytopenia, hypertriglyceridemia and hyperferritinemia. Initial diagnosis of kala-azar could not be confirmed because of the absence of clinical evidence, negativity of bone marrow aspiration or specific serology for visceral leishmaniasis. Repeated marrow aspiration, performed due lack of clinical response, revealed histiocytes showing haemophagocytosis consistent with haemophagocytic lymphohistocytosis (HLH) and appropriate treatment was started. She continued to have high-grade fever, and a third bone marrow aspiration ultimately revealed presence of Leishmania amastigotes with evidence of active haemophagocytosis. The girl was treated with liposomal amphotericin (AmBisome) for 5 days, following which she recovered rapidly with definitive remission.
2857	Quantitative analysis of cell-free Epstein-Barr virus genome copy number in patients with EBV-associated hemophagocytic lymphohistiocytosis.	11908724	To determine whether the EBV genome content in serum or plasma reflects clinical features and outcome in EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), we quantified the cell-free EBV genome copy number by real-time PCR in 38 patients with EBV-HLH, and compared this to the values from 15 patients with infectious mononucleosis (IM). The median (range) cell-free EBV genome copy number at diagnosis was 3.0 x 10(3) (undetectable -5.5 x 10(7)) copies/ml in EBV-HLH, which was significantly higher than the 6.6 x 10(1) (undetectable -1.0 x 10(3)) copies/ml in IM (P = 0.0008). We serially analyzed cell-free EBV genome copy number in 10 cases of EBV-HLH up to 4 months from diagnosis. In four patients who achieved remission, the EBV genome became undetectable soon after starting therapy. In the remaining six patients who responded poorly to therapy, the EBV genome copy number in the serum or plasma remained at high levels except for one case. In addition, we confirmed that the EBV genome became undetectable after hematopoietic stem cell transplantation in 4 EBV-HLH cases. These results suggest that the quantitative analysis of cell-free EBV genome copy number is useful for evaluating disease activity and for predicting the response to therapy in EBV-HLH.
2858	Expression of the helix-loop-helix gene id3 in the zebrafish embryo.	11900982	Proteins of the Extramacrochaetae and Id subfamily of Helix-Loop-Helix (HLH) proteins are negative regulators of bHLH transcription factors. We cloned a cDNA from zebrafish which encodes a member of the id3 subfamily. High levels of transcripts accumulated in the germ ring and in the embryonic shield. Towards the end of gastrulation, Id3 was highly expressed in the anterior prechordal plate and hypoblast. At later stages, id3 expression was turned on and off in a large variety of tissues within short periods of time. These include the lateral mesoderm, the cornea, the lens, the brain, the neural crest, the retina and the fins.
2859	The ISOBM TD-7 Workshop on hCG and related molecules. Towards user-oriented standardization of pregnancy and tumor diagnosis: assignment of epitopes to the three-dimensional structure of diagnostically and commercially relevant monoclonal antibodies directed against human chorionic gonadotropin and derivatives.	11893904	The ISOBM TD-7 hCG Workshop was established to characterize the molecular epitope structure and specificities of a panel of diagnostically relevant monoclonal antibodies (MAbs) directed against human chorionic gonadotropin (hCG) and its derivatives, and to consider how this information could be used to improve comparability of immunoassay results for these analytes. In this multicenter study, 27 MAbs have been characterized in detail as to their main and fine specificities by direct binding-, competitive- and sandwich-RIA, -ELISA, BIAcore and Western blotting. Antigens used in the study included the upcoming first WHO reference reagents for immunoassay, i.e. nick-free hCG (hCG), nicked hCG (hCGn), hCG alpha-subunit (hCGalpha), hCG beta-subunit (hCGbeta), nicked hCG beta-subunit (hCGbetan), hCG beta-core fragment (hCGbetacf), synthetic peptides of hCGbeta C-terminal peptide (hCGbetaCTP), and homologous hormones, luteinizing hormone (LH) and subunits (LHbeta) from various species. Correct classification of blinded internal controls demonstrated the reliability of the MAb referencing approach. Three-dimensional molecular epitope assignment was possible in many instances by comparing immunoreactivity of the ISOBM MAbs (n = 27) to a large panel of MAbs (n = 18) previously well characterized in the Innsbruck (P.B.) and Paris (J.M.B.) laboratories. All three major antibody specificities (alpha, n = 1; beta, n = 21; alphabeta, n = 5) were represented in the TD-7 MAb panel. HCGbeta MAbs could further be subdivided into (i) those recognizing hCGbeta only (epitopes: beta(6), n = 1; beta(7), n = 2; beta(14), n = 1) and (ii) those recognizing hCGbeta + hCG (beta1, beta2, beta4, beta5, n = 10; beta8 and beta9, n = 9). Members of the latter group were specific either for hCG + hCGbeta + hCGbetacf (beta1, n = 3) or hCG + hCGbeta + hCGbetaCTP (beta8, n = 6; beta9, n = 1) or in addition to hCG + hCGbeta + hCGbetacf recognized hLH/hLHbeta to a minor (beta2, n = 3; beta4, n = 3) or similar degree (beta5, n = 1). Epitopes were (i) located on the first and third loops protruding from the cystine knot of hCGbeta (beta2-beta6, aa hCGbeta20-25 and 68-77), (ii) presumably centered around the knot itself (beta1), or (iii) on hCGbetaCTP (epitope beta8 = hCGbeta141-144, beta9 = hCGbeta113-116). The ISOBM panel of MAbs represents all major epitope specificities suitable for the design of specific sandwich immunoassays. High analyte variability in serum and urine during the course of pregnancy and tumor development favors certain epitope combinations. For routine diagnostic purposes, assays recognizing a broad spectrum of hCG/hCGbeta variants such as hCG + hCGn + hCGbeta + hCGbetan + hCGbetacf + -CTPhCG + -CTPhCGbeta may be useful. Low cross-reactivity against related glycoprotein hormones (e.g. hLH) and their derivatives is mandatory. These criteria are best met by combinations of MAbs directed against epitopes located around the cystine knot (beta1) and against those encompassing the top of loops 1 and 3 on hCGbeta (beta2, beta4). The first WHO reference reagents for immunoassay of hCG and hCG-related molecules being prepared by the IFCC should facilitate characterization of what assays for 'hCG' are measuring. The next step towards improving between-laboratory comparability of measurements of hCG/hCG derivatives in pregnancy and oncology is provided by results of this TD-7 Workshop.
2860	Hypothalamic bHLH transcription factors are novel candidates in the regulation of energy balance.	11886445	The basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.
2861	Stenting of the arterial duct and banding of the pulmonary arteries: basis for combined Norwood stage I and II repair in hypoplastic left heart.	11877362	Outcome of patients with hypoplastic left heart (HLH) is mainly influenced by the successful first-step palliation according to the Norwood procedure. An alternative approach is heart transplantation (HTX). The feasibility of ductal stenting in newborns with duct-dependent systemic blood flow and bilateral pulmonary artery banding has been reported. But it remains to be elucidated whether this approach allows a new strategy for patients with HLH.In patients with various forms of HLH (n=11) and prostaglandin E-1 administration, ductal stenting was performed with balloon expandable Jo stents or Saxx stents. Bilateral pulmonary artery banding was surgically accomplished 1 to 3 days after the transcatheter procedure. Unrestricted blood flow through the interatrial septum was secured by balloon dilatation atrial septotomy, as required. Interventional procedures were performed with no mortality. Stent and ductal patency were achieved for up to 331 days. Two patients underwent HTX, and 8 patients had a palliative 1-stage procedure with reconstruction of the aortic arch and bidirectional cavopulmonary connection at the age of 3.5 to 6 months. There were 2 deaths. One patient with preoperative right heart failure died after the reconstructive surgery, and 1 patient died 4 months after ductal stenting and bilateral banding awaiting HTX.The present study is the first clinical trial showing that stenting the duct followed by bilateral pulmonary artery banding in newborns with HLH allows the combination of neoaortic reconstruction, which is part of first-stage palliation of HLH, with the establishment of a bidirectional cavopulmonary connection. Additionally, it allows the chance for HTX after extended waiting periods.
2862	Requirement for ERK1/2 activation in the regulation of progesterone production in human granulosa-lutein cells is stimulus specific.	11861509	This study was conducted to determine whether the ERK1/2 family of MAPKs can be modulated by physiological regulators of the human corpus luteum, and whether this activation is important for progesterone secretion in human granulosa-lutein (hGL) cells. Human LH (hLH), hCG, and agents that indirectly elevate cAMP [cholera toxin, forskolin, (Bu)(2)cAMP], time- and dose-dependently activated ERK1/2 in hGL cells. ERK1/2 activation was reduced by preincubation with PKA inhibitors, including myristoylated PKI, suggesting that cAMP mediates ERK1/2 activation. Two structurally distinct inhibitors of MAPK kinase (MEK), PD 98059 and U 0126, abrogated hLH/hCG-induced ERK1/2 activation, but had no effect on hLH-, hCG-, or 22R-hydroxycholesterol-stimulated progesterone secretion. In contrast, both inhibitors blocked cholera toxin-, forskolin-, and (Bu)(2)cAMP-induced ERK1/2 phosphorylation concomitant with a reduction in progesterone secretion. The known luteotropin, PGE(2), promoted MEK- and cAMP-dependent activation of ERK1/2, and inhibitors of either MEK or PKA decreased PGE(2)-induced progesterone synthesis. Our findings demonstrate that the requirement for ERK1/2 activation as a regulator of progesterone synthesis in hGL cells is stimulus dependent, and that the MEK inhibitor-sensitive step is distal to cAMP generation, but proximal to the conversion of cholesterol to pregnenolone.
2863	Pneumocystis carinii pneumonitis in haemophagocytic lymphohistiocytosis.	11853350	We report on a 10-y-old boy who developed Pneumocystis carinii pneumonitis (PCP) as the dominant symptom at the onset of haemophagocytic lymphohistiocytosis (HLH). PCP is a common infection in patients with combined primary immunodeficiencies or acquired immunodeficiency syndrome but it has rarely been observed at the onset of HLH. Typically, HLH presents as a febrile syndrome associated with cytopenia and hepatosplenomegaly. Repeated bone marrow aspirates, spleen or lymph node biopsies are sometimes required to reveal haemophagocytosis. Because of the significant immunosuppression during treatment of HLH, prophylaxis of PCP with co-trimoxazole is recommended. However, de-arranged immune response in HLH renders the patients susceptible to opportunistic infections, even before the introduction of immunosuppressants.We suggest that in patients with unclear respiratory symptoms, it is worth considering a differential diagnosis of HLH.
2864	cDNA array analysis of pineal gene expression reveals circadian rhythmicity of the dominant negative helix-loop-helix protein-encoding gene, Id-1.	11849369	The pineal gland is a major output of the endogenous vertebrate circadian clock, with melatonin serving as the output signal. In many species, elevated nocturnal melatonin production is associated with changes in pineal gene expression. In the current study, cDNA array analysis was used in an attempt to identify additional genes that exhibit day/night differential expression in the rat pineal gland. This revealed 38 candidate genes, including Id-1 (inhibitor of DNA binding and differentiation). Id-1 encodes a helix-loop-helix (HLH) protein that lacks a basic DNA binding domain and could affect pineal physiology via a dominant negative trans-acting regulatory activity. For this reason Id-1 was selected for further analysis. Id-1 was expressed in a major population of pineal cells and the Id-1 protein was associated with a nuclear complex. The levels of Id-1 mRNA and protein exhibit approximately six-fold day/night rhythms. In contrast, the related genes Id-2 and Id-3 do not exhibit marked day/night differences in pineal expression. Rhythmic Id-1 expression is primarily limited to a C-terminally extended splice variant of Id-1, which would restrict the functional output of the rhythm to protein binding partners of this isoform of Id-1. Our findings add to the body of evidence indicating that transcriptional regulators play a role in neuroendocrine rhythms, and extend this by introducing the concept of a dominant negative HLH involvement. The rhythm in Id-1 in the pineal gland provides an experimental opportunity to identify Id-1-binding partners which may also be involved in Id-1 activity in other functional contexts.
2865	Perforin defects of primary haemophagocytic lymphohistiocytosis in Japan.	11841437	The perforin gene was analysed in 15 Japanese patients with primary haemophagocytic lymphohistiocytosis (HLH). Perforin gene defects were found in two out of eight patients with familial HLH (FHL), and one out of seven without affected siblings. Four novel mutations were identified. Compound heterozygous mutations (one FHL and one sporadic HLH) and only one allele mutation (one FHL) were defined. Flow cytometry revealed no perforin expression in CD8+ or CD56+ cells from a surviving patient with a mutation. The frequency of mutation was at least 20% of FHL in Japan. Flow cytometry for intracellular perforin may be useful for the screening of FHL2.
2866	Id proteins in cell cycle control and cellular senescence.	11840324	The Id family of helix-loop-helix (HLH) proteins are thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcription factors. Although it has been suggested for some time that Id is involved in cell cycle regulation, little is known about the molecular mechanism of this control. Recent studies, however, have revealed that Id binds to important cell cycle regulatory proteins other than bHLH proteins. Two such proteins, pRB (retinoblastoma tumour suppressor protein) family proteins and Ets-family transcription factors are known to play key roles in cell cycle regulation, transformation and tumour suppression. Through the characterization of these pathways we will begin to understand the mechanisms by which Id controls normal and abnormal cell cycle progression.
2867	The regulation and function of the Id proteins in lymphocyte development.	11840323	Helix-loop-helix (HLH) proteins are essential factors for lymphocyte development and function. One class of HLH proteins, the E-proteins, regulate many aspects of lymphocyte maturation, survival, proliferation, and differentiation. E-proteins are negatively regulated by another class of HLH proteins known as the Id proteins. The Id proteins function as dominant negative inhibitors of E-proteins by inhibiting their ability to bind DNA. Here we discuss the function and regulation of the Id proteins in lymphocyte development.
2868	Emc, a negative HLH regulator with multiple functions in Drosophila development.	11840322	Expression and functional analyses of Emc have demonstrated that it is a prototype for a protein required for multiple processes in development. Initially characterized as a negative regulator of sensory organ development, it was later found to regulate many other developmental processes and cell proliferation. Its ability to block the function of bHLH proteins by forming heterodimers, which are ineffective in DNA binding, accounts for the role of Emc in preventing the acquisition of several cell fates which are under the control of bHLH proteins. However, while maintaining this repressive molecular mechanism, emc also appears to act as a positive regulator of differentiation.
2869	The basic helix-loop-helix domain of the E47 transcription factor requires other protein regions for full DNA binding activity.	11820794	Most transcription factors are believed to be composed of independently functioning modules [A. D. Frankel and P. S. Kim (1991) Cell 65, 717-719]. Basic helix-loop-helix (bHLH) transcription factors appear to fit this model, with the HLH domains mediating dimerization, the basic regions mediating DNA binding, and other modules controlling other functions such as transcriptional activation. We tested predictions of this model using forced dimers of bHLH proteins including E47 homodimers and MyoD:E47 heterodimers and found that protein dimers containing complete bHLH domains but lacking other regions of E47 have only 20% of the DNA binding ability and transcriptional transactivation activity of wild-type dimers. These results demonstrate that the bHLH domains do not function as completely independent DNA binding modules. In addition, these results demonstrate that the transcriptional activation domains from a single bHLH protein are sufficient to activate transcription.
2870	Blocking HES1 expression initiates GABAergic differentiation and induces the expression of p21(CIP1/WAF1) in human neural stem cells.	11809764	Mammalian neural stem cells can develop into a variety of neuronal and glial cell types. This involves a highly coordinated process of differentiation in which the Notch signaling pathway and the system of helix-loop-helix (HLH) transcriptional regulators play a key role. By exercising control over proliferation, initiation of differentiation, neurite outgrowth, and synaptogenesis, the network of HLH transcription factors regulates the fate of neural stem cells and progenitors. Here we show that the HLH transcription factor HES1 regulates the proliferation of human neural stem cells and that blocking its expression stimulates the expression of cyclin-dependent kinase inhibitor p21(CIP1/WAF1). Furthermore, we demonstrate that the suppression of HES1 expression initiates differentiation of neural stem cells into neurons, the majority of which develop the GABAergic phenotype. These findings underscore the importance of the HLH network, and HES1 in particular, in guiding the phenotypic development of neural stem cells.
2871	Dermo-1, a multifunctional basic helix-loop-helix protein, represses MyoD transactivation via the HLH domain, MEF2 interaction, and chromatin deacetylation.	11809751	Dermo-1 is a multifunctional basic helix-loop-helix (bHLH) transcription factor that has been shown to be a potent negative regulator for gene transcription and apoptosis. To understand the molecular mechanisms that mediate the function of Dermo-1, we generated a series of Dermo-1 mutants and used a MyoD-mediated transcriptional activation model to characterize the roles of its N-terminal, bHLH, and C-terminal structural domains in transcriptional repression. Both the C-terminal and HLH domains of Dermo-1 were essential for its repression of MyoD-mediated transactivation. Dermo-1 repressed, in a dose-dependent fashion, the transactivation activity of myocyte enhancer factor 2 (MEF2), a protein known to cooperate with MyoD in activating E-box-dependent gene expression. Both the N- and C-terminal domains of Dermo-1, but not the bHLH domain, were required for the inhibition of MEF2, suggesting that Dermo-1 inhibits both MyoD- and MEF2-dependent transactivation but through different mechanisms. Dermo-1 interacted directly with MEF2 and selectively repressed the MEF2 transactivation domain. An overall increase of histone acetylation induced by trichostatin A treatment reduced Dermo-1 transcriptional repression activity, suggesting that histone deacetylation is involved in Dermo-1-mediated transcriptional repression. Together, these results suggest that MEF2 is an important target in Dermo-1-mediated transcriptional repression and provide initial evidence of the involvement of histone acetylation in Dermo-1 transcriptional repression.
2872	Crosstalk between Myc and activating transcription factor 2 (ATF2): Myc prolongs the half-life and induces phosphorylation of ATF2.	11781825	Myc is a key regulator of cell growth, differentiation and apoptosis, and affects cell fate decisions by activating as well as by inhibiting the expression of cellular genes. Myc is a member of the basic region-helix-loop-helix-leucine zipper (b-HLH-Zip) class of transcription factors, which heterodimerizes with the Max protein and recognizes a consensus Myc binding motif. Stimulation of gene expression by Myc is thought to be mediated by direct binding of Myc-Max heterodimers to specific target genes. So far, only a few genes have been identified as direct binding targets of Myc, raising the possibility that Myc affects gene expression also by indirect mechanisms. In this work we present evidence that v-Myc encoded by the avian retrovirus MC29 stimulates activating transcription factor 2 (ATF2)-dependent transcription. Analysis of the effect of Myc on ATF2 shows that v-Myc prolongs the half-life of ATF2 and induces the phosphorylation of N-terminal sites of ATF2 (Thr-69 and Thr-71) which have previously been identified as the target sites of stress-activated protein kinases and implicated in the regulation of ATF2 activity. Taken together, our results suggest that v-Myc can affect gene expression indirectly by modulating the activity of ATF2.
2873	Expression of ID genes in differentiated elements of human male germ cell tumors.	11763316	The ID genes are members of a family of genes that encode helix-loop-helix (HLH)-containing proteins. The Id proteins, unlike other HLH proteins, lack an adjacent DNA binding domain and hence act as dominant negative regulators of HLH transcription factors that have been implicated in control of cellular differentiation. Although the role of Id genes in murine development has been documented, their roles in human embryogenesis remain unknown. In this study, human male germ cell tumors (GCTs) were used as a model for examining the expression of the ID genes in various histologies that are reflective of different temporal phases of human development. In seminomas, little or no expression of IDI, ID2, and ID3 was detected, consistent with the uncommitted germ cell-like phenotype of this tumor histology. Likewise, GCTs with histologies reflective of extraembryonic and embryonic patterns of differentiation exhibited patterns of expression of the three ID genes often similar to those noted during murine development. It was also evident, as revealed by ID expression patterns, that despite the overall aberrant spatial differentiation patterns displayed by these tumors, some tissue-tissue interactions reminiscent of those observed during normal embryogenesis are retained. Thus, adult male GCTs offer a unique system in which the role of genes such as the IDs can be studied in human embryogenesis.
2874	Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.	11760832	Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.
2875	Hemophagocytic lymphohistiocytosis (HLH) presenting on the 3rd day of life.	11758143	Hemophagocytic lymphohistiocytosis (HLH) embraces the frequently indistinguishable conditions, namely familial hemophagocytic lymphohistiocytosis (FHLH), sporadic hemophagocytic lymphohistiocytosis (SHLH) and virus associated hemophagocytic syndrome (VAHS). The disease is very rare and invariably lethal. Evidence suggests that the disease may be due to an inherited defect in immunoregulation that predisposes to an uncontrolled proliferation of activated histiocytes in response to a stimulus such as viral infection. We report here a 3-day-old neonate with confirmed HLH who had a stormy course and a fatal outcome to the disease process, in spite of early chemotherapy. To our knowledge, we believe this is the youngest reported case of HLH from Middle East. No familial or infective cause could be attributed.
2876	Modulation of basic helix-loop-helix transcription complex formation by Id proteins during neuronal differentiation.	11756408	It is assumed that the Id helix-loop-helix (HLH) proteins act by associating with ubiquitously expressed basic HLH (bHLH) transcription factors, such as E47 and E2-2, which prevents these factors from forming functional hetero- or homodimeric DNA binding complexes. Several tissue-specific bHLH proteins, including HASH-1, dHAND, and HES-1, are important for development of the nervous system. Neuroblastoma tumors are derived from the sympathetic nervous system and exhibit neural crest features. In differentiating neuroblastoma cells, HASH-1 is down-regulated, and there is coincident up-regulation of the transcriptional repressor HES-1, which is known to bind the HASH-1 promoter. We found that the three Id proteins expressed in neuroblastoma cells (Id1, Id2, and Id3) were down-regulated during induced differentiation, indicating that Id proteins help keep the tumor cells in an undifferentiated state. Studying interactions, we noted that all four Id proteins could dimerize with E47 or E2-2, but not with HASH-1 or dHAND. However, the Id proteins did complex with HES-1, and increased levels of Id2 reduced the DNA binding activity of HES-1. Furthermore, HES-1 interfered with Id2/E2-2 complex formation. The ability of Id proteins to affect HES-1 activity is of particular interest in neuronal cells, where regulation of HES-1 is essential for the timing of neuronal differentiation.
2877	Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members.	11756153	Mutations in the perforin gene have been described in some patients with hemophagocytic lymphohistiocytosis (HLH), but the role of perforin defects in the pathogenesis of HLH remains unclear. Four-color flow cytometric analysis was used to establish normal patterns of perforin expression for control subjects of all ages, and patterns of perforin staining in cytotoxic lymphocytes (natural killer [NK] cells, CD8(+) T cells, CD56(+) T cells) from patients with HLH and their family members were studied. Eleven unrelated HLH patients and 19 family members were analyzed prospectively. Four of the 7 patients with primary HLH showed lack of intracellular perforin in all cytotoxic cell types. All 4 patients showed mutations in the perforin gene. Their parents, obligate carriers of perforin mutations, had abnormal perforin-staining patterns. Analysis of cytotoxic cells from the other 3 patients with primary HLH and remaining family members had normal percentages of perforin-positive cytotoxic cells. On the other hand, the 4 patients with Epstein-Barr virus-associated HLH typically had depressed numbers of NK cells but markedly increased proportions of CD8(+) T cells with perforin expression. Four-color flow cytometry provides diagnostic information that, in conjunction with evidence of reduced NK function, may speed the identification of life-threatening HLH in some families and direct further genetic studies of the syndrome.
2878	Fate alteration of neuroepithelial cells from neurogenesis to astrocytogenesis by bone morphogenetic proteins.	11755226	Bone morphogenetic proteins (BMPs), a class of cytokines belonging to the transforming growth factor-beta superfamily, have been shown to play a wide variety of roles during development including those in the central nervous system. We here report that BMP2, BMP4 and BMP7 have an equivalent potential to inhibit neurogenesis and concomitantly induce astrocytogenesis of mouse fetal neuroepithelial cells. We further show that these BMPs activate a promoter of the gene for negative helix-loop-helix (HLH) factor, Id1, which is known to inhibit the function of such neurogenic transcription factors as Mash1 and neurogenin. These results suggest that BMP2, BMP4 and BMP7 alternate the fate of neuroepithelial cells from neuronal type to astrocytic one via a common mechanism involving negative HLH factor.
2879	Expression of Id helix-loop-helix proteins in colorectal adenocarcinoma correlates with p53 expression and mitotic index.	11751402	Id helix-loop-helix (HLH) proteins function as regulators of cell growth and differentiation and when overexpressed can induce malignant transformation. In a series of 34 cases of primary human colorectal adenocarcinoma, immunoreactivity for Id1, Id2, and Id3 was found to be significantly elevated in tumor compared with normal mucosa (P = 0.001 for Id1 and Id2; P = 0.002 for Id3). No elevation of Id expression was observed in 17 cases of adenoma. Expression of Id1 and to a lesser extent of Id2 was correlated with mitotic index (P = 0.005 for Id1; P = 0.042 for Id2) in human adenocarcinomas, and expression of all three Id proteins was correlated with p53 immunoreactivity (a marker of mutational 'inactivation' of p53 function; P = 0.002 for Id1; P = 0.006 for Id2; P = 0.016 for Id3). In normal intestinal mucosa of p53-null mice and in spontaneous tumors arising in Min+/- mice, expression of all three Id proteins was also found to be up-regulated. Antisense oligonucleotide blockade of Id protein expression inhibited the proliferation of human adenocarcinoma cells. Enforced, ectopic expression of the E47 basic HLH (bHLH) protein in human adenocarcinoma cell lines efficiently sequestered endogenous Id proteins as Id-bHLH heterodimers, as shown by coimmunoprecipitation and subcellular colocalization studies. This led to growth arrest of the cells. Enforced overexpression of a mutant E47 protein, deficient in transactivation and DNA binding function, also partially inhibited cell growth. Taken together, these data imply that deregulated expression of Id proteins in colorectal adenocarcinoma arises at least in part as a consequence of loss of p53 function and contributes to the uncontrolled proliferation of tumor cells in colorectal cancer.
2880	Upregulation of the HLH Id gene family in neural progenitors and glial cells of the rat spinal cord following contusion injury.	11746449	Spinal cord injury (SCI) leads to a complex sequence of cellular responses, including astrocyte activation, oligodendrocyte death, and ependymal cell proliferation. Inhibitors of DNA binding (Id1, Id2, Id3) belong to a helix-loop-helix (HLH) gene family. Id genes have been implicated in playing a vital role in the proliferation of many cell types, including astrocytes and myoblasts. In the present study, the expression of Id family members in spinal cord after contusion injury was investigated by in situ hybridization. Id1, Id2, and Id3 mRNA expression was upregulated 5 mm rostral and caudal to the lesion center, and reached maximal levels 3 days after SCI. In addition, cell populations expressing Id1, Id2, and Id3 mRNA were maximally increased 3 days after SCI. The increase in Id2 and Id3 mRNA expression and Id2 and Id3 mRNA+ cells was still observed at 8 days. The Id mRNA expressing cells were phenotyped by combining immunostaining of cell-specific markers with in situ hybridization. Glial fibrillary acidic protein (GFAP)+ astrocytes were found to express all three Id mRNA, whereas S-100alpha+ astrocytes only expressed high levels of Id2 and Id3 mRNA. Cells having a neural progenitor morphology and the marker nestin appeared after SCI and they expressed Id1, Id2, and Id3 mRNA. Interestingly, some Rip+ oligodendrocytes located in the areas close to the central canal expressed Id3 mRNA after injury. In conclusion, Id genes are upregulated in a time-dependent manner in astrocytes, oligodendrocytes, and neural progenitor subpopulations after SCI, suggesting that they play major roles in cellular responses following SCI.
2881	Asn(78) and His(81) form a destabilizing locus within the Max HLH-LZ homodimer.	11741584	The human Max protein lies at the center of the Myc/Max/Mad family of transcription factors. Its role at the center of this regulatory network is dependent on the helix-loop-helix leucine zipper (HLH-LZ) dimerization domain. The Max LZ contains three residues that deviate from the pattern of hydrophobic amino acids normally present at the interface of LZ dimers: Asn(78), His(81) and Asn(92). In contrast to interfacial Asn residues in other LZ proteins, we have shown that Asn(92) does not act to destabilize the homodimer. Here we describe thermal denaturation experiments performed on Asn(78) and His(81) mutants demonstrating that these residues are involved in actively destabilizing the Max homodimer.
2882	Effect of maternal undernutrition during pregnancy on early ovarian development and subsequent follicular development in sheep fetuses.	11732987	Gonad development in female sheep fetuses is thought to occur in a number of key stages. The aim of this study was to determine the effects of maternal undernutrition, applied at one or more of these critical stages, on fetal ovarian development. Groups of ewes (n = 11-19) were fed rations providing either 100% (high; H) or 50% (low; L) of energy requirements for live weight maintenance during selected 'windows' during gestation. Control ewes (HH and HHH) were fed the H ration from mating until they were killed at days 50, 65 (HH) or 110 (HHH) of gestation, whereas ewes of other groups were fed the L ration for the periods between day 0 and day 30 of gestation (LH and LHH), day 31 and day 50 or 65 of gestation (HL and HLH), day 65 and day 110 of gestation (HHL) or day 0 of gestation until the animals were killed (LL and LLL). At day 50 of gestation, there was no effect of nutritional treatment on mean fetal mass but compared with HH animals, mean fetal ovarian mass was significantly lower in HL (P < 0.05) and LL (P < 0.001) animals. At day 65 of gestation, there were significantly fewer germ cells (P < 0.05) at the resting, diplotene stage of initial meiosis in LL animals than there were in HH animals, indicating delayed germ cell maturation and onset of meiosis. Qualitative assessment of proliferative cell nuclear antigen immunostaining indicated that, at day 50 of gestation, staining was located predominantly in the germ cells, whereas by day 65 of gestation, staining was confined predominantly to somatic cells. Undernutrition in each one of these windows was associated with delayed ovarian follicular development (P < 0.05-0.001) as measured by development of the granulosa cell layer at day 110 of gestation. This study demonstrates that undernutrition before and during folliculogenesis can delay fetal follicular development.
2883	Fusion of ETV6 to fibroblast growth factor receptor 3 in peripheral T-cell lymphoma with a t(4;12)(p16;p13) chromosomal translocation.	11731410	Fusions of the ETV6/TEL gene to receptor or protein tyrosine kinases (TKs), such as PDGFRbeta, JAK2, ABL, ABL2, TRKC, and Syk, have been reported in various hematological malignancies. Expression of the resultant chimeric proteins is believed to lead to constitutive TK activity through activation by the helix-loop-helix (HLH) domain of ETV6. We identified a novel ETV6 partner gene, fibroblast growth factor receptor 3 (FGFR3), in a patient with peripheral T-cell lymphoma (PTCL) with a t(4;12)(p16;p13) translocation. The ETV6-FGFR3 transcript showed a fusion of exon 5 of ETV6 to exon 10 of FGFR3, resulting in an open reading frame for a chimeric protein consisting of the HLH domain of ETV6 and the TK domains of FGFR3. This is the first report of ETV6 and FGFR3 involvement in PTCL.
2884	Early patterning of the prospective midbrain-hindbrain boundary by the HES-related gene XHR1 in Xenopus embryos.	11731236	The molecular mechanisms that govern early patterning of anterior neuroectoderm (ANE) for the prospective brain region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1). XHR1 is specifically expressed in the midbrain-hindbrain boundary (MHB) region at the tailbud stage. The localized expression of XHR1 was detected as early as the early gastrula stage in the presumptive MHB region, an area just anterior to the involuting dorsal mesoderm that is demarcated by the expression of the gene Xbra. Expression of XHR1 was detected much earlier than that of other known MHB genes, XPax-2 and En-2, and also before the formation of the expression boundary between Xotx2 and Xgbx-2, suggesting that the early patterning of the presumptive MHB is independent of Xotx2 and Xgbx-2. Instead, the location of XHR1 expression appears to be determined in relation to the Xbra expression domain, since reduced or ectopic expression of Xbra altered the XHR1 expression domain according to the location of Xbra expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1 in the MHB region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1. Furthermore, ectopically expressed wild-type XHR1 near the MHB region enhanced En-2 expression only in the MHB region but not in the region outside the MHB. These data suggest that XHR1 is required, but not sufficient by itself, to initiate MHB marker gene expression. Based on these data, we propose that XHR1 demarcates the prospective MHB region in the neuroectoderm in Xenopus early gastrulae.
2885	Prominent dyserythropoiesis in four cases of haemophagocytic lymphohistiocytosis.	11729218	Haemophagocytic lymphohistiocytosis (HLH) is a disease characterised by peripheral blood pancytopenia secondary to haemophagocytosis of formed blood cells by activated histiocytes. The demonstration of haemophagocytosis may be difficult and the diagnosis may require repeated tissue sampling (including bone marrow, cerebrospinal fluid, lymph nodes, spleen, and liver) and the demonstration of associated clinical or laboratory features. This report describes pronounced dyserythropoiesis in the bone marrow aspirates in four patients with HLH, including familial and secondary cases. In three patients, bone marrow haemophagocytosis was inconspicuous or absent, and the prominent dyserythropoiesis may have suggested an alternative diagnosis. The dyserythropoiesis observed should be added to the constellation of clinical and laboratory features associated with HLH.
2886	TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3.	11711431	Transforming growth factor-beta (TGF-beta) is a potent inhibitor of skeletal muscle differentiation, but the molecular mechanism and signaling events that lead to this inhibition are poorly characterized. Here we show that the TGF-beta intracellular effector Smad3, but not Smad2, mediates the inhibition of myogenic differentiation in MyoD-expressing C3H10T1/2 cells and C2C12 myoblasts by repressing the activity of the MyoD family of transcriptional factors. The Smad3-mediated repression was directed at the E-box sequence motif within muscle gene enhancers and the bHLH region of MyoD, the domain required for its association with E-protein partners such as E12 and E47. The repression could be overcome by supplying an excess of E12, and covalent tethering of E47 to MyoD rendered the E-box-dependent transcriptional activity refractory to the effects of Smad3 and TGF-beta. Smad3 physically interacted with the HLH domain of MyoD, and this interaction correlated with the ability of Smad3 to interfere with MyoD/E protein heterodimerization and binding of MyoD complexes to oligomerized E-box sites. Together, these results reveal a model for how TGF-beta, through Smad3-mediated transcriptional repression, inhibits myogenic differentiation.
2887	Contribution of the helix-loop-helix factor Id2 to regulation of vascular smooth muscle cell proliferation.	11706002	Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loop-helix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enhanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression.
2888	Interleukin-18 in hemophagocytic lymphohistiocytosis.	11699209	Hemophagocytic lymphohistiocytosis (HLH) is characterized by dysregulated hyperactivation of macrophages and T helper 1 (Th1) cells accompanied by excessive secretion of inflammatory cytokines. Although TNF-alpha and IFN-gamma are known to be important factors for the development of the disease, the mechanism of their overproduction has not been clarified, yet. We measured serum IL-18 levels of patients with HLH to investigate the possible significance of IL-18 in its pathophysiology, especially in IFN-gamma production. IL-18 levels were significantly increased in all patients with HLH compared with healthy controls. A significant correlation was observed between IL-18 and IFN-gamma levels. In addition to IFN-gamma and soluble Fas ligand (sFasL), IL-18 levels significantly correlated with disease activity. IL-18 may play important roles in the pathogenesis of HLH, particularly through induction of Th1 cells. In addition, IL-18 measurement may not only be useful for the diagnosis, but also for the evaluation of disease activity.
2889	Development of a fluorescence based high throughput assay for antagonists of the human chorionic gonadotropin receptor extracellular domain: analysis of peptide inhibitors.	11689111	A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.
2890	Linkage of M-CSF signaling to Mitf, TFE3, and the osteoclast defect in Mitf(mi/mi) mice.	11684011	Osteoclasts are multinucleated hematopoietic cells essential for bone resorption. Macrophage colony-stimulating factor (M-CSF) is critical for osteoclast development and function, although its nuclear targets in osteoclasts are largely unknown. Mitf and TFE3 are two closely related helix-loop-helix (HLH) transcription factors previously implicated in osteoclast development and function. We demonstrate that cultured Mitf(mi/mi) osteoclasts are immature, mononuclear, express low levels of TRAP, and fail to mature upon M-CSF stimulation. In addition, M-CSF induces phosphorylation of Mitf and TFE3 via a conserved MAPK consensus site, thereby triggering their recruitment of the coactivator p300. Furthermore, an unphosphorylatable mutant at the MAPK consensus serine is specifically deficient in formation of multinucleated osteoclasts, mimicking the defect in Mitf(mi/mi) mice. These results identify a signaling pathway that appears to coordinate cytokine signaling with the expression of genes vital to osteoclast development.
2891	The role of infections in primary hemophagocytic lymphohistiocytosis: a case series and review of the literature.	11595993	There is a paucity of literature addressing infection-related morbidity and mortality in children with primary hemophagocytic lymphohistiocytosis (HLH), a rare condition characterized by abnormal proliferation of macrophages, hypercytokinemia, and T cell immunosuppression. Therefore, a retrospective chart review was done of patients diagnosed with primary HLH over a 15-year period. Significant infections present at diagnosis, during the course of illness, and just prior to death or at autopsy were noted. Of the 18 children identified with primary HLH, an infectious agent was documented at the initial presentation of HLH in 5. Significant infections occurred during therapy in 10 (56%) of 18. Of the 12 fatal cases, invasive infection was the cause of death in 8 children, and 6 of these deaths were directly attributable to invasive fungal infection. Significant infections were common during therapy in children with primary HLH, and fungal infections were an important cause of mortality in this group.
2892	Recent developments in the management of haemophagocytic lymphohistiocytosis.	11585022	Over the past two decades, the underlying pathophysiology of haemophagocytic lymphohistiocytosis (HLH) (synonyms: haemophagocytic syndrome, macrophage activation syndrome) has been well recognised. Cytokine storm plays a major role, which derives from an inappropriate immune reaction caused by proliferating and activated T-cell or natural killer (NK) cells associated with macrophage activation and inadequate apoptosis of immunogenic cells. Many biological parameters reflecting activity of disease or response to treatment have been identified, in particular, serum ferritin has been confirmed to be one of the markers for HLH. The common types of HLH consist of non-hereditary (acquired) infection-associated disease such as Epstein-Barr virus (EBV)-haemophagocytic lymphohistiocytosis (HLH) and hereditary (familial) disease such as FHL, in which, at the molecular level, dysfunctional perforin was clarified. Regarding the therapeutic strategies, prompt differential diagnosis of underlying disease is essential and choice of treatment should be based on the risk (low or high) of prognosis, where either cyclosporin A, steroids or iv. immunoglobulin (IVIG) may be indicated as initial treatment for low-risk patients, with etoposide-containing regimens for high-risk patients. Significant improvement of prognosis has been obtained by incorporating intensive supportive care at the disease onset and prompt introduction of immunosuppressants to control cytokine storm. Subsequent immunochemotherapy and haemopoietic stem cell transplantation have contributed significantly to further improve survival of hereditary and refractory HLH patients.
2893	Metabolism of gonadotropins: comparisons of the primary structures of the human pituitary and urinary LH beta cores and the chimpanzee CG beta core demonstrate universality of core production.	11572797	The gonadotropins are a family of closely related heterodimeric glycoprotein hormones homologous in structure to disulfide-knot growth factors. Metabolic proteolytic processing in vivo of this disulfide cross-linked region results in urinary excretion of a residual highly stable core structure. The primary structure of the pituitary form of the hLH beta core was reported earlier, but it has proved difficult to isolate the urinary core, although antibodies to the pituitary core demonstrated its presence. By conventional and immunoaffinity methods, the urinary core has been isolated and its structure determined by both chemical and mass spectrometric methods. The urinary hLH beta core is the same as the pituitary-extracted hLH beta core, beta 6-40 disulfide bridged to beta 55-93, except that the pituitary core is more heterogeneous containing also beta 49-93. These findings imply a dual origin of urinary cores, both directly from a secreting tissue and by kidney processing of circulating hormone. We also found that pregnant chimpanzees excrete a CG beta core with a primary structure identical to that of the human CG beta core of pregnancy. In conclusion, gonadotropin core generation and urinary excretion of nearly identical gonadotropin metabolites is common among primates. Although possible biological functions of these core fragments remain unproven, they have diagnostic utility because of their stability and abundance.
2894	In vivo function of a differentiation inhibitor, Id2.	11569914	Cell differentiation is an essential process for the development of various cell types that constitute multicellular organisms. During development, the large family of factors bearing a helix-loop-helix (HLH) motif participates profoundly in this process and these factors serve as good experimental tools for investigating mechanisms underlying tissue-specific differentiation. The HLH family includes both positive and negative regulators of cell differentiation: basic HLH (bHLH)-type transcription factors and Id proteins, respectively. Following an exciting decade focusing on bHLH factors, advances achieved in studies of the inhibitory factors in the last couple of years have placed them in the front line of the research on differentiation and proliferation control. Here, we present and discuss recent results obtained using Id2-deficient mice, which manifest intriguing phenotypes in various systems.
2895	A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action.	11560930	Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98-112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loop-helix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.
2896	Liver failure with marked hyperferritinemia: 'ironing out' the diagnosis.	11544539	Hemophagocytic lymphohistiocytosis (HLH) may manifest as neonatal liver failure characterized by hepatosplenomegaly, profound coagulopathy, ascites and hyperbilirubinemia. Marked hyperferritinemia may be present in these patients, mimicking perinatal hemochromatosis. Tissue specimens are critical in distinguishing these two diseases and in directing management. Clinical recognition and diagnosis of HLH can be difficult but are crucial for appropriate therapy and genetic counselling. Liver transplantation is absolutely contraindicated for patients with HLH but may be the only life-saving treatment modality for patients with perinatal hemochromatosis.
2897	Differential cellular targets of Epstein-Barr virus (EBV) infection between acute EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection.	11535525	Unusual Epstein-Barr virus (EBV) infection into T or natural killer cells plays a pivotal role in the pathogenesis of acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV). The precise frequency and localization of EBV genome in lymphocyte subpopulations especially within T-cell subpopulations are unclear in these EBV-related disorders. This study analyzed the frequency of EBV-infected cells in circulating lymphocyte subpopulations from 4 patients with acute EBV-HLH and 4 with CAEBV. EBV- encoded small RNA-1 in situ hybridization examination of peripheral blood lymphocytes showed a significantly higher frequency of EBV-infected cells of 1.0% to 13.4% in EBV-HLH and 1.6% to 25.6% in CAEBV, respectively. The patterns of EBV infection in lymphocyte subpopulations were quite different between acute EBV-HLH and CAEBV. EBV infection was predominant in CD8(+) T cells in all EBV-HLH patients, whereas the dominant EBV-infected cell populations were non-CD8(+) lymphocyte subpopulations in CAEBV patients. Phenotypical analysis revealed that EBV-infected cell populations from both EBV-HLH and CAEBV were activated. There was no predominance of any EBV substrain of latent membrane protein-1, EBV-associated nuclear antigen (EBNA)-1, and EBNA-2 genes between the 2 abnormal EBV-associated disorders, and self-limited acute infectious mononucleosis. These results showing differential virus-cell interactions between acute EBV-HLH and CAEBV indicated different pathogenic mechanisms against EBV infection between the 2 EBV-associated diseases, which accounts for the difference in clinical manifestations between the 2 diseases.
2898	Oligoclonal expansion of alphabeta T lymphocytes in Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with abnormal karyotypes.	11520570	We observed a fatal case of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) with an abnormal karyotype. Increased levels of alphabeta T cells of the patient were investigated using an inverse polymerase chain reaction (PCR) of the T-cell receptor variable region gene, followed by Jbeta-PCR and single-strand conformation polymorphism (SSCP) to confirm the clonality of specific alphabeta-T cell subsets. A high frequency (>15%) was recognized in Vbeta9 at onset, but not in any Vbeta and Valpha families 2 weeks after chemotherapy. High levels (>20%) of some Jbeta genes were detected in all Vbeta families investigated, and the predominant bias of the Jbeta2 gene relative to the Jbeta1 gene (86.1% versus 13.9% at onset, and 77.4% versus 23.5% after chemotherapy) was recognized in pan-alphabeta T cells. When each Vbeta-Jbeta fragment was compared among the samples at onset and after chemotherapy by SSCP analysis, several distinct bands were observed that indicate a clonal evolution. Thus, the findings suggest that some of the alphabeta T cell clones could be associated with abnormal karyotypes in EBV-HLH. The present findings provide molecular evidence of the presence of oligoclonal T cells in pan-alphabeta-T cells and clonal evolution during a short clinical course in EBV-HLH with abnormal karyotypes.
2899	Acute encephalopathy as a primary manifestation of haemophagocytic lymphohistiocytosis.	11508922	Haemophagocytic lymphohistiocytosis (HLH) is characterized anatomically by an infiltration of multiple tissues with lymphocytes and haemophagocytic histiocytes. First symptoms are usually hepatosplenomegaly, pancytopenia, and intractable fever. Up to 73% of those with HLH develop CNS involvement during the disease course. The peculiarity of the two patients presented here, a 20-month-old Italian female and a 4-year-old Moroccan female, is that the initial presenting neurological symptoms mimicked an encephalitis, anticipating the typical systemic symptoms by 1 and 4 months. They developed progressive encephalopathy accompanied by status epilepticus, one child developed a secondary hydrocephalus. In both children it was not possible to detect an underlying infection or malignant disease and there were no other cases in the family that suggested a familial form of HLH. Diagnosis and initiation of treatment was delayed because of the initial encephalopathic clinical picture and the late onset of the typical systemic features. As early diagnosis allows better therapeutical approaches, haemophagocytic lymphohistiocytosis should be considered in children with persistent or progressive findings of encephalopathy, especially in the absence of identification of a plausible pathogen.
2900	Cross-regulatory interaction between Stra13 and USF results in functional antagonism.	11498797	Transcription factors belonging to the basic helix-loop-helix (bHLH) family are critical regulators of cellular proliferation and differentiation. The functional activity of these proteins can be regulated by heterodimerization through the HLH domain, as a result of formation of functional or non-functional heterodimers. The presence of a leucine zipper in bHLH-leucine zipper (bHLHZip) proteins, however, prevents such heterodimeric interactions via the HLH domain between bHLH and bHLHZip proteins. To identify cellular proteins that directly interact with and modulate transcriptional repression mediated by the bHLH protein Stra13, we carried out a yeast two hybrid screen. The bHLHZip protein USF (Upstream Stimulatory factor) was identified as a Stra13 interacting protein. We demonstrate a direct interaction between Stra13 and USF that is dependent upon the C-terminal repression domain of Stra13 and the DNA-binding domain of USF. Stra13 and USF also colocalize and functionally interact in mammalian cells. Co-expression of USF abrogates Stra13-mediated repression of target genes and conversely, Stra13 inhibits DNA-binding and USF-mediated transactivation. Taken together, our data demonstrate that Stra13 and USF interact physically and functionally, and identify a novel mode of cross regulatory interaction between members of the bHLH and bHLHZip families that abrogates their functional activity.
2901	Successful umbilical cord blood transplantation from an unrelated donor for a patient with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	11477448	We report a case of a 5-year-old girl with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) who underwent cord blood (CB) stem cell transplantation (CBSCT) from an unrelated donor. The patient presented with persistent high-grade fever and hepatosplenomegaly. Because the disease was refractory to immunochemotherapy according to the HLH94 protocol, she received 2.0 x 10(7) CB nucleated cells/kg body weight (BW) after conditioning with BU/CY/etoposide. No acute GVHD developed, using FK506 for prophylaxis. The neutrophil count reached >0.5 x 10(9)/l by day 21 and the platelet count reached >50 x 10(9)/l by day 84. The patient recovered well with sequelae of neurological deficits more than 10 months after receiving CBSCT, without showing evidence of HLH or chronic GVHD. Real-time PCR proved applicable for estimation of the EBV load in PBMC of the patient. We conclude that CBSCT may be indicated for some cases of refractory EBV-HLH, who have no HLA-matched siblings and are therefore dependent on unrelated marrow donors.
2902	Bilateral subdural effusions related to disease activity in familial hemophagocytic lymphohistiocytosis in an 8-month-old infant.	11464995	An 8-month-old girl had classic features of hemophagocytic lymphohistiocytosis (HLH). A presumptive diagnosis of familial hemophagocytic lymphohistiocytosis was made on the basis of her age and the presence of parental consanguinity. In view of abnormal neurologic findings at presentation, a magnetic resonance imaging scan was performed and showed bilateral proteinaceous subdural effusions. These resolved within 1 week of commencement of chemotherapy for the primary condition. These subdural effusions were the only objective documentations of central nervous system involvement, along with an increased cerebrospinal fluid protein level. We also report other radiologic findings of HLH, which are of use in strengthening this diagnosis in individuals in whom the diagnosis is strongly suspected.
2903	Invertebrate myogenesis: looking back to the future of muscle development.	11448630	Recent studies in invertebrates have provided important mechanistic insights into several general aspects of muscle development. Two new genes have been identified that are involved in muscle fusion in Drosophila and a novel maternal component was shown to be responsible for myogenic determination in an ascidian. In addition, genetic analyses of nematode and Drosophila homologues of factors known to be myogenic regulators in other species yielded surprising findings about both the evolutionary conservation and divergence of these functions. Drosophila myogenesis has become a highly informative model for understanding the interplay between the signaling and transcriptional networks that underlie cell-fate specification during embryonic development.
2904	Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a.	11427735	The Id family of helix-loop-helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G(1)-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1 inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.
2905	Measurement of serum and peritoneal fluid LH concentrations as a diagnostic tool for human endometriosis.	11427164	A rapid, sensitive enzymeimmunoassay for the measurement of LH concentrations in serum and peritoneal fluid samples of healthy women and women with endometriosis is reported. The ligand (LH) was captured by a readily available, widely used and well-characterized monoclonal antibody (mAb, 518B7) generated against the beta subunit of bovine LH. This mAb, although specific for LH, shows very little species specificity and detects LH by radioimmunoassay in humans. A polyclonal antiserum raised in rabbits against hCG was conjugated to horseradish peroxidase and was used as the second antibody signal. This anti-hCG antiserum crossreacts with LH. The enzymeimmunoassay uses the standard human LH (hLH) preparations (NIADDK-hLH-I-3, AFP-827OB) and results are based on the relative concentrations of LH in serum and peritoneal fluid. Total assay time was < 3 h. The range of the standard curve was 0.002-0.500 ng LH per well and the lowest concentration of hLH that could be distinguished from zero concentration was 0.15 +/- 0.02 ng ml(-1) serum and 0.058 +/- 0.021 ng ml(-1) peritoneal fluid. Clinical diagnostic parameters for the LH enzymeimmunoassay showed a sensitivity of 85.71%, specificity 92.50%, efficiency 88.54%, positive predictive value 94.11% and negative predictive value 82.22%. The study was retrospective. Serum LH concentrations of women with endometriosis were 13.67 +/- 7.21 ng ml(-1), whereas serum LH concentrations of women in the control group were 4.52 +/- 2.03 ng ml(-1). One-way ANOVA showed significant differences (P < 0.001) between women with endometriosis and control groups. Women in the control group had peritoneal fluid LH values of 5.65 +/- 2.43 ng ml(-1), whereas peritoneal fluid LH values of 64.06 +/- 16.44 ng ml(-1) were obtained in women with endometriosis (P < 0.001). A cycle-dependent pattern of serum and peritoneal fluid LH concentration was observed in women in the control group, which was not observed in the peritoneal fluid of the group with endometriosis. The application of this assay to serum or peritoneal fluid samples provides the attractive possibility that it could be included in the panel of markers used for diagnosis of endometriosis.
2906	Familial hemophagocytic lymphohistiocytosis: how late can the onset be?	11410413	Most patients with familial hemophagocytic lymphohistiocytosis (HLH) develop the disease within the first two years of age. In a minority of cases a later occurrence has been reported, with an upper age limit of eight years. A significant concordance of the age at onset within each family has also been observed.We report four cases of families with HLH diagnosed at an unusually late age, comprised between between 9 and 17 years; in each of these families another child developed the disease in infancy. The natural killer activity of the patients was depleted; nevertheless, we had indirect evidence that, in at least two families, mutations of the perforin gene were not causing the disease.Such a late onset is very unusual and suggests that there is a subgroup of families with HLH in which the disease may present early or late in different members. Thus in some families with HLH the siblings might remain at risk of developing the disease for several years. Their actual risk cannot be defined until the genetic mutation is identified in each family and assessed in each member.
2907	High-level bacterial expression of a natively folded, soluble extracellular domain fusion protein of the human luteinizing hormone/chorionic gonadotropin receptor in the cytoplasm of Escherichia coli.	11394638	We have expressed the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor as a fusion protein with thioredoxin in the cytoplasm of an Escherichia coli strain that contains mutations in both the thioredoxin reductase and glutathione reductase genes. The chimeric protein isolated following induction of expression is purified in a soluble form and binds hCG with an affinity approximating that of native receptor. This truncated form of the receptor displays the same specificity as intact hLH/CG receptor and does not bind human follicle stimulating hormone. This cytoplasmically produced protein is expressed at levels that exceed 10 mg/L. Expression of properly folded extracellular domain of the hLH/CG receptor in the cytoplasm of E. coli allows the facile and economic purification of large quantities of material. This will facilitate the determination of the structure of the hormone-binding domain of the glycoprotein receptor as well as the production of epitope-specific antibodies.
2908	Oligomerization mediated by a helix-loop-helix-like domain of baculovirus IE1 is required for early promoter transactivation.	11390606	IE1 is a principal transcriptional regulator of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is stimulated when early viral promoters are cis linked to homologous-region (hr) enhancer sequences of AcMNPV. This transcriptional enhancement is correlated with the binding of IE1 as a dimer to the 28-bp palindromic repeats comprising the hr enhancer. To define the role of homophilic interactions in IE1 transactivation, we have mapped the IE1 domains required for oligomerization. We report here that IE1 oligomerizes by a mechanism independent of enhancer binding, as demonstrated by in vitro pull-down assays using fusions of IE1 (582 residues) to the C terminus of glutathione S-transferase. In vivo oligomerization of IE1 was verified by immunoprecipitation of IE1 complexes from extracts of plasmid-transfected SF21 cells. Analyses of a series of site-directed IE1 insertion mutations indicated that a helix-loop-helix (HLH)-like domain extending from residue 543 to residue 568 is the primary determinant of oligomerization. Replacement of residues within the hydrophobic face of the putative dimerization domain disrupted IE1 homophilic interactions and caused loss of IE1 transactivation of hr-dependent promoters in plasmid transfection assays. Thus, oligomerization is required for IE1 transcriptional stimulation. HLH mutations also reduced IE1 stability and abrogated transactivation of non-hr-dependent promoters. These data support a model wherein IE1 oligomerizes prior to DNA binding to facilitate proper interaction with the symmetrical recognition sites within the hr enhancer and thereby promote the transcription of early viral genes.
2909	CD14(dim)/CD16(bright) monocytes in hemophagocytic lymphohistiocytosis.	11385615	Hemophagocytic lymphohistiocytosis (HLH) is an extremely rare and highly lethal chronic inflammatory disease, which is mediated by proinflammatory cytokines. In the peripheral blood of a boy suffering from HLH, a chronic expansion of CD14(dim)/CD16(bright) inflammatory monocytes was detected. Compared with CD14(bright) monocytes, their immunophenotype correlated with more mature monocytic cells differentiating to macrophages: they showed lower expression of CD11b, CD64 and CD35. Such CD14(dim)/CD16(bright) monocytes produce the inflammatory cytokines IL-1beta, IL-6 and TNF-alpha. They fit in well with the pathophysiological concept of HLH as an inflammatory state of lymphocytes and of the monocyte/macrophage system. In the presented patient the percentage of these circulating inflammatory monocytes decreased over time during clinical response to immunosuppressive therapy. This finding may indicate that CD14(dim)/CD16(bright) monocytes represented the degree of inflammation in this extremely rare and highly lethal disease.
2910	Requirement for etoposide in the treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.	1135295811786591	We sought to identify the clinical variables most critical to successful treatment of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH).Among the factors tested were age at diagnosis (< 2 years or > or = 2 years), time from diagnosis to initiation of treatment with or without etoposide-containing regimens, timing of cyclosporin A (CSA) administration during induction therapy, and the presence or absence of etoposide.By Kaplan-Meier analysis, the overall survival rate for the entire cohort of 47 patients, most of whom had moderately severe to severe disease, was 78.3% +/- 6.7% (SE) at 4 years. The probability of long-term survival was significantly higher when etoposide treatment was begun less than 4 weeks from diagnosis (90.2% +/- 6.9% v 56.5% +/- 12.6% for patients receiving this agent later or not at all; P <.01, log-rank test). Multivariate analysis with the Cox proportional hazards model demonstrated the independent prognostic significance of a short interval from EBV-HLH diagnosis to etoposide administration (relative risk of death for patients lacking this feature, 14.1; 95% confidence interval, 1.16 to 166.7; P =.04). None of the competing variables analyzed had significant predictive strength in the Cox model. However, concomitant use of CSA with etoposide in a subset of patients appears to have prevented serious complications from neutropenia during the first year of treatment.We conclude that early administration of etoposide, preferably with CSA, is the treatment of choice for patients with EBV-HLH.
2911	Specific Pax-6/microphthalmia transcription factor interactions involve their DNA-binding domains and inhibit transcriptional properties of both proteins.	11350962	Pax-6 and microphthalmia transcription factor (Mitf) are required for proper eye development. Pax-6, expressed in both the neuroretina and pigmented retina, has two DNA-binding domains: the paired domain and the homeodomain. Mice homozygous for Pax-6 mutations are anophthalmic. Mitf, a basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factor associated with the onset and maintenance of pigmentation, identifies the retinal pigmented epithelium during eye development. Loss of Mitf function results in the formation of an ectopic neuroretina at the expense of the dorsal retinal pigmented epithelium. In the present study, we investigated the interaction between Pax-6 and Mitf. In transient transfection-expression experiments, we found that transactivating effects of Pax-6 and Mitf on their respective target promoters were strongly inhibited by co-transfection of both transcription factors. This repression was due to direct protein/protein interactions involving both Pax-6 DNA-binding domains and the Mitf b-HLH-LZ domain. These results suggest that Pax-6/Mitf interactions may be critical for retinal pigmented epithelium development.
2912	DNA binding properties of basic helix-loop-helix fusion proteins of Tal and E47.	11350595	The basic helix-loop-helix (bHLH) transcription factor Tal has been shown to form heterodimers with the ubiquitously expressed bHLH transcription factor E47 and thereby modulate gene expression. The absence of homodimeric Tal-DNA complexes had been attributed to the inability of Tal to homodimerize, but subsequent studies have shown that the bHLH region of Tal does homodimerize. In order to correlate the contributions of both the basic region and the helix-loop-helix (HLH) domain to the lack of DNA binding by Tal homodimers, mutant and fusion proteins based on Tal and E47 were designed and synthesized. Size-exclusion chromatography established that all mutant and fusion proteins were dimeric. Point mutations were made within the basic region of Tal based on residues within E47 that are essential for DNA binding, but an affinity for DNA was not observed. Even complete replacement of the basic region in Tal with the basic region of E47, in an E47-Tal fusion protein, did not confer DNA binding upon the protein. However, when the dimerization domain in Tal was replaced with its E47 counterpart, in a Tal-E47 fusion protein, sequence specific DNA binding was observed with an apparent dissociation constant of 3.6 x 10(-9) M2. Furthermore, circular dichroism studies showed that the basic region of Tal in the Tal-E47 fusion protein underwent a random coil to helix transition in the presence of a specific DNA probe. These experimental observations indicate that the inability of Tal homodimers to recognize DNA stems from a misalignment of its basic region with respect to the HLH domain, rather than an intrinsic inability of the Tal basic region to bind DNA.
2913	Bone marrow transplantation in hemophagocytic lymphohistiocytosis.	11342360	Two important syndromes of hemophagocytic lymphohistiocytosis (HLH) have to be considered in infants and young children with recurrent fever, organomegaly and cytopenias. Familial hemophagocytic lymphohistiocytosis (FHLH) is a genetically heterogeneous autosomal recessive disease with histiocytic and lymphocytic infiltrations in multiple organs and is currently curable only by bone marrow transplantation (BMT). Secondary HLH most commonly results from viral infections and some patients may be cured by treating the causative organism, others will need chemotherapy and immunosuppression. Since infections can also trigger disease episodes in FHLH, making the correct diagnosis can prove difficult. The published experience of BMT in HLH is reviewed. Taken together, cure of the majority of patients with HLH by matched related BMT, unrelated or haploidentical BMT is possible. Incomplete resolution of disease activity does not necessarily impede a successful outcome. Central nervous system involvement will eventually develop in many HLH patients and may cause considerable morbidity. Appropriate early treatment and a timely BMT will hopefully decrease mortality rates and improve neurodevelopmental outcome in this disease.
2914	Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus.	11336510	During primary neurogenesis in Xenopus, a cascade of helix--loop--helix (HLH) transcription factors regulates neuronal determination and differentiation. While XNeuroD functions at a late step in this cascade to regulate neuronal differentiation, the factors that carry out terminal differentiation are still unknown. We have isolated a new Xenopus member of the Ebf/Olf-1 family of HLH transcription factors, Xebf3, and provide evidence that, during primary neurogenesis, it regulates neuronal differentiation downstream of XNeuroD. In developing Xenopus embryos, Xebf3 is turned on in the three stripes of primary neurons at stage 15.5, after XNeuroD. In vitro, XEBF3 binds the EBF/OLF-1 binding site and functions as a transcriptional activator. When overexpressed, Xebf3 is able to induce ectopic neurons at neural plate stages and directly convert ectodermal cells into neurons in animal cap explants. Expression of Xebf3 can be activated by XNeuroD both in whole embryos and in animal caps, indicating that this new HLH factor might be regulated by XNeuroD. Furthermore, in animal caps, XNeuroD can activate Xebf3 in the absence of protein synthesis, suggesting that, in vitro, this regulation is direct. Similar to XNeuroD, but unlike Xebf2/Xcoe2, Xebf3 expression and function are insensitive to Delta/Notch-mediated lateral inhibition. In summary, we conclude that Xebf3 functions downstream of XNeuroD and is a regulator of neuronal differentiation in Xenopus.
2915	[Neurological form of onset in haemophagocytic lymphohistiocytosis].	11333388	Haemophagocyte lymphohistiocytosis (HLH) is a hematological disorder, autosomal recessive and in which there is benign proliferation of histiocytes with intense phagocytic activity of hematopoietic cells. The clinical features include fever, pancytopenia, coagulation disorders, liver dysfunction, the presence of histiocytes and haemophagocytes in the bone marrow, lymph nodes, spleen and liver. The nervous system is always involved and sooner or later patients develop a nervous system disorder with variable symptoms which may include irritability, disorders of consciousness, convulsions, ataxia, nystagmus or signs of intracranial hypertension.Onset of the disease showing purely neurological features is rare. We therefore describe the case of an 8 month old baby with HLH with a purely neurological condition involving irritability, horizontal rapid eye movements and vertical saccadic movements of both eyes and focal convulsive seizures. Initial complementary examinations were normal, except for study of the CSF with a lowered protein level and cells (monocytes). Finding hepatosplenomegaly and pallor, together with the laboratory investigations, made it advisable to do a bone marrow punch biopsy to detect haemophagocytes which would be diagnostic of HLH. In spite of chemotherapy there was rapid neurological deterioration, with alterations of the white matter and hydrocephaly which required insertion of a ventriculo-peritoneal shunt. The patient died when he was 10 months old.The cases of HLH in which cerebromeningeal disorders alone precede systemic symptoms are extremely rare. Hence the interest in reporting this case, so that it may be borne in mind in other cases of acute neurological onset. In this case initially there was encephalitis alone, but this was rapidly followed by systemic complications.
2916	BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis.	11331769	We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-beta, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix-loop-helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.
2917	ID gene expression varies with lineage during differentiation of pluripotential male germ cell tumor cell lines.	11320653	Human male germ cell tumors (GCTs) comprise an excellent model system for understanding the molecular events controlling cellular differentiation and lineage decision. Pluripotential embryonal carcinoma cell lines derived from GCTs can be induced to undergo terminal differentiation along specific lineages dependent upon the differentiating agent. We report here that one such cell line, NTera2/clone D1 (NT2/D1), previously shown to undergo differentiation along a neuronal lineage by all-trans-retinoic acid (RA), can be induced along a distinct non-neuronal lineage by the mammalian morphogens, bone morphogenetic proteins-2 and -4 (BMP-2 and -4). Very little is known regarding the molecular events that govern such human lineage decisions. In this study, the role of the ID (inhibitor of differentiation and DNA-binding) family of genes that act as inhibitors of the function of helix-loop-helix (HLH) transcriptional activators involved in lineage commitment was investigated using two pluripotential GCT cell lines as a model system. In the differetiation programs studied, Id1 was noted to decline, an event often associated with the decrease in proliferative rate occurring during differentiation. However, differences in the expression of ID2 and ID3 family members were detected between the programs. Notably, an increase in Id3 during RA-induced differentiation of NT2/D1 cells was observed, while Id2 levels increased during BMP-2 and -4 treatment of NT2/D1 cells and during the induction of an endodermal-like differentiation program in the cell line, 27X-1. The pluripotential male GCT cell lines comprise a unique system in which the roles of specific genes such as the ID family of genes in human cell differentiation and lineage decision can be studied.
2918	Identification of new translocations involving ETV6 in hematologic malignancies by fluorescence in situ hybridization and spectral karyotyping.	11319801	TEL/ETV6 is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners; 35 different chromosome bands have been involved in ETV6 translocations, of which 13 have been cloned. To identify additional ETV6 partner genes and to characterize the chromosomal abnormalities more fully, we studied bone marrow samples from patients known to have rearrangements of 12p, using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). FISH analysis was done with 14 probes located on 12p12.1 to 12p13.3. Nine ETV6 rearrangements were identified using FISH. The aberrations include t(1;12)(p36;p13), t(4;12)(q12;p13) (two patients), t(4;12)(q22;p13), t(6;12)(p21;p13), der(6)t(6;21)(q15;q?)t(12;21)(p13;q22), t(6;12)(q25;p13), inv(12)(p13q24), and t(2;2;5;12;17)(p25;q23;q31;p13;q12). Six new ETV6 partner bands were identified: 1p36, 4q22, 6p21, 6q25, 12q24, and 17q12. Our present data as well previous data from us and from other researchers suggest that ETV6 is involved in 41 translocations. The breakpoints in ETV6 were upstream from the exons coding for the HLH (helix-loop-helix) domain in six cases. Although cytogenetic analysis identified 12p abnormalities in all cases, FISH and SKY detected new and unexpected chromosomal rearrangements in many of them. Thus, complete characterization of the samples was achieved by using all three techniques in combination.
2919	Identification of a transcription factor, an 80-kDa protein that interacts with the HLH recognition motif of the rat p53 promoter.	11310562	The p53 promoter has been shown to contain a number of potential regulatory motifs. It was previously reported that the upstream stimulating factor (USF) played a central role in regulating the p53 expression. The USF binding site, E-box, is located around 40 bp upstream of the major transcription start site. In this study, it was confirmed that the E-box binds to proteins by DNase I footprinting assay. In the electrophoretic mobility shift assay (EMSA), two retarded bands were detected. One band was abolished by the competition of USF consensus oligonucleotide, but the other band was not. This result indicated that a factor, other than USF, was bound to the E-box. The molecular masses of the binding proteins were determined by a Southwestern-blotting assay. As a result, 46- and 80-kDa proteins were detected. The 46-kDa protein was eliminated by the competition of USF consensus oligonucleotide. Also, the Southwestern-blotting assay with 32P-labeled USF consensus oligonucleotide showed only a 46-kDa protein. Therefore, the 46-kDa protein was USF. These results showed that USF and the 80-kDa protein were bound to the E-box. In addition, it was proved by in vitro transcription assay that this 80-kDa protein had a basal transcriptional activity.
2920	High-grade prostate cancer is associated with low serum testosterone levels.	11304729	The aim of this study was to assess whether low serum testosterone levels in men with newly diagnosed prostate cancer have an association to the endocrine status, prostate-specific antigen (PSA) levels, Gleason score, and androgen receptor expression.Besides a full clinical work-up, the following hormones were quantified in men with newly diagnosed prostate cancer by serum analysis: total testosterone, human luteinising hormone (hLH), human follicle stimulating hormone (hFSH), estradiol, and dehydroepiandrostendione (DHEA). In a subgroup of men, androgen receptor expression was determined immunohistochemically.One hundred and fifty six patients (65.7 +/- 8.5 yrs) with a mean PSA of 29.8 ng/ml (median: 7.4 ng/ml) were analysed. Fifty-two patients (33%) had a partial androgen deficiency (serum testosterone < 3.0 ng/ml). These men had lower hLH (3.3 vs. 5.9 mIU/ml), hFSH (6.2 vs. 8.4 mIU/ml), and estradiol (18.8 vs. 29.1 pg/ml) serum levels. Mean Gleason score was higher (7.4 vs. 6.2) in men with a low serum testosterone, PSA-levels were lower (25.3 vs. 31.9 ng/ml). Mean testosterone levels decreased from 4.1 +/- 1.7 ng/ml in patients with Gleason scores < or = 5 to 2.8 +/- 2.7 ng/ml with Gleason scores > or = 8. Androgen receptor expression was higher in patients with low serum testosterone.Patients with high Gleason score prostate cancer have lower testosterone and estradiol serum levels. The fact that gonadotropins were lower in parallel suggests a tumor-mediated suppression of the hypothalamic-pituitary-gonadal hormone axis particularly in men with high Gleason score tumours.
2921	Notch pathway: making sense of suppressor of hairless.	1130126611301248	Suppressor of Hairless (Su(H)) is a DNA-binding protein component of the Notch signalling pathway, thought to be required, with a fragment of the Notch receptor, for target gene activation. Recent studies show that this is only one side of the story: target gene enhancers may be regulated by Su(H) in a variety of different ways.
2922	A leucine zipper protein of mitochondrial origin.	11295448	Sequence-specific DNA-binding proteins are characterised by short coiled-coil structural domains classified as zinc finger/RING finger, leucine zipper (L-Zip) or helix-loop-helix (HLH) motifs. The L-Zip proteins are defined by a pattern of at least four leucine (L) residues repeated every seventh amino acid that mediates protein dimerisation through the formation of parallel alpha-helical dimers. Usually the zipper is incorporated into a helix-loop-helix conformation called the basic helix-loop-helix-leucine zipper (bHLH/Zip). To date, all of the several hundred proteins reported as containing the L-Zip and/or bHLH/Zip motifs are nuclear-encoded. No leucine zipper polypeptide has, hitherto, been reported as mitochondrial in origin. Here we report such a polypeptide, the nicotinamide dehydrogenase subunit 4L (nad4L). We first identified this in human blood flukes of the genus Schistosoma (phylum Platyhelminthes; class Trematoda) but show that this is a common feature in other eucaryotes as well. Therefore, in addition to their well recognised role in oxidative phosphorylation, nad4L proteins may be pivotally involved in a range of other biological processes such as transcription and/or replication activation or as signal transmitters in communication with the nucleus and other cellular organelles. This may indicate a link between transcription regulation and respiration in mitochondria. We have also identified L-Zip-like motifs in nuoK, the procaryotic equivalent of the nad4L mitochondrial protein.
2923	Roles of Sox factors in neural determination: conserved signaling in evolution?	11291862	Neural differentiation in amphibian embryos is initiated by the neural inducers emanating from the Spemann-Mangold organizer. The fate of uncommitted ectoderm is determined by graded BMP activity along the dorsal-ventral axis. Several transcriptional regulators acting in early neural differentiation have been identified, including Sox, Zic, Pou, HLH and Fox factors. In this paper, I review recent molecular studies on neural determination, focusing mainly on Sox factors. I also discuss the possible conservation of regulatory factors in neural differentiation, comparing Xenopus and Drosophila counterparts.
2924	Dimerization of sterol regulatory element-binding protein 2 via the helix-loop-helix-leucine zipper domain is a prerequisite for its nuclear localization mediated by importin beta.	11283257	The sterol regulatory element-binding protein 2 (SREBP-2), a transcription factor of the basic helix-loop-helix-leucine zipper (bHLH-Zip) family, is synthesized in the form of a membrane-attached precursor molecule. When cells are deprived of sterols, a two-step proteolytic processing releases the transcriptionally active N-terminal segment of SREBP-2, thereby allowing it to enter the nucleus. In previous studies, we showed that the nuclear import of SREBP-2 occurs via the direct interaction of importin beta with the HLH-Zip domain. In this study, in order to more completely understand the intracellular dynamics of SREBP-2, we focused on the manner by which importin beta recognizes SREBP-2 at the initial step of the import. It was found that the active form of SREBP-2 exists as a stable dimer in solution and that the substitution of leucine residues for alanine in the leucine zipper motif disrupted the dimerization. It was also demonstrated that this mutant protein did not enter the nucleus either in vivo or in vitro. Solution binding assays, which involved the chemical cross-linking of wild-type or mutated SREBP-2 with importin beta, revealed that the import-active complex appeared to be composed of a dimeric form of SREBP-2 and importin beta. In addition, the SREBP-2 binding domain of importin beta corresponded to an overlapping but not identical region for importin alpha binding, which may explain how importin beta is able to recognize the dimeric HLH-Zip directly. These results indicate that dimerization is a prerequisite process for the nuclear import of SREBP-2 mediated by importin beta.
2925	Regulation of the helix-loop-helix proteins, E2A and Id3, by the Ras-ERK MAPK cascade.	11175815	Activation of mitogen-activated protein kinase (MAPK) pathways leads to cellular differentiation and/or proliferation in a wide variety of cell types, including developing thymocytes. The basic helix-loop-helix (bHLH) proteins E12 and E47 and an inhibitor HLH protein, Id3, play key roles in thymocyte differentiation. We show here that E2A DNA binding is lowered in primary immature thymocytes consequent to T cell receptor (TCR)-mediated ligation. Whereas expression of E2A mRNA and protein are unaltered, Id3 transcripts are rapidly induced upon signaling from the TCR. Activation of Id3 transcription is regulated in a dose-dependent manner by the extracellular signal-regulated kinase (ERK) MAPK module. These observations directly connect the ERK MAPK cascade and HLH proteins in a linear pathway.
2926	Proneural enhancement by Notch overcomes Suppressor-of-Hairless repressor function in the developing Drosophila eye.	11267869	The receptor protein Notch plays a conserved role in restricting neural-fate specification during lateral inhibition. Lateral inhibition requires the Notch intracellular domain to coactivate Su(H)-mediated transcription of the Enhancer-of-split Complex. During Drosophila eye development, Notch plays an additional role in promoting neural fate independently of Su(H) and E(spl)-C, and this finding suggests an alternative mechanism of Notch signal transduction.We used genetic mosaics to analyze the proneural enhancement pathway. As in lateral inhibition, the metalloprotease Kuzbanian, the EGF repeat 12 region of the Notch extracellular domain, Presenilin, and the Notch intracellular domain were required. By contrast, proneural enhancement became constitutive in the absence of Su(H), and this led to premature differentiation and upregulation of the Atonal and Senseless proteins. Ectopic Notch signaling by Delta expression ahead of the morphogenetic furrow also caused premature differentiation.Proneural enhancement and lateral inhibition use similar ligand binding and receptor processing but differ in the nuclear role of Su(H). Prior to Notch signaling, Su(H) represses neural development directly, not indirectly through E(spl)-C. During proneural enhancement, the Notch intracellular domain overcomes the repression of neural differentiation. Later, lateral inhibition restores the repression of neural development by a different mechanism, requiring E(spl)-C transcription. Thus, Notch restricts neurogenesis temporally to a narrow time interval between two modes of repression.
2927	Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans.	11243807	Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.
2928	Bone marrow changes mimicking myelodysplasia in patients with hemophagocytic lymphohistiocytosis.	11185994	In hemophagocytic lymphohistiocytosis (HLH), cytokine-induced pancytopenia is a common finding and is associated with hypoplastic and hypocellular bone marrow and abundant hemophagocytosis. To date, neutrophil nuclear segmentation abnormalities have not been clarified in HLH patients. We report a study of bone marrow from 6 cases of HLH that showed abnormal granulocytes, dyserythropoietic changes, and micromegakaryocytes mimicking the findings in myelodysplasia at the onset of disease. Pelger-Huët anomalies were particularly noted in all cases. The increased levels of cytokines in these cases may have caused cellular damage leading to the morphological changes in the bone marrow of these HLH patients. The impact of these findings on pathophysiology and prognosis in HLH patients remains to be determined.
2929	Drosophila melanogaster casein kinase II interacts with and phosphorylates the basic helix-loop-helix proteins m5, m7, and m8 derived from the Enhancer of split complex.	11208814	Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic (alpha) and regulatory (beta) subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, we have screened a D. melanogaster embryo cDNA library for proteins that interact with DmCKIIalpha. One of the cDNAs isolated in this screen encodes m7, a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex (E(spl)C), which regulates neurogenesis. m7 interacts with DmCKIIalpha but not with DmCKIIbeta, suggesting that this interaction is specific for the catalytic subunit of DmCKII. In addition to m7, we demonstrate that DmCKIIalpha also interacts with two other E(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specificity observed for the interaction of DmCKIIalpha with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus site for phosphorylation by CKII within a subdomain unique to these three proteins. Accordingly, these three proteins are phosphorylated by DmCKIIalpha, as well as by the alpha2beta2 holoenzyme purified from Drosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser(159) of m8 with either Ala or Asp abolishes phosphorylation, identifying this residue as the site of phosphorylation. We also demonstrate that m8 forms a direct physical complex with purified DmCKII, corroborating the observed two-hybrid interaction between these proteins. Finally, substitution of Ser(159) of m8 with Ala attenuates interaction with DmCKIIalpha, whereas substitution with Asp abolishes the interaction. These studies constitute the first demonstration that DmCKII interacts with and phosphorylates m5, m7, and m8 and suggest a biochemical and/or structural basis for the functional equivalency of these bHLH proteins that is observed in the context of neurogenesis.
2930	Aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) activity is unaltered by phosphorylation of a periodicity/ARNT/single-minded (PAS)-region serine residue.	11179451	The aryl hydrocarbon nuclear translocator (ARNT) protein belongs to the family of basic helix-loop-helix (HLH)-periodicity/ARNT/single-minded [Per/ARNT/Sim (PAS)] transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other basic HLH-PAS proteins. To date, it has been shown that both the HLH and PAS domains are required for aryl hydrocarbon receptor (AhR) ARNT heterodimerization and that phosphorylation of ARNT is also required for this heterodimerization. Presently, regulation of ARNT with respect to phosphorylation is poorly understood. In an earlier study, murine ARNT was shown to be a phosphoprotein, to display charge heterogeneity, and to have a shift in its predominant isoforms after heterodimerization with the AhR. It was hypothesized that this shift may represent a change in ARNT phosphorylation status. Metabolic [(32)P]orthophosphate labeling of human ARNT-transfected COS-1 cells, in conjunction with phosphoamino acid analysis, Edman degradation, and phosphopeptide mapping, demonstrated that ARNT is predominantly phosphorylated on serine residues and that serine 348 (S348) in the PAS domain is phosphorylated. Alanine and glutamic acid substitutions were used to demonstrate that loss of phosphorylation at this site did not influence AhR-mediated xenobiotic response elements-driven or ARNT-mediated class B E-box-driven signaling. Additionally, the phosphorylation pattern of ARNT was unaltered after AhR heterodimerization. Although phosphorylation of S348 did not modulate AhR-ARNT or ARNT-ARNT signaling, phosphorylation of this PAS-region serine residue may be important in other ARNT-mediated gene expression systems.
2931	Congenital alveolar capillary dysplasia with misalignment of pulmonary veins associated with hypoplastic left heart syndrome.	11178633	Three full-term infants died in the first month of life with hypoplastic left heart syndrome (HLH) and persistent pulmonary hypertension (PPH). At postmortem examination, they were found to have alveolar capillary dysplasia with misalignment of pulmonary veins (ACD with MPV). The association of HLH syndrome, and ACD with MPV with intestinal malrotation and/or obstruction, is unique. Decreased blood flow in the ascending aorta in fetuses with left outflow tract obstruction might cause vasoconstriction of pulmonary arterioles to maintain cerebral perfusion. Vasoconstriction early during embryogenesis might lead to decreased growth and development of alveolar capillaries and pulmonary veins. This results in pulmonary hypertension, and the arterial blood is forced to bypass the deficient capillary bed and can drain only via the anomalous bronchial veins.
2932	Analysis of sequence signature defining functional specificity and structural stability in helix-loop-helix proteins.	11170202	Specific functional properties of many proteins directing developmental responses via transcriptional regulation are orchestrated by their characteristic helix-loop-helix (HLH) structural motif. The entire HLH motif in all these proteins assumes a common conformation irrespective of their individual biological effects. The motif controls the affinity of HLH proteins for homo- or heterodimerization, permitting mixing and matching of regulatory factors, and thereby expanding the functional repertoire. Systematic analysis of molecular contacts at the dimer interface using the models built for the functional dimers combined with the pattern of conserved/nonconserved residues within different categories of HLH proteins helped in understanding the differential role played by different residues at the dimer interface for expressing corresponding functions. The residues associated with the self and partner interactions were identified, and the signature residues contributing toward dimeric stability and functional specificity were defined. It is evident that most of the residues involved in self interactions are common among all the HLH proteins. However, while certain residues involved in partner interactions are common among all the HLH proteins, certain others are common within a category, and still others vary widely defining specificity signature at different levels.
2933	Bifunctional hCG analogs adopt different conformations in LH and FSH receptor complexes.	11162891	Human reproduction requires specific interactions between follitropin (hFSH) and its receptor (FSHR) and between lutropin (hLH) or choriogonadotropin (hCG) and the lutropin receptor (LHR). Substitution of hFSH residues between hCG beta-subunit cysteines 11-12 creates a bifunctional analog that binds both receptors. To understand the basis of this observation, we used antibody probes to compare the conformations of bifunctional analogs before and after they were complexed with each receptor. Introduction of hFSH residues between cysteines 11-12 changed a distant conformation-sensitive region created by the juxtaposition of the subunit aminotermini. This region, found not to contact either receptor, was altered further when bifunctional ligands bound FSHR. All other surfaces, detected in LHR complexes, were also recognized in FSHR complexes, an indication that bifunctional ligands bind both receptors in similar orientations. These observations suggest that unlike hCG or hFSH, bifunctional gonadotropins can acquire "lutropin" and "follitropin" conformations, a phenomenon accentuated by receptor contacts.
2934	Hemophagocytic lymphohistiocytosis due to germline mutations in SH2D1A, the X-linked lymphoproliferative disease gene.	11159547	The hemophagocytic lymphohistiocytoses (HLH) comprise a heterogeneous group of disorders characterized by dysregulated activation of T cells and macrophages. Although some patients with HLH harbor perforin gene mutations, the cause of the remaining cases is not known. The phenotype of HLH bears a strong resemblance to X-linked lymphoproliferative disease (XLP), an Epstein-Barr virus (EBV)-associated immunodeficiency resulting from defects in SH2D1A, a small SH2 domain-containing protein expressed in T lymphocytes and natural killer cells. Here it is shown that 4 of 25 male patients with HLH who were examined harbored germline SH2D1A mutations. Among these 4 patients, only 2 had family histories consistent with XLP. On the basis of these findings, it is suggested that all male patients with EBV-associated hemophagocytosis be screened for mutations in SH2D1A. Patients identified as having XLP should undergo genetic counseling, and be followed long-term for development of lymphoma and hypogammaglobulinemia.
2935	Modulation of WHSC2 expression in human endothelial cells.	11150502	WHSC2, a novel gene recently isolated within the critical region of Wolf-Hirschhorn syndrome, is expressed in endothelial cells. WHSC2 is downregulated by HIV-1 Tat, whereas it is not modulated by angiogenic and pro-differentiative molecules. WHSC2 encodes a basic polypeptide of 528 amino acids. The in vitro translated protein shows a molecular weight of 57 kDa. WHSC2 has two nuclear translocation sequences which actively mediate its transport to the nucleus, as shown in whsc2-GFP-transfected NIH-3T3. We also found a helix-loop-helix (HLH) motif in region 130-185. Since members of the HLH family control differentiation and cell cycle progression, we hypothesize that WHSC2 may function as a transcriptional repressor.
2936	Id helix-loop-helix proteins antagonize pax transcription factor activity by inhibiting DNA binding.	11134340	The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family.
2937	Development-related increase in cortisol biosynthesis by human granulosa cells.	11134135	Antiinflammatory mechanisms are important in ovulation and may be regulated by cortisol (F). We previously showed that after administration of human (h)CG for ovulation induction, luteinized granulosa cells (LGC) abundantly express 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) messenger RNA but not 11betaHSD type 2 (11betaHSD2) messenger RNA. 11ssHSD1 is responsible for the reversible formation of antiinflammatory F from its inactive precursor cortisone (E), whereas 11betaHSD2 unidirectionally converts F to E through 11-oxidation. This pattern of gene expression predicts that LGC from periovulatory follicles would show increased activation of E to F, compared with granulosa cells from immature follicles (IGC), and that follicular fluid concentrations of E and F would alter accordingly. To test this hypothesis, we isolated IGC, thecal cells (TC), and follicular fluid, from ovaries of cyclic women, removed during surgery for benign gynecological disease. LGC and follicular fluid were aspirated from periovulatory follicles, 35 h after hCG injection, in patients undergoing in vitro fertilization treatment. In an 11betaHSD assay based on interconversion of tritiated E and F by cell suspensions in vitro, IGC (% conversion, 0.6 +/- 0.4, mean +/- SEM) and collagenase-dispersed TC (0.2 +/- 0.1%) were unable to convert E to F, whereas LGC (36.3 +/- 3.7%) were highly efficient at this reaction. Immature granulosa cells, LGC, and (to a lesser extent) TC were all able to convert F to E. Correspondingly, follicular fluid concentrations of total F and F:E ratios were significantly higher in periovulatory follicles, compared with immature follicles. Culturing IGC for 48 h in the presence of hFSH resulted in increased 11betaHSD1 reductase activity, paralleling stimulation of estrogen (aromatase activity) and progesterone biosynthesis. Similar treatment with hLH did not influence 11betaHSD1 reductase activity, except in a patient with more mature IGC, which also showed a significant increase in E-to-F conversion, as well as progesterone synthesis in response to hLH. These data confirm that 11betaHSD activity in the human ovary is developmentally regulated and gonadotropin responsive, favoring metabolism of F to E in immature follicles and E to F in periovulatory follicles. Increased formation of F by LGC in periovulatory follicles is consistent with an antiinflammatory function for this glucocorticoid at ovulation.
2938	SAGE analysis of neuroblastoma reveals a high expression of the human homologue of the Drosophila Delta gene.	11107116	Serial Analysis of Gene Expression (SAGE) is an efficient method to establish a complete mRNA expression profile of a tissue.We applied SAGE to identify expression of developmental control genes in neuroblastoma. Results. The human homologue of the Drosophila Delta gene Delta like-1 (DLK1) was shown to have an unusually high expression in a SAGE library of the SK-N-FI neuroblastoma cell line. Northern blot analysis confirmed high DLK1 expression in SK-N-FI and several other neuroblastoma cell lines. Signalling between Delta and its receptor Notch controls many differentiation steps in Drosophila and man, including neural crest cell fate decision.Our data therefore suggest a role for the Delta-Notch pathway in neuroblast differentiation.
2939	Interactions between chip and the achaete/scute-daughterless heterodimers are required for pannier-driven proneural patterning.	11090617	The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip was previously identified as a cofactor of the dorsal selector Apterous, and we show here that both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax.
2940	The basic helix-loop-helix transcription factors LIN-32 and HLH-2 function together in multiple steps of a C. elegans neuronal sublineage.	11076762	bHLH transcription factors function in neuronal development in organisms as diverse as worms and vertebrates. In the C. elegans male tail, a neuronal sublineage clonally gives rise to the three cell types (two neurons and a structural cell) of each sensory ray. We show here that the bHLH genes lin-32 and hlh-2 are necessary for the specification of multiple cell fates within this sublineage, and for the proper elaboration of differentiated cell characteristics. Mutations in lin-32, a member of the atonal family, can cause failures at each of these steps, resulting in the formation of rays that lack fully-differentiated neurons, neurons that lack cognate rays, and ray cells defective in the number and morphology of their processes. Mutations in hlh-2, the gene encoding the C. elegans E/daughterless ortholog, enhance the ray defects caused by lin-32 mutations. In vitro, LIN-32 can heterodimerize with HLH-2 and bind to an E-box-containing probe. Mutations in these genes interfere with this activity in a manner consistent with the degree of ray defects observed in vivo. We propose that LIN-32 and HLH-2 function as a heterodimer to activate different sets of targets, at multiple steps in the ray sublineage. During ray development, lin-32 performs roles of proneural, neuronal precursor, and differentiation genes of other systems.
2941	Hemophagocytic syndromes and infection.	11076718	Hemophagocytic lymphohistiocytosis (HLH) is an unusual syndrome characterized by fever, splenomegaly, jaundice, and the pathologic finding of hemophagocytosis (phagocytosis by macrophages of erythrocytes, leukocytes, platelets, and their precursors) in bone marrow and other tissues. HLH may be diagnosed in association with malignant, genetic, or autoimmune diseases but is also prominently linked with Epstein-Barr (EBV) virus infection. Hyperproduction of cytokines, including interferon-gamma and tumor necrosis factor-alpha, by EBV- infected T lymphocytes may play a role in the pathogenesis of HLH. EBV-associated HLH may mimic T-cell lymphoma and is treated with cytotoxic chemotherapy, while hemophagocytic syndromes associated with nonviral pathogens often respond to treatment of the underlying infection.
2942	Overlapping roles of two Hox genes and the exd ortholog ceh-20 in diversification of the C. elegans postembryonic mesoderm.	11060243	Members of the Hox family of homeoproteins and their cofactors play a central role in pattern formation of all germ layers. During postembryonic development of C. elegans, non-gonadal mesoderm arises from a single mesoblast cell M. Starting in the first larval stage, M divides to produce 14 striated muscles, 16 non-striated muscles, and two non-muscle cells (coelomocytes). We investigated the role of the C. elegans Hox cluster and of the exd ortholog ceh-20 in patterning of the postembryonic mesoderm. By examining the M lineage and its differentiation products in different Hox mutant combinations, we found an essential but overlapping role for two of the Hox cluster genes, lin-39 and mab-5, in diversification of the postembryonic mesoderm. This role of the two Hox gene products required the CEH-20 cofactor. One target of these two Hox genes is the C. elegans twist ortholog hlh-8. Using both in vitro and in vivo assays, we demonstrated that twist is a direct target of Hox activation. We present evidence from mutant phenotypes that twist is not the only target for Hox genes in the M lineage: in particular we show that lin-39 mab-5 double mutants exhibit a more severe M lineage defect than the hlh-8 null mutant.
2943	ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis.	11058077	The ubiquitously expressed family of ID helix-loop-helix (HLH) proteins function as dominant negative regulators of basic HLH (bHLH) transcriptional regulators that drive cell lineage commitment and differentiation in metazoa. Recent data from cell line and in vivo studies have implicated the functions of ID proteins in other cellular processes besides negative regulation of cell differentiation. ID proteins play key roles in the regulation of lineage commitment, cell fate decisions and in the timing of differentiation during neurogenesis, lymphopoiesis and neovascularisation (angiogenesis). They are essential for embryogenesis and for cell cycle progression, and they function as positive regulators of cell proliferation. ID proteins also possess pro-apoptotic properties in a variety of cell types and function as cooperating or dominant oncoproteins in immortalisation of rodent and human cells and in tumour induction in Id-transgenic mice. In several human tumour types, the expression of ID proteins is deregulated, and loss- and gain-of-function studies implicate ID functions in the regulation of tumour growth, vascularisation, invasiveness and metastasis. More recent biochemical studies have also revealed an emerging 'molecular promiscuity' of mammalian ID proteins: they directly interact with and modulate the activities of several other families of transcriptional regulator, besides bHLH proteins.
2944	Neural hyperplasia induced by RNA interference with m4/malpha gene activity.	11044604	The E(spl) complex (E(spl)-C) contains three different classes of genes that are downstream of Notch signaling. The bHLH genes mediate the Notch signal by repressing proneural gene activity, for example during the singularization of mechanosensory organ precursor cells (SOPs). Genes of the second class, the E(spl) m4/malpha family, antagonize this process if overexpressed. Here we show that this is based on dominant-negative effects since RNA interference gives neurogenic phenotypes indistinguishable from E(spl)-C mutations. Furthermore, a third member of the m4/malpha gene family, named bbu/tom, behaves differently with respect to RNA expression patterns, its regulation by Notch signaling and loss of function phenotypes.
2945	Epstein-Barr virus (EBV)-induced B-cell proliferative disorder after chemotherapy in a patient with hemophagocytic lymphohistiocytosis with associated EBV-induced T-cell proliferation.	11042520	We report a case of Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD) which developed after chemotherapy for hemophagocytic lymphohistiocytosis (HLH), who had no history of immunodeficiency or familial X-linked LPD. In HLH, the presence of EBV in T-cells was confirmed by a combination of in situ hybridization (ISH) and immunostaining. Southern blot analysis using EBV-TR and immunoglobulin JH probes revealed oligoclonal proliferation of B-cells in each organ involved by abnormal B-lymphoid cells at autopsy. Combined ISH and immunostaining disclosed the presence of EBV in proliferating B-cells. Cytokine analysis during the period of T-cell activation in HLH revealed marked elevation of interferon (IFN) gamma, interleukin (IL)-10 and soluble IL-2 receptor (sIL-2R) and mild to moderate increases of tumor necrosis factor (TNF)-alpha were observed, while IFN gamma, IL-10 and sIL-2R were elevated initially during the HLH phase, which then decreased as LPD developed and B-cell proliferation predominated. Immunosuppressive chemotherapy for HLH may then have allowed latent EBV in B lymphocytes to induce transformation and oligoclonal proliferation of B-cells, finally resulting in LPD. Mechanisms of EBV-induced cell proliferation remain unclear, but alteration of various cytokines may be responsible for it.
2946	Outcome of clonal hemophagocytic lymphohistiocytosis: analysis of 32 cases.	11042518	We studied the impact of clonality, determined by analysis of Epstein-Barr virus genome termini, T-cell receptor genes and clonal chromosomal abnormality, on the clinical outcome in 32 patients with hemophagocytic lymphohistiocytosis (HLH). Of the cases studied, 23 cases were EBV-clonal, 15 cases were TCR-clonal and 7 cases were cytogenetically clonal. Thirty patients were treated with immuno-chemotherapy and/or multiagents' chemotherapy and 4 received bone marrow transplantation. All 7 cases, in which cytogenetically abnormal clones were identified, were fatal (3-year survival by Kaplan-Meier analysis; 14%, 95%CI: 0-40%). None of these 7 cases received bone marrow transplantation. On the other hand, the 3-year survival of 23 clonal EBV-positive HLH cases including 4 cytogenetically abnormal cases was 64 % (95%CI: 42-84%), while that of 15 TCR-clonal cases was 53% (95%CI: 26-78%). Our observations suggest that cytogenetically abnormal cases are at extremely high risk, requiring intensive immuno-chemotherapy followed by prompt and timely allogeneic bone marrow transplantation.
2947	Regulation of Id gene expression during embryonic stem cell-derived hematopoietic differentiation.	11027551	To elucidate the role of helix-loop-helix (HLH) Id proteins in hematopoietic differentiation, we used a model of embryonic stem (ES) cell differentiation in vitro which gives access not only to hematopoietic myeloid progenitor cells but also to the more primitive blast colony-forming cell (BL-CFC), the in vitro equivalent of the hemangioblast that gives rise to blast cell colonies in the presence of VEGF. We first demonstrated that ES cell-derived blast cell colonies could be used as a model to study hematopoietic differentiation and maturation. We next established the expression profile of Id genes in this model. Transcripts of the four Id genes were present in ES cells. Id1, Id3 and Id4 gene expression was down-regulated during the development of blast cell colonies while that of Id2 was maintained. Thus, Id1, Id3, and Id4 proteins are candidates for being negative regulators of hematopoiesis in the model of hematopoietic ES cell differentiation in vitro.
2948	Evidence that helix-loop-helix proteins collaborate with retinoblastoma tumor suppressor protein to regulate cortical neurogenesis.	11027225	The retinoblastoma tumor suppressor protein (pRb) family is essential for cortical progenitors to exit the cell cycle and survive. In this report, we test the hypothesis that pRb collaborates with basic helix-loop-helix (bHLH) transcription factors to regulate cortical neurogenesis, taking advantage of the naturally occurring dominant-inhibitory HLH protein Id2. Overexpression of Id2 in cortical progenitors completely inhibited the induction of neuron-specific genes and led to apoptosis, presumably as a consequence of conflicting differentiation signals. Both of these phenotypes were rescued by coexpression of a constitutively activated pRb mutant. In contrast, Id2 overexpression in postmitotic cortical neurons affected neither neuronal gene expression nor survival. Thus, pRb collaborates with HLHs to ensure the coordinate induction of terminal mitosis and neuronal gene expression as cortical progenitors become neurons.
2949	Regulation of Drosophila wing vein patterning: net encodes a bHLH protein repressing rhomboid and is repressed by rhomboid-dependent Egfr signalling.	11023875	The stereotyped pattern of veins in the Drosophila wing is generated in response to local EGF signalling. Mutations in the rhomboid (rho) gene, which encodes a sevenpass membrane protein required to enhance signalling transmitted by the EGF receptor (Egfr), inhibit vein development and disrupt the vein pattern. By contrast, net mutations produce ectopic veins in intervein regions. We have cloned the net gene and show that it encodes a basic HLH protein that probably acts as a transcriptional repressor. net and rho are expressed in mutually exclusive patterns during the development of the wing imaginal disc. Lack of net activity causes rho expression to expand, and vice versa. Furthermore, ectopic expression of net or rho results in their mutual repression and thus suppresses vein formation or generates tube-like wings composed of vein-like tissue. Egfr signalling and net exert mutually antagonising activities during the specification of vein versus intervein fate. While Egfr signalling represses net transcription, net exhibits a two-tiered control by repressing rho transcription and interfering with Egfr signalling downstream of Rho. Our results further suggest that net is required to maintain intervein development by restricting Egfr signalling, which promotes vein development, to the Net-free vein regions of the wing disc.
2950	Growth state-dependent binding of USF-1 to a proximal promoter E box element in the rat plasminogen activator inhibitor type 1 gene.	11010817	Induced PAI-1 gene expression in renal epithelial (NRK-52E, clone EC-1) cells occurs as part of the immediate-early response to serum. PAI-1 transcripts are maximally expressed early in G(1) (within 4 h of serum addition to quiescent EC-1 cells) and then subsequently decline to basal levels prior to entry into DNA synthetic phase. Comparative analysis of PAI-1 mRNA abundance and de novo-synthesized thiolated RNA in quiescent cells, as well as at 4 h (early G(1)) and 20 h (late G(2)) postserum addition, in conjunction with RNA decay measurements indicated that PAI-1 gene regulation upon growth activation was predominantly transcriptional. An E box motif (CACGTG), important in the induced expression of some growth state-dependent genes, mapped to nucleotides -160 to -165 upstream of the transcription start site in the PAI-1 proximal promoter. Mobility-shift assessments, using a 18-bp deoxyoligonucleotide construct containing the E box within the context of PAI-1-specific flanking sequences, confirmed binding of EC-1 nuclear protein(s) to this probe and, specifically, to the E box hexanucleotide site. The specificity of this protein-probe interaction was verified by competition analyses with double-stranded DNA constructs that included E box deoxyoligonucleotides with non-PAI-1 flanking bases, mutant E box sequences incapable of binding NRK nuclear proteins, and unrelated (i.e., AP-1) target motifs. Extract immunodepletion and supershift/complex-blocking experiments identified one PAI-1 E box-binding protein to be upstream stimulatory factor-1 (USF-1), a member of the HLH family of transcription factors. Mutation of the CACGTG site to TCCGTG in an 18-bp PAI-1 probe inhibited the formation of USF-1-containing complexes confirming that an intact E box motif at -160 to -165 bp in the PAI-1 promoter and, in particular, the CA residues at -165 and -164 are essential for USF-1 binding. Incorporation of this 2 bp change into a reporter construct containing 764 bp of the proximal PAI-1 "promoter" ligated to a CAT gene effectively reduced (by 74%) CAT activity in cycling cells. An intact E box motif at nucleotides -160 to -165 in the PAI-1 promoter, thus, is an important functional element in the regulation of PAI-1 transcriptional activity in renal cells.
2951	Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans.	10996789	Dystrophin is the product of the gene that is mutated in Duchenne muscular dystrophy (DMD), a progressive neuromuscular disease for which no treatment is available. Mice carrying a mutation in the gene for dystrophin (mdx mice) display only a mild phenotype, but it is aggravated when combined with a mutation in the MyoD gene. The nematode worm Caenorhabditis elegans has a dystrophin homologue (dys-1), but null mutations in dys-1 do not result in muscle degeneration.We generated worms carrying both the dys-1 null mutation cx18, and a weak mutation, cc561ts, of the C. elegans MyoD homologue hlh-1. The double mutants displayed a time-dependent impairment of locomotion and egg laying, a phenotype not seen in the single mutants, and extensive muscle degeneration. This result allowed us to look for genes that, when misexpressed, could suppress the dys-1; hlh-1 phenotype. When overexpressed, the dyc-1 gene - whose loss-of-function phenotype resembles that of dys-1 - partially suppressed the dys-1; hlh-1 phenotype. The dyc-1 gene encodes a novel protein sharing similarities with the mammalian neural nitric oxide synthase (nNOS)-binding protein CAPON, and is expressed in the muscles of the worm.As a C. elegans model for dystrophin-dependent myopathy, the dys-1; hlh-1 worms should permit the identification of genes, and ultimately drugs, that would reverse the muscle degeneration in this model.
2952	Neuron-specific enolase in hemophagocytic lymphohistiocytosis: a potential indicator for macrophage activation?	10979210	To determine the pathogenesis of hemophagocytic lymphohistiocytosis (HLH), serum levels of neuron-specific enolase (NSE) and cytokine profiles were investigated. Serum concentrations of NSE and several cytokines were measured by immunoassays, and the association was evaluated in 18 HLH patients. Serum NSE levels increased (> 10 ng/mL) in 27/29 samples (93%) during the active febrile phase, the mean level of which (35.9 ng/mL) was much higher than that during the remission phase (11.2 ng/mL) (P = .001). The peak levels of NSE in 11 patients who required cytotoxic agents were higher than those in 7 patients without chemotherapy, 64.6 +/- 49.4 and 17.9 +/- 12.9, respectively (P = .265). The NSE levels correlated positively with the levels of interferon (IFN)-gamma (Pearson's correlation coefficient [r] = 0.408, P = .044), soluble interleukin-2 receptor (sIL-2R) (r = 0.464, P = .048), lactate dehydrogenase (r = 0.830, P < .00001), aspartate aminotransferase (r = 0.531, P = .003), and ferritin (r = 0.715, P < .00001), and correlated negatively with platelet count (r = -0.422, P = .021), but not with other parameters, including tumor necrosis factor-alpha, IL-1 beta, IL-18, soluble Fas ligand and C-reactive protein. Multiple regression analysis indicated that the correlation of NSE with platelet count was independent of other correlations. Sequential NSE changes well reflected the clinical course of patients. Immunohistochemical staining revealed an appreciable number of NSE-positive histiocytes in bone marrow specimens with florid hemophagocytosis. These results suggest that the circulating NSE originated from macrophages stimulated with IFN-gamma/sIL-2R, and partly from the destruction of platelets. Serum NSE level may be a useful marker for predicting the disease progression of HLH.
2953	Advances in the management of hemophagocytic lymphohistiocytosis.	10979202	Hemophagocytic lymphohistiocytosis (HLH) is a prototype of the hemophagocytic syndrome and occurs most often in children. Progress in cytokine research has now made it possible to show that HLH occurs as a consequence of uncontrolled, dysregulated cellular immune reactivity caused by a number of different underlying diseases. Three major risk groups of HLH can be identified: (1) familial HLH (FHL), (2) Epstein-Barr virus-associated HLH (EBV-HLH), and (3) life-threatening infection-associated or underlying disease-unknown HLH in infancy. Diagnostic criteria now exist that allow the differential diagnosis of these groups, which is important because distinct therapeutic measures are advised for each group. FHL patients require immediate application of immunochemotherapy with a core combination of corticosteroids and etoposide together with monitoring of central nervous system disease by early and repeated magnetic resonance imaging of the brain, followed by timely stem cell transplantation (SCT). EBV-HLH should also be treated with a combination of corticosteroids and etoposide. Aggressive or relapsed cases should be treated with cyclosporin A and, if necessary, with more intensive chemotherapy, such as that used for non-Hodgkin's lymphoma. SCT may also be needed in these refractory cases. In cases of herpes simplex virus, adenovirus 7, and other pathogen-undetermined HLH in early infancy, it is of great importance to administer appropriate antiviral or antibacterial agents. The most important point to make regarding HLH treatment is that the underlying cause of HLH must be promptly established to enable the rapid application of the appropriate therapy. Currently, 30% to 40% of HLH cases have a poor outcome. It is necessary for hematologists to cooperate with specialists in other fields so that early diagnosis, which is critical for improvements in outcome, can be made.
2954	Treatment strategies for Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH).	10975382	In Epstein-Barr virus (EBV) infection, the virus immortalizes B lymphocytes and cytotoxic T lymphocytes (CTLs) are directed toward both latent and lytic viral antigens expressed on EBV-infected B-cells. Various EBV-associated diseases occur as a result of this disruption of immune surveillance. In the majority of EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) cases, the major cell types containing EBV DNA are not B-cells, but clonally proliferating T-cells or NK-cells. Proliferation of these cells produces severe immune reactions in the host, and the clinical features related to massive cytokine production at the onset of disease are unique and distinct from other EBV-associated diseases. In the treatment of EBV-HLH, therapeutic infusion of EBV-specific CTLs appears to be ineffective, and eradication of EBV-containing cells is useful but not sufficient to save lives, because of high incidence of acute mortality due to cytokine-induced multiple organ failure and neutropenia-associated opportunistic infections. The optimal treatment strategy for this disease consists of three steps: (1) control of cytokine storm including coagulopathy and multiple organ failure, (2) control of opportunistic infections, and (3) eradication of clonally proliferating EBV-containing T- or NK- cells by immunochemotherapy and, if necessary, hemopoietic stem cell/bone marrow transplantation (SCT/BMT).
2955	Helix-loop-helix transcription factors in electrically active and inactive skeletal muscles.	10951440	The muscle-specific helix-loop-helix (HLH) transcription factors myoD, myogenin, MRF4, and myf-5 are called the muscle regulatory factor family (MRF). Levels of MRFs are strongly regulated by muscle electrical activity and are thought to control downstream genes that are important for muscle phenotype such as the acetylcholine receptor (AChR) and possibly genes connected to muscle metabolic properties. The MRFs interact with ubiquitously expressed HLH factors such as E-proteins and Id-proteins to form heterodimers. In the present paper, we report the effects of paralysis obtained by nerve impulse block with tetrodotoxin (TTX) and denervation on messenger ribonucleic acid (mRNA) levels for Id-1, E47, myogenin, AChR alpha-subunit and beta-actin. Both Id-1 and E47 showed twofold increases in absence of nerve evoked electrical activity. These changes in the ubiquitously expressed HLH factors might have important functional implications for downstream gene expression, but in comparison, myogenin mRNA was increased 10-fold. We conclude that myogenin and the other muscle-specific MRFs remain the transcription factors with the strongest activity dependence that has so far been described in muscle.
2956	Haematological associations of Epstein-Barr virus infection.	10942621	Epstein-Barr virus (EBV) is one of eight human herpesviruses and is ubiquitous. Primary infection with EBV in childhood is generally silent, but often causes overt diseases such as infectious mononucleosis (IM) and lymphoproliferative disorders (LPD). The latter occurs in immunologically compromised individuals. Historically, EBV has been thought to be aetiologically linked to human malignancies such as EBV genome-positive Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC). Furthermore, studies using recent developments in molecular and immunological diagnostic approaches have suggested that this virus has a causative role in a spectrum of human diseases of previously unknown pathogenesis, including chronic active EBV infection syndrome (CAEBV), EBV-related haemophagocytic lymphohistiocytosis (HLH), and certain disorders such as EBV genome-positive T-cell lymphoma, natural killer (NK) cell leukaemia/lymphoma, Hodgkin's disease (HD) and gastric carcinoma. This chapter reviews recent progress regarding EBV-associated diseases.
2957	Human homologue of maid: A dominant inhibitory helix-loop-helix protein associated with liver-specific gene expression.	10915743	The helix-loop-helix (HLH) family of transcriptional regulatory proteins are key regulators in numerous developmental processes. The class I HLH proteins, such as E12 are ubiquitously expressed. Class II HLH proteins, such as MyoD, are expressed in a tissue-specific manner. Class I and II heterodimers can bind to E-boxes (CANNTG) and regulate lineage commitments of embryonic cells. In an attempt to identify partners for the E12 protein that may exert control during liver development, we performed the yeast 2-hybrid screen using an expression complementary DNA library from human fetal liver. A novel dominant inhibitory HLH factor, designated HHM (human homologue of maid), was isolated and characterized. HHM is structurally related to the Id family and was highly expressed in brain, pituitary gland, lung, heart, placenta, fetal liver, and bone marrow. HHM physically interacted with E12 in vitro and in mammalian cells. Comparison of the dominant inhibitory effects of HHM and Id2 on the binding of E12/MyoD dimer to an E-box element revealed a weaker inhibition by HHM. However, HHM but not Id2 specifically inhibited the luciferase gene activation induced by hepatic nuclear factor 4 (HNF4) promoter. The HHM was transiently expressed during stem-cell-driven regeneration of the liver at the stage in which the early basophilic foci of hepatocytes started to appear. These results suggest that HHM is a novel type of dominant inhibitory HLH protein that might modulate liver-specific gene expression.
2958	Effects of time intervals and tone durations on auditory stream segregation.	10909253	Adult listeners rated the difficulty of hearing a single coherent stream in a sequence of high (H) and low (L) tones that alternated in a repetitive galloping pattern (HLH-HLH-HLH...). They could hear the gallop when the sequence was perceived as a single stream, but when it segregated into two substreams, they heard H-H- ... in one stream and L-L- ... in the other. The onset-to-onset time of the tones, their duration, the interstimulus interval (ISI) between tones of the same frequency, and the frequency separation between H and L tones were varied. Subjects' ratings on a 7-point scale showed that the well-known effect of speed's increasing stream segregation is primarily due to its effect on the ISI between tones in the same frequency region. This has implications for several theories of streaming.
2959	Endocrine patterns in patients with benign and malignant prostatic diseases.	10906738	The known importance of the endocrine system, particularly of steroid hormones, for development of the prostate gland and the fact that steroid hormones act as immunmodulators prompted us to compare hypophyseal, adrenal, and gonadal hormones, including cortisol, in patients with benign and malignant prostatic diseases.Patients with newly diagnosed, untreated prostate cancer (PC) (n = 75) and, as a control population, those with untreated lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH) (n = 159) entered this prospective study. In all patients, the following parameters were obtained by serum analysis: prostate-specific antigen (PSA), human luteinizing hormone (hLH), human follicle-stimulating hormone (hFSH), testosterone, estradiol (E2), cortisol, and dehydroepiandrosterone-sulphate (DHEA-S). Serum samples were collected of fasting patients between 7. 30-10.00 AM.Age was comparable in both groups (PC: 65.6 +/- 7.6 years (mean +/- standard deviation) vs. controls: 64.9 +/- 8. 1 years; P = 0.56). HFSH (PC: 6.6 +/- 3.9 mIU/ml; controls: 8.4 +/- 6.4 mIU/ml; P = 0.04), hLH (PC: 5.3 +/- 4.8mIU/ml; controls: 7.6 +/- 6.2 mIU/ml; P = 0.009), and estradiol (PC: 25.8 +/- 12.7 pg/ml; controls: 32.6 +/- 12.6 pg/ml; P = 0.0003) were significantly lower in PC patients than controls. Cortisol (PC: 16.7 +/- 4.2 microg/dl; controls: 13.5 +/- 4.3 microg/dl; P < 0.0001) was significantly higher in cases. The difference for cortisol and estradiol concentrations between PC patients and controls held true in all life-decades. Serum concentrations for DHEA-S and testosterone were comparable between PC and control patients. In PC patients, none of the endocrine parameters correlated to serum PSA or clinical/pathological stage.Patients with newly diagnosed, untreated PC yielded significantly higher cortisol and lower estradiol serum concentrations than controls. The known effect of cortisol on the immune status warrants further studies.
2960	Two different activities of Suppressor of Hairless during wing development in Drosophila.	10903180	The Notch pathway plays a crucial and universal role in the assignation of cell fates during development. In Drosophila, Notch is a transmembrane protein that acts as a receptor of two ligands Serrate and delta. The current model of Notch signal transduction proposes that Notch is activated upon binding its ligands and that this leads to the cleavage and release of its intracellular domain (also called Nintra). Nintra translocates to the nucleus where it forms a dimeric transcription activator with the Su(H) protein. In contrast with this activation model, experiments with the vertebrate homologue of Su(H), CBF1, suggest that, in vertebrates, Nintra converts CBF1 from a repressor into an activator. Here we have assessed the role of Su(H) in Notch signalling during the development of the wing of Drosophila. Our results show that, during this process, Su(H) can activate the expression of some Notch target genes and that it can do so without the activation of the Notch pathway or the presence of Nintra. In contrast, the activation of other Notch target genes requires both Su(H) and Nintra, and, in the absence of Nintra, Su(H) acts as a repressor. We also find that the Hairless protein interacts with Notch signalling during wing development and inhibits the activity of Su(H). Our results suggest that, in Drosophila, the activation of Su(H) by Notch involve the release of Su(H) from an inhibitory complex, which contains the Hairless protein. After its release Su(H) can activate gene expression in absence of Nintra.
2961	The enhancer of split complex of Drosophila includes four Notch-regulated members of the bearded gene family.	10903170	During Drosophila development, transcriptional activation of genes of the Enhancer of split Complex (E(spl)-C) is a major response to cell-cell signaling via the Notch (N) receptor. Although the structure and function of the E(spl)-C have been studied intensively during the past decade, these efforts have focused heavily on seven transcription units that encode basic helix-loop-helix (bHLH) repressors; the non-bHLH members of the complex have received comparatively little attention. In this report, we analyze the structure, regulation and activity of the m1, m2 and m6 genes of the E(spl)-C. We find that E(spl)m2 and E(spl)m6 encode divergent members of the Bearded (Brd) family of proteins, bringing to four (m(alpha), m2, m4 and m6) the number of Brd family genes in the E(spl)-C. We demonstrate that the expression of both m2 and m6 is responsive to N receptor activity and that both genes are apparently direct targets of regulation by the N-activated transcription factor Suppressor of Hairless. Consistent with this, both are expressed specifically in multiple settings where N signaling takes place. Particularly noteworthy is our finding that m6 transcripts accumulate both in adult muscle founder cells in the embryo and in a subset of adepithelial (muscle precursor) cells associated with the wing imaginal disc. We show that overexpression of either m2 or m6 interferes with N-dependent cell fate decisions in adult PNS development. Surprisingly, while misexpression of m6 impairs lateral inhibition, overexpression of m2 potentiates it, suggesting functional diversification within the Brd protein family. Finally, we present our initial studies of the structure, expression and regulation of the newest member of the Brd gene family, Ocho, which is located in the recently identified Bearded Complex.
2962	A cysteine residue in the helix-loop-helix domain of Id2 is critical for homodimerization and function.	10891368	Id proteins are negative regulators of basic helix-loop-helix (bHLH) transcription factors. In this study, we compared the expression of Id2 mRNA in proliferating (fetal) and nonproliferating (adult) alveolar epithelial cells (AECs). The expression of Id2 was higher in adult AECs than in the corresponding fetal cells, suggesting that Id2 might play a functional role in developmental regulation of lung epithelial cell proliferation. By screening a mouse embryo cDNA library in the yeast two-hybrid system, Id2 was identified as a self-associating protein. Structural analysis by deletion and site-directed mutagenesis demonstrated that the HLH domain and a cysteine residue within the HLH domain are essential for Id2 homodimerization. Furthermore, in vitro synthesized Id2 homodimers became monomers under reducing conditions, indicating that the formation of an intermolecular disulfide bond is critical for Id2 homodimerization. Transient transfection assays in A549 cells showed that wild-type Id2 down-regulated the activity of the cyclin A promoter by 70%, while mutating the cysteine critical for Id2 homodimerization abolished the inhibitory effect of wild-type Id2.
2963	Fatal Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with clonal karyotype abnormality.	10846833	We report a case of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) with clonal karyotype abnormality. A 5-year-old boy was admitted to our hospital with persistent high-grade fever, hepatomegaly, and pancytopenia. Laboratory data disclosed a coagulation abnormality and severe liver damage. Clonal proliferation of EBV-infected cells was detected in the bone marrow by Southern hybridization, and bone marrow cells exhibited clonal chromosomal abnormality. Although the patient was treated with immunochemotherapy according to the HLH94 protocol, the disease recurred during the induction therapy, and the patient died of disseminated intravascular coagulopathy. Considering this aggressive and fatal clinical course, it is important to take intensive therapeutic measures if karyotype abnormality is noted in the treatment of EBV-HLH patients.
2964	Su(H)-independent activity of hairless during mechano-sensory organ formation in Drosophila.	10842054	Formation of mechano-sensory organs in Drosophila involves the selection of neural precursor cells (SOPs) mediated by the classical Notch pathway in the process of lateral inhibition. Here we show that the subsequent cell type specifications rely on distinct subsets of Notch signaling components. Whereas E(spl) bHLH genes implement SOP selection, they are not required for later decisions. Most remarkably, the Notch signal transducer Su(H) is essential to determine outer but not inner cell fates. In contrast, the Notch antagonist Hairless, thought to act upon Su(H), influences strongly the entire cell lineage demonstrating that it functions through targets other than Su(H) within the inner lineage. Thereby, Hairless and numb may have partly redundant activities. This suggests that Notch-dependent binary cell fate specifications involve different sets of mediators depending on the cell type considered.
2965	Negative regulation of selected bHLH proteins by eHAND.	10837146	The bHLH protein eHAND plays an important role in the development of extraembryonic, mesodermal, and cardiac cell lineages, presumably through heterodimerization with other HLH proteins and DNA binding. In this study, we have identified a novel transcriptional activity of eHAND. In transient transfection assays, eHAND is a potent inhibitor of activation by some but not all bHLH proteins. eHAND can prevent E-box DNA binding by these bHLH proteins. Interestingly, eHAND can also strongly inhibit transactivation activity by a MyoD approximately E47 tethered dimer, which suggests a distinct mechanism of action. eHAND also inhibits MyoD-dependent skeletal muscle cell differentiation and expression of the muscle-specific myosin heavy chain protein. In addition, we show that eHAND can repress activity of the natural p75LNGFR promoter, whose expression overlaps that of eHAND and dHAND. The inhibitory activity of eHAND may be attributed to multiple mechanisms, such as the ability to act as a corepressor, the presence of a repression domain, and its ability to sequester E proteins in an inactive complex. Based upon its inhibitory effect on bHLH proteins and cellular differentiation, we propose that eHAND may function by several mechanisms to promote placental giant cell proliferation by negatively regulating the activities of the bHLH protein MASH-2.
2966	The basic helix-loop-helix transcription factors myogenin and Id2 mediate specific induction of caveolin-3 gene expression during embryonic development.	10835421	Caveolin-3 protein is the only member of the caveolin family that shows a unique muscle-specific expression pattern, and loss of its functional activity causes muscular dystrophy. Caveolin-3 mRNA levels are dramatically increased during the formation of myotubes in the C2C12 cell line. In this study, we characterized the human caveolin-3 5'-flanking region. Promoter analyses demonstrate that the proximal E box element serves as a myogenin binding site and is both necessary and sufficient to control caveolin-3 gene transcription. Transient transfection assays indicated that overexpression of myogenin activates caveolin-3 reporter gene expression, whereas Id2 overexpression inhibited caveolin-3 promoter activation by myogenin. A mutant Id2 protein lacking the HLH domain was not capable of suppressing myogenin-mediated activation. Determination of caveolin-3 transcript distribution patterns in vivo revealed that mRNA was first detectable at day 10 of gestation in the developing somites and heart. Caveolin-3 protein in myoblasts and myotubes was expressed in both the plasma membrane and vesicular structures. During skeletal myogenesis the level of Id2, an inhibitor of differentiation, decreases, allowing the induced basic helix-loop-helix transcription factor myogenin to form transcriptionally active heterodimers that bind to the caveolin-3 promoter and thereby mediate its transcription.
2967	Molecular analysis of latent membrane protein 1 in patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Japan.	10830744	Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is considered to be an oncoprotein because it is crucial for B-lymphocyte transformation. Since a 30 base pair (bp) deletion in the carboxy-terminal portion of the LMP1 gene was found in a CAO cell line derived from nasopharyngeal carcinoma containing EBV, an association between EB viral genetic alteration and tumorigenicity has been postulated. In this study we have analyzed LMP1 DNA isolated from 10 Japanese patients with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). In all HLH patients, we found the 30 bp deletion and 4-8-tandem repeats of the sequence DNGPQDPDNTD in the LMP1 gene. Furthermore, detailed amino acid (aa) sequence analysis revealed that 7 aa substitutions identical to those found in CAO-LMP1 but not in B95.8 cell line-LMP1 were found in all the HLH cases. NF-kappaB assay revealed that HLH-LMP1 activated NF-kappaB significantly more than that of B95.8-LMP1 (p=0.032). We conclude that EBV from all of our HLH cases shared common genetic characteristics with EBV obtained from the CAO cell line, which is distinct from the wild-type EBV isolated from the B95.8 cell line. These data suggest that the mutational changes of the LMP1 gene may play an important role in the pathogenesis of these fatal EBV-related disorders.
2968	The steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle differentiation.	10817756	Nuclear receptor-mediated activation of transcription involves coactivation by cofactors collectively denoted the steroid receptor coactivators (SRCs). The process also involves the subsequent recruitment of p300/CBP and PCAF to a complex that synergistically regulates transcription and remodels the chromatin. PCAF and p300 have also been demonstrated to function as critical coactivators for the muscle-specific basic helix-loop-helix (bHLH) protein MyoD during myogenic commitment. Skeletal muscle differentiation and the activation of muscle-specific gene expression is dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF-2, which function in a cooperative manner. We examined the functional role of one SRC, GRIP-1, in muscle differentiation, an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding GRIP-1 is expressed in proliferating myoblasts and post-mitotic differentiated myotubes, and that protein levels increase during differentiation. Exogenous/ectopic expression studies with GRIP-1 sense and antisense vectors in myogenic C2C12 cells demonstrated that this SRC is necessary for (1) induction/activation of myogenin, MEF-2, and the crucial cell cycle regulator, p21, and (2) contractile protein expression and myotube formation. Furthermore, we demonstrate that the SRC GRIP-1 coactivates MEF-2C-mediated transcription. GRIP-1 also coactivates the synergistic transactivation of E box-dependent transcription by myogenin and MEF-2C. GST-pulldowns, mammalian two-hybrid analysis, and immunoprecipitation demonstrate that the mechanism involves direct interactions between MEF-2C and GRIP-1 and is associated with the ability of the SRC to interact with the MADS domain of MEF-2C. The HLH region of myogenin mediates the direct interaction of myogenin and GRIP-1. Interestingly, interaction with myogenic factors is mediated by two regions of GRIP-1, an amino-terminal bHLH-PAS region and the carboxy-terminal region between amino acids 1158 and 1423 (which encodes an activation domain, has HAT activity, and interacts with the coactivator-associated arginine methyltransferase). This work demonstrates that GRIP-1 potentiates skeletal muscle differentiation by acting as a critical coactivator for MEF-2C-mediated transactivation and is the first study to ascribe a function to the amino-terminal bHLH-PAS region of SRCs.
2969	Relationships between extramacrochaetae and Notch signalling in Drosophila wing development.	10804180	The function of extramacrochaetae is required during the development of the Drosophila wing in processes such as cell proliferation and vein differentiation. extramacrochaetae encodes a transcription factor of the HLH family, but unlike other members of this family, Extramacrochaetae lacks the basic region that is involved in interaction with DNA. Some phenotypes caused by extramacrochaetae in the wing are similar to those observed when Notch signalling is compromised. Furthermore, maximal levels of extramacrochaetae expression in the wing disc are restricted to places where Notch activity is higher, suggesting that extramacrochaetae could mediate some aspects of Notch signalling during wing development. We have studied the relationships between extramacrochaetae and Notch in wing development, with emphasis on the processes of vein formation and cell proliferation. We observe strong genetic interaction between extramacrochaetae and different components of the Notch signalling pathway, suggesting a functional relationship between them. We show that the higher level of extramacrochaetae expression coincides with the domain of expression of Notch and its downstream gene Enhancer of split-m(beta). The expression of extramacrochaetae at the dorso/ventral boundary and in boundary cells between veins and interveins depends on Notch activity. We propose that at least during vein differentiation and wing margin formation, extramacrochaetae is regulated by Notch and collaborates with other Notch-downstream genes such as Enhancer of split-m(beta).
2970	Secretion of inhibin B by human prepubertal testicular cells in culture.	10802526	Inhibin B is a secretory product of Sertoli cells of the human testis. It has been reported that serum levels of inhibin B in infant boys, peaking at 3 months of age, exceed levels in adult men. The aim of this study was to evaluate inhibin B secretion in primary prepubertal mixed testicular cell cultures, prepared from testes collected at necropsy.Cell cultures were divided into three age groups on the basis of differences in testicular histology: group 1 (n = 7), 1- to 10-day-old newborns, group 2 (n = 7), 1- to 9-month-old infants, and group 3 (n = 8), 12- to 84-month-old children. Cells were maintained in culture for 6 days, harvested and counted. In some samples, during the last 4 days, cells were stimulated with 10ng/ml highly purified human (h) LH (n = 9), 2 ng/ml recombinant human (rh) FSH (n = 9) or 50 ng/ml rhGH (n = 4). On day 6, the secretion of inhibin B and testosterone into the medium was estimated in triplicate. Inhibin B was determined by ELISA and testosterone by RIA.Median (range) inhibin B secretion was 465 (225-1007), 275 (107-298), and 58 (15-184) pg/million cells.24h in groups 1, 2 and 3 respectively. A logarithmic transformation of these values was performed to normalize data. Mean+/-s.d. of transformed inhibin B secretion in group 1 was significantly higher than in group 2 or 3 (P<0.005) and the values for groups 1 and 2 were significantly higher than that for group 3 (P< 0.005). No significant correlation between testosterone and inhibin B secretion into the medium was found when the 22 culture samples were analyzed as a whole. Inhibin B secretion was significantly increased after stimulation with highly purified hLH, rhFSH and rhGH (P < 0.05) and a significant positive correlation between inhibin B and testosterone was found under both hLH and rhFSH stimulation.It is concluded that cells collected from newborns have the highest capacity to secrete inhibin B in vitro, and that this capacity decreases with age during the first years of life. Since no data are available on serum inhibin levels in newborns, it is possible that concentrations at 3 months of age do not represent a post-natal peak but a declining level of high newborn values. As expected, FSH stimulated inhibin B secretion in culture. LH stimulation was probably mediated by paracrine factors secreted by interstitial cells. Finally, our results add new evidence of the involvement of GH in testicular maturation.
2971	Hemophagocytic lymphohistiocytosis.	10798121	Hemophagocytic lymphohistiocytosis (HLH) is a rare condition in children associated with immunodeficiency, life threatening infections and malignancy. Infection associated hemophagocytosis responds well to appropriate antimicrobioal therapy and rarely to steroids when the infective agent is suspected to be of viral origin.
2972	Haemophagocytic lymphohistiocytosis in X-linked severe combined immunodeficiency.	10792291	Haemophagocytic lymphohistiocytosis (HLH) is characterized by destruction of haematopoietic elements, and is associated with a variety of manifestations including immune abnormalities. We describe an infant with HLH who had no evidence of infection or malignancy. He had markedly reduced natural killer (NK) and T-cell numbers and mitogen responses, consistent with severe combined immune deficiency. Western blot and flow cytometry analyses revealed an absence of interleukin (IL)-2 receptor gamma (gamma common) chain expression and a transition (C --> T) at nucleotide 684 in the gamma common gene. This novel case highlights the need for a thorough evaluation of immunological phenotype and genotype in patients with HLH.
2973	The Id4 HLH protein and the timing of oligodendrocyte differentiation.	10790366	An intracellular timer is thought to help control the timing of oligodendrocyte differentiation. We show here that the expression of the helix-loop-helix gene Id4 in oligodendrocyte precursor cells decreases in vivo and in vitro with a time course expected if Id4 is part of the timer. We also show that Id4 expression decreases prematurely when the precursor cells are induced to differentiate by mitogen withdrawal. Both Id4 mRNA and protein decrease together under all of these conditions, suggesting that the control of Id4 expression is transcriptional. Finally, we show that enforced expression of Id4 stimulates cell proliferation and blocks differentiation induced by either mitogen withdrawal or treatment with thyroid hormone. These findings suggest that a progressive fall in Id4 transcription is part of the intracellular timer that helps determine when oligodendrocyte precursor cells withdraw from the cell cycle and differentiate.
2974	Spatially restricted factors cooperate with notch in the regulation of Enhancer of split genes.	10790334	Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologues in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, we have analysed the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes and made comparisons to E(spl)m8. We find that although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. We conclude that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators and, since the activation of target genes is critical in determining the developmental consequences of Notch activity, provide a framework for understanding Notch function in different developmental contexts.
2975	Rapid identification of key amino-acid-DNA contacts through combinatorial peptide synthesis.	10780928	Basic helix-loop-helix (bHLH) transcription factors are characterized by a conserved four-helix bundle that recognizes a specific hexanucleotide DNA sequence in the major groove. Previous studies have shown that amino acids in the basic region make base-specific contacts, whereas the HLH region is responsible for dimerization. Structural data suggest that portions of the loop region may be proximal to the DNA; however, the role of the loop in DNA-binding affinity and specificity has not been investigated.Protein-DNA recognition by the Drosophila bHLH transcription factor Deadpan was probed using combinatorial solid-phase peptide synthesis methods. A series of bHLH peptide libraries that modulate amino acid content and length in the loop region was screened with DNA and peptide affinity columns, and analyzed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). A functional bHLH peptide with reduced loop length was found, and Lys80 was unambiguously identified as the sole loop residue critical for DNA binding. Unnatural amino acids were substituted at this position to assess contributions of the terminal amino group and the alkyl chain length to DNA-binding affinity and specificity.Using combinatorial solid-phase peptide synthesis methods and MALDI-MS, we were able to rapidly identify a key amino acid involved in DNA binding by a bHLH protein. Our approach provides a powerful alternative to current recombinant DNA methods to identify and probe the energetics of protein-DNA interactions.
2976	Caenorhabditis elegans twist plays an essential role in non-striated muscle development.	10769229	The basic helix-loop-helix (bHLH) transcription factor Twist plays a role in mesodermal development in both invertebrates and vertebrates. In an effort to understand the role of the unique Caenorhabditis elegans Twist homolog, hlh-8, we analyzed mesodermal development in animals with a deletion in the hlh-8 locus. This deletion was predicted to represent a null allele because the HLH domain is missing and the reading frame for the protein is disrupted. Animals lacking CeTwist function were constipated and egg-laying defective. Both of these defects were rescued in transgenic mutant animals expressing wild-type hlh-8. Observing a series of mesoderm-specific markers allowed us to characterize the loss of hlh-8 function more thoroughly. Our results demonstrate that CeTwist performs an essential role in the proper development of a subset of mesodermal tissues in C. elegans. We found that CeTwist was required for the formation of three out of the four non-striated enteric muscles born in the embryo. In contrast, CeTwist was not required for the formation of the embryonically derived striated muscles. Most of the post-embryonic mesoderm develops from a single lineage. CeTwist was necessary for appropriate patterning in this lineage and was required for expression of two downstream target genes, but was not required for the expression of myosin, a marker of differentiation. Our results suggest that mesodermal patterning by Twist is an evolutionarily conserved function.
2977	[Familial hemophagocytic lymphohistiocytosis: diagnosis, treatment and pathophysiological mechanisms].	10765622	Familial hemophagocytic lymphohistiocytosis (FHL) is an invariably fatal disease typically seen in infancy and early childhood, with a median survival without therapy of two months. It is characterized by prolonged fever, hepatosplenomegaly, cytopenia, and deficient NK-cell activity and T-cell cytotoxic capacity. Severe neurological symptoms as well as coagulation disorders and abnormalities in liver function and lipid status may also develop. Since the mid 1980's there has been a remarkable increase in our understanding of this disease. In a large-scale international collaborative effort mediated through the Histiocyte Society, diagnostic criteria and an international treatment protocol (HLH-94) based on immunochemotherapy and BMT have been developed. A large proportion of affected children can now be cured and, moreover, successful chemotherapy in utero of FHL has been achieved. It has been shown that the symptoms and signs are mediated through a pronounced hypercytokinemia. Previous suggestions that FHL may be caused by a deficiency in apoptosis were recently confirmed when perforin gene defects were described, which may well explain the disastrous lymphohistiocytic accumulation and subsequent T-cell activation.
2978	Splenectomy in haemophagocytic lymphohistiocytosis: report of histopathological changes with CD19+ B-cell depletion and therapeutic results.	10759706	The pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in children without a known familial pattern of inheritance is often difficult to establish. Splenic enlargement, one of the main clinical findings in this disorder, has led to the use of splenectomy for uncontrollable coagulopathy, persistent cytopenia or both. This procedure is also thought to be a useful tool in making a differential diagnosis in cases of the immunochemotherapy-resistant HLH. We report here five cases of splenectomized childhood HLH, in which subsets of mononuclear spleen cells were analysed either by flow cytometry or immunohistochemistry, and the results were compared with those from cases of hereditary spherocytosis (controls). There was a statistically significant depletion of CD19+ B cells in the HLH cases (3.8 +/- 3.2% vs. 52.6 +/- 4.5%, P < 0. 0001) associated with an increase of T cells in three cases and of natural killer cells in another. The histopathological findings included atrophic white pulps, B-cell depletion with fibrosis and haemosiderosis in all five cases. Despite temporary therapeutic benefits, three of the HLH patients had a rapidly deteriorating post-splenectomy course and all three eventually died. These results demonstrate striking depletion of B cells in the enlarged spleens of children with HLH, which may be an intrinsic feature of HLH pathogenesis. Further study is needed to establish the therapeutic value of splenectomy in this disease.
2979	Integration of FGF and TWIST in calvarial bone and suture development.	10751173	Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.
2980	Functional isoforms of IkappaB kinase alpha (IKKalpha) lacking leucine zipper and helix-loop-helix domains reveal that IKKalpha and IKKbeta have different activation requirements.	10733566	The activity of the NF-kappaB family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IkappaB subunits. Site-specific serine phosphorylation of IkappaBs by two IkappaB kinases (IKKalpha [also known as CHUK] and IKKbeta) targets them for proteolysis. IKKalpha and -beta have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic alpha-helical domains. Here, we describe the properties of two novel cellular isoforms of IKKalpha: IKKalpha-DeltaH and IKKalpha-DeltaLH. IKKalpha-DeltaH and IKKalpha-DeltaLH are differentially spliced isoforms of the IKKalpha mRNA lacking its HLH domain and both its LZip and HLH domains, respectively. IKKalpha is the major RNA species in most murine cells and tissues, except for activated T lymphocytes and the brain, where the alternatively spliced isoforms predominate. Remarkably, IKKalpha-DeltaH and IKKalpha-DeltaLH, like IKKalpha, respond to tumor necrosis factor alpha stimulation to potentiate NF-kappaB activation in HEK293 cells. A mutant, catalytically inactive form of IKKalpha blocked IKKalpha-, IKKalpha-DeltaH-, and IKKalpha-DeltaLH-mediated NF-kappaB activation. Akin to IKKalpha, its carboxy-terminally truncated isoforms associated with the upstream activator NIK (NF-kappaB-inducing kinase). In contrast to IKKalpha, IKKalpha-DeltaLH failed to associate with either itself, IKKalpha, IKKbeta, or NEMO-IKKgamma-IKKAP1, while IKKalpha-DeltaH complexed with IKKbeta and IKKalpha but not with NEMO. Interestingly, each IKKalpha isoform rescued HEK293 cells from the inhibitory effects of a dominant-negative NEMO mutant, while IKKalpha could not. IKKalpha-DeltaCm, a recombinant mutant of IKKalpha structurally akin to IKKalpha-DeltaLH, was equally functional in these assays, but in sharp contrast, IKKbeta-DeltaCm, a structurally analogous mutant of IKKbeta, was inactive. Our results demonstrate that the functional roles of seemingly analogous domains in IKKalpha and IKKbeta need not be equivalent and can also exhibit different contextual dependencies. The existence of cytokine-inducible IKKalpha-DeltaH and IKKalpha-DeltaLH isoforms illustrates potential modes of NF-kappaB activation, which are not subject to the same in vivo regulatory constraints as either IKKalpha or IKKbeta.
2981	SURVEY AND SUMMARY: Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes.	10710415	Basic helix-loop-helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes. The HLH domain dictates dimerization to create homo- and heterodimers. Dimerization juxtaposes the basic regions of the two monomers to create a DNA interaction surface that recognizes the consensus sequence called the E-box, 5'-CANNTG-3'. Several bHLH proteins have been identified in the yeast Saccharomyces cerevisiae using traditional genetic methodologies. These proteins regulate diverse biological pathways. The completed sequence of the yeast genome, combined with novel methodologies allowing whole-genome expression studies, now offers a unique opportunity to study the function of these bHLH proteins. It is the purpose of this review to summarize the current knowledge of bHLH protein function in yeast.
2982	gridlock, an HLH gene required for assembly of the aorta in zebrafish.	10710309	The first artery and vein of the vertebrate embryo assemble in the trunk by migration and coalescence of angioblasts to form endothelial tubes. The gridlock (grl) mutation in zebrafish selectively perturbs assembly of the artery (the aorta). Here it is shown that grl encodes a basic helix-loop-helix (bHLH) protein belonging to the Hairy/Enhancer of the split family of bHLH proteins. The grl gene is expressed in lateral plate mesoderm before vessel formation, and thereafter in the aorta and not in the vein. These results suggest that the arterial endothelial identity is established even before the onset of blood flow and implicate the grl gene in assignment of vessel-specific cell fate.
2983	The molecular basis of lung morphogenesis.	10704888	To form a diffusible interface large enough to conduct respiratory gas exchange with the circulation, the lung endoderm undergoes extensive branching morphogenesis and alveolization, coupled with angiogenesis and vasculogenesis. It is becoming clear that many of the key factors determining the process of branching morphogenesis, particularly of the respiratory organs, are highly conserved through evolution. Synthesis of information from null mutations in Drosophila and mouse indicates that members of the sonic hedgehog/patched/smoothened/Gli/FGF/FGFR/sprouty pathway are functionally conserved and extremely important in determining respiratory organogenesis through mesenchymal-epithelial inductive signaling, which induces epithelial proliferation, chemotaxis and organ-specific gene expression. Transcriptional factors including Nkx2.1, HNF family forkhead homologues, GATA family zinc finger factors, pou and hox, helix-loop-helix (HLH) factors, Id factors, glucocorticoid and retinoic acid receptors mediate and integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Signaling by the IGF, EGF and TGF-beta/BMP pathways, extracellular matrix components and integrin signaling pathways also directs lung morphogenesis as well as proximo-distal lung epithelial cell lineage differentiation. Soluble factors secreted by lung mesenchyme comprise a 'compleat' inducer of lung morphogenesis. In general, peptide growth factors signaling through cognate receptors with tyrosine kinase intracellular signaling domains such as FGFR, EGFR, IGFR, PDGFR and c-met stimulate lung morphogenesis. On the other hand, cognate receptors with serine/threonine kinase intracellular signaling domains, such as the TGF-beta receptor family are inhibitory, although BMP4 and BMPR also play key inductive roles. Pulmonary neuroendocrine cells differentiate earliest in gestation from among multipotential lung epithelial cells. MASH1 null mutant mice do not develop PNE cells. Proximal and distal airway epithelial phenotypes differentiate under distinct transcriptional control mechanisms. It is becoming clear that angiogenesis and vasculogenesis of the pulmonary circulation and capillary network are closely linked with and may be necessary for lung epithelial morphogenesis. Like epithelial morphogenesis, pulmonary vascularization is subject to a fine balance between positive and negative factors. Angiogenic and vasculogenic factors include VEGF, which signals through cognate receptors flk and flt, while novel anti-angiogenic factors include EMAP II.
2984	The ETV6-NTRK3 gene fusion encodes a chimeric protein tyrosine kinase that transforms NIH3T3 cells.	10702799	The congenital fibrosarcoma t(12;15)(p13;q25) rearrangement splices the ETV6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophin-3 receptor gene on chromosome 15q25. Resultant ETV6-NTRK3 fusion transcripts encode the helix - loop - helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. We show here that ETV6-NTRK3 homodimerizes and is capable of forming heterodimers with wild-type ETV6. Moreover, ETV6-NTRK3 has PTK activity and is autophosphorylated on tyrosine residues. To determine if the fusion protein has transforming activity, NIH3T3 cells were infected with recombinant retroviral vectors carrying the full-length ETV6-NTRK3 cDNA. These cells exhibited a transformed phenotype, grew macroscopic colonies in soft agar, and formed tumors in severe combined immunodeficient (SCID) mice. We hypothesize that chimeric proteins mediate transformation by dysregulating NTRK3 signal transduction pathways via ligand-independent dimerization and PTK activation. To test this hypothesis, we expressed a series of ETV6-NTRK3 mutants in NIH3T3 cells and assessed their transformation activities. Deletion of the ETV6 HLH domain abolished dimer formation with either ETV6 or ETV6-NTRK3, and cells expressing this mutant protein were morphologically non-transformed and failed to grow in soft agar. An ATP-binding mutant failed to autophosphorylate and completely lacked transformation activity. Mutants of the three NTRK3 PTK activation-loop tyrosines had variable PTK activity but had limited to absent transformation activity. Of a series of signaling molecules well known to bind to wild-type NTRK3, only phospholipase-Cgamma (PLCgamma) associated with ETV6-NTRK3. However, a PTK active mutant unable to bind PLCgamma did not show defects in transformation activity. Our studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity. Oncogene (2000) 19, 906 - 915.
2985	Identification and chromosomal localization of murine ORC3, a new member of the mouse origin recognition complex.	10702681	A new member of the murine origin recognition complex (ORC) related to Saccharomyces cerevisiae ORC3 has been cloned. Transcription of ORC3 is not suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. The transcription level of the ORC3 gene is constantly high in all phases of the cell cycle. Murine ORC3 protein contains a putative nuclear localization signal and a non-basic helix-loop-helix motif. Both motifs are conserved in eukaryotes. A potential dimerization partner of ORC3p in the murine ORC complex is ORC1p which also contains an HLH motif. This HLH motif is also highly conserved in all eukaryotic ORC1 proteins. Comparison of murine ORC3p with other ORC3-related proteins shows high amino acid homology and motif conservation leading to the conclusion that ORC3p is part of the initiation machinery conserved in eukaryotes. The mouse ORC3 gene Orc3 was assigned to mouse chromosome 4A3 by fluorescence in situ hybridization (FISH) analysis.
2986	Combined immunoprecipitation and agglutination for the detection of the heterodimeric molecule: human chorionic gonadotropin as a study model.	10698471	Double-particle reversed passive hemagglutination (RPHA) was performed for the detection of an intact heterodimeric form of human chorionic gonadotropin (intact hCG) composed in Profasi hCG (P-hCG). This technique relied on two mixed types of human O RBC, which were individually coated with two distinct monoclonal antibodies that recognized alpha or beta subunit of hCG, i.e. ALC-1 and BEL-5, respectively. The positive hemagglutination result was achieved by this technique. However, in the BEL-5 coated single-particle control system, positive results for both P-hCG and beta subunit hCG solution were realized. The occurrence of beta-multimer hCG was a causative molecule revealed by the hemagglutination inhibition technique. Thereby, the novel method called "combined immunoprecipitation and agglutination" was developed to overcome this problem. The free beta subunit together with the betamultimer hCG were eliminated from other forms presented in P-hCG after using the ALC-1 coated particles. The precipitated particles, which captured the heterodimer hCG molecule, reacted further with soluble BEL-5 to subsequently form a trellis. A positive result was obtained only with P-hCG, but not with beta subunit hCG or hLH. This study is inferable as a model for the detection of heterodimeric molecule by an elementary method.
2987	mSin3A regulates murine erythroleukemia cell differentiation through association with the TAL1 (or SCL) transcription factor.	10688671	Activation of the TAL1 (or SCL) gene is the most frequent gain-of-function mutation in T-cell acute lymphoblastic leukemia (T-ALL). TAL1 belongs to the basic helix-loop-helix (HLH) family of transcription factors that bind as heterodimers with the E2A and HEB/HTF4 gene products to a nucleotide sequence motif termed the E-box. Reported to act both as an activator and as a repressor of transcription, the mechanisms underlying TAL1-regulated gene expression are poorly understood. We report here that the corepressor mSin3A is associated with TAL1 in murine erythroleukemia (MEL) and human T-ALL cells. Interaction mapping showed that the basic-HLH domain of TAL1 was both necessary and sufficient for TAL1-mSin3A interaction. TAL1 was found, in addition, to interact with the histone deacetylase HDAC1 in vitro and in vivo, and a specific histone deacetylase inhibitor, trichostatin A (TSA), relieved TAL1-mediated repression of an E-box-containing promoter and a GAL4 reporter linked to a thymidine kinase minimal promoter. Further, TAL1 association with mSin3A and HDAC1 declined during dimethyl sulfoxide-induced differentiation of MEL cells in parallel with a decrease in mSin3A abundance. Finally, TSA had a synergistic effect with enforced TAL1 expression in stimulating MEL cells to differentiate, while constitutive expression of mSin3A inhibited MEL cell differentiation. These results demonstrate that a corepressor complex containing mSin3A and HDAC1 interacts with TAL1 and restricts its function in erythroid differentiation. This also has implications for this transcription factor's actions in leukemogenesis.
2988	Management of severe neutropenia with cyclosporin during initial treatment of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis.	10674906	Severe neutropenia (absolute neutrophil count <500/gl) is probably due to the combined effects of dysregulated cytokine production and chemotherapeutic agents, and is one of the risk factors in the initial treatment of patients with Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH). We report here 9 cases of neutropenic HLH, of which 8 were treated with cyclosporin (CSA, 2-6 mg/kg/day; continuous infusion, or 6 mg/kg/day; per os, for periods ranging from 9 days to >8 weeks) in the initial neutropenic phase during induction treatment using corticosteroids and etoposide. Five of the 6 cases, in which CSA treatment was started early (before the second week of induction), survived the critical period with recovery of neutrophil counts within a week. The remaining 3 cases, in which CSA was introduced later or not at all, died of infection. Based on these results, we recommend a prompt short-term CSA infusion during neutropenic episodes in the most common treatment regimen of etoposide and corticosteroids in patients with HLH. Improved neutrophil recovery as a result of CSA treatment makes it possible to continue immunochemotherapy safely and obtain improved patient outcomes.
2989	Thymocyte selection is regulated by the helix-loop-helix inhibitor protein, Id3.	10661402	E2A, HEB, E2-2, and daughterless are basic helix-loop-helix (bHLH) proteins that play key roles in multiple developmental pathways. The DNA binding activity of E2A, HEB, and E2-2 is regulated by a distinct class of inhibitor HLH proteins, the Id gene products. Here, we show that Id3 is required for major histocompatability (MHC) class I- and class II-restricted thymocyte positive selection. Additionally, H-Y TCR-mediated negative selection is severely perturbed in Id3 null mutant mice. Finally, we show that E2A and Id3 interact genetically to regulate thymocyte development. These observations identify the HLH inhibitory protein Id3 as an essential component required for proper thymocyte maturation.
2990	Reciprocal Id expression and myelin gene regulation in Schwann cells.	10656257	Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.
2991	Expression of helix-loop-helix type negative regulators of differentiation during limb regeneration in urodeles and anurans.	10646803	The urodele is capable of regenerating its limb by forming a blastema even in the adult. By contrast, the anuran, which is phylogenetically close to the urodele, loses this ability during metamorphosis and forms blastema-like tissues that develop only into a spike-like structure in the adult. In order to compare the molecular mechanism of the formation and maintenance of the blastema between the urodele and anuran, the genes encoding helix-loop-helix (HLH) type negative regulators of differentiation were characterized for both the Japanese newt, Cynops pyrrhogaster, and African clawed frog, Xenopus laevis. Cynops homologs of Id2, Id3, and HES1 and Xenopus Id2 were identified. To learn the roles of these genes in regeneration, their expression was examined. The expression of Id2 and Id3 was low in unamputated limbs, but was up-regulated in blastemas of both adult newt and Xenopus. Interestingly, transcripts of the two Id genes showed specific localizations in the blastema and the expression patterns were very similar in both species through the early to medium bud stage. Id2 was expressed predominantly in the blastemal epidermis, and Id3 was expressed equally in the blastemal epidermis and mesenchyme including cells in precartilage condensations. HES1 expression was up-regulated in the newt blastemal epidermis. It was thought that the up-regulation of these genes in the epidermis was related to the proliferation of the cells and that increased expression of these genes in the mesenchyme was related to the undifferentiated state of the blastemal cells. These results and considerations strongly suggested that the state of differentiation is similar in the early to medium bud blastema of both urodeles and anurans. The expression of Id3 remained high through to the digits stage in newts. In contrast, its expression in Xenopus decreased in spike-like regenerates, which correspond to palette-digits stage of newt regenerates. From these results, it was suggested that the blastema redifferentiates earlier in the frog than in the newt, and therefore the timing of redifferentiation of the cartilage is crucial for complete regeneration.
2992	Id-2 regulates critical aspects of human cytotrophoblast differentiation, invasion and migration.	10631176	During early human placental development, the conceptus attaches itself to the uterus through cytotrophoblast invasion. Invasive cytotrophoblast cells differentiate from precursor villous cytotrophoblasts, but the essential regulating factors in this process are unknown. Basic helix-loop-helix (bHLH) transcription factor dimers are essential regulators of mouse trophoblast development. We therefore examined the importance of this family of factors in the human placenta. In many cell lineages, bHLH factors are sequestered by members of the Id family, HLH proteins that lack the basic DNA binding domain (Inhibitor of DNA binding proteins (Id-1 to Id-4)). During differentiation of some tissues, Id expression declines, allowing bHLH factors to dimerize, bind DNA and trans-activate lineage-specific genes. To begin to study the role of bHLH transcription factors in human placental development, we first characterized Id expression in cytotrophoblast cells. The cells expressed Id-3 constitutively; Id-2 was downregulated, at the mRNA and protein levels, as the cells differentiated in culture and in situ, respectively. In cases when cytotrophoblast differentiation was compromised (in placentas from women with preeclampsia, or in cells grown under hypoxic conditions in culture), Id-2 expression was maintained. To assess the functional relevance of these correlations, we used an adenovirus vector to maintain Id-2 protein expression in cultured cytotrophoblasts. Compared to control (lacZ-expressing) cells, cytotrophoblasts transduced to constitutively express Id-2 retained characteristics of undifferentiated cells: (alpha)1 integrin expression was low and cyclin B expression was retained. Furthermore, invasion through Matrigel was partially inhibited and migration was strikingly enhanced in Id-2-expressing cells. These results suggest that Id-2 and the bHLH factors that it partners play important roles in human cytotrophoblast development.
2993	Control of endodermal endocrine development by Hes-1.	10615124	Development of endocrine cells in the endoderm involves Atonal and Achaete/Scute-related basic helix-loop-helix (bHLH) proteins. These proteins also serve as neuronal determination and differentiation factors, and are antagonized by the Notch pathway partly acting through Hairy and Enhancer-of-split (HES)-type proteins. Here we show that mice deficient in Hes1 (encoding Hes-1) display severe pancreatic hypoplasia caused by depletion of pancreatic epithelial precursors due to accelerated differentiation of post-mitotic endocrine cells expressing glucagon. Moreover, upregulation of several bHLH components is associated with precocious and excessive differentiation of multiple endocrine cell types in the developing stomach and gut, showing that Hes-1 operates as a general negative regulator of endodermal endocrine differentiation.
2994	Differences in urinary excretion patterns of the hLH beta core fragment in premenopausal, perimenopausal, and postmenopausal women.	10614675	The heterodimeric luteinizing hormone beta core fragment (hLH beta cf) is a highly stable urinary analyte reflective of circulating hLH. It is measured easily because of its high molar content and has none of the multiple isoforms and subunit dissociation problems of LH urinary measurements. As part of a long-term effort to develop new biochemical assays to stage women during the perimenopausal transition, we have examined the patterns of urinary excretion of this metabolite of hLH in premenopausal, perimenopausal, and postmenopausal women.We measured the concentration of the hLH beta cf in 10 consecutive first morning void urine specimens from premenopausal, perimenopausal, and postmenopausal women. Day 1 of collection was the first day of menses in the cycling women.Postmenopausal women exhibited a widely fluctuating pattern of LH beta core fragment excretion, which is not correlated with hLH measured by immunofluorometric assay or with follicle-stimulating hormone measured by immunofluorometric assay. The postmenopausal group was easily distinguished from premenopausal women on the basis of an area-under-the-curve concentration function. Perimenopausal women displayed intermediate hLH beta cf concentrations; some clearly were in postmenopausal ranges, and others were in the premenopausal ranges.The pattern of excretion and concentrations of the hLH beta cf is significantly different between premenopausal and postmenopausal women. Perimenopausal women exhibited intermediate changes. The capability to measure this type of stable urinary metabolite as a reflection of changes in dynamics of its parent circulating hormone offers new possibilities in the development and application of large-scale testing that does not require blood sampling.
2995	Antagonism of notch signaling activity by members of a novel protein family encoded by the bearded and enhancer of split gene complexes.	10603347	Cell-cell signaling through the Notch receptor is a principal mechanism underlying cell fate specification in a variety of developmental processes in metazoans, such as neurogenesis. In this report we describe our investigation of seven members of a novel gene family in Drosophila with important connections to Notch signaling. These genes all encode small proteins containing predicted basic amphipathic (&agr;)-helical domains in their amino-terminal regions, as described originally for Bearded; accordingly, we refer to them as Bearded family genes. Five members of the Bearded family are located in a newly discovered gene complex, the Bearded Complex; two others reside in the previously identified Enhancer of split Complex. All members of this family contain, in their proximal upstream regions, at least one high-affinity binding site for the Notch-activated transcription factor Suppressor of Hairless, suggesting that all are directly regulated by the Notch pathway. Consistent with this, we show that Bearded family genes are expressed in a variety of territories in imaginal tissue that correspond to sites of active Notch signaling. We demonstrate that overexpression of any family member antagonizes the activity of the Notch pathway in multiple cell fate decisions during adult sensory organ development. These results suggest that Bearded family genes encode a novel class of effectors or modulators of Notch signaling.
2996	Regulation of gene expression by glucose.	10604195	Fatty acid synthase (EC 2.3.1.85) is an enzyme involved in the lipogenic pathway allowing fatty acid synthesis from glucose. Glucose up-regulates the transcription of the fatty acid synthase gene in both adipocytes and hepatocytes, with insulin having only an indirect role. The signal metabolite could be glucose-6-phosphate rather than glucose itself. The glucose response element of the fatty acid synthase gene has not yet been precisely identified, although a -2 kb region of the fatty acid synthase promoter is sufficient to confer nutritional responsiveness to a reporter gene. ADD1/SREBP1, a b-HLH-LZ transcription factor belonging to the sterol regulatory element-binding protein family might be involved in the transduction of the glucose effect. Finally, the stimulatory effect of glucose on the expression of the fatty acid synthase gene is inhibited by the activation of AMP-activated protein kinase. Interestingly enough, AMP-activated protein kinase is structurally and functionally related to the yeast SNF1 protein kinase complex which is essential for the transcriptional activation of glucose-repressed genes in Saccharomyces cerevisiae.
2997	Recurrent viral associated hemophagocytic syndrome in a child with Langerhans cell histiocytosis.	10598673	Langerhans cell histiocytosis (LCH) with subsequent viral-associated hemophagocytic syndrome (VAHS) or secondary hemophagocytic lymphohistiocytosis (HLH) is extremely rare. A 15-month-old girl with disseminated LCH experienced three episodes of VAHS during maintenance therapy. Viral infection, with influenza A, herpes simplex, and adenovirus, respectively, was documented at each episode. She recovered each time after interruption of maintenance therapy. The occurrence of fever and pancytopenia in patients with chemotherapy-treated LCH can be associated with VAHS and not with relapsing LCH.
2998	Establishment of distinct MyoD, E2A, and twist DNA binding specificities by different basic region-DNA conformations.	10594029	Basic helix-loop-helix (bHLH) proteins perform a wide variety of biological functions. Most bHLH proteins recognize the consensus DNA sequence CAN NTG (the E-box consensus sequence is underlined) but acquire further functional specificity by preferring distinct internal and flanking bases. In addition, induction of myogenesis by MyoD-related bHLH proteins depends on myogenic basic region (BR) and BR-HLH junction residues that are not essential for binding to a muscle-specific site, implying that their BRs may be involved in other critical interactions. We have investigated whether the myogenic residues influence DNA sequence recognition and how MyoD, Twist, and their E2A partner proteins prefer distinct CAN NTG sites. In MyoD, the myogenic BR residues establish specificity for particular CAN NTG sites indirectly, by influencing the conformation through which the BR helix binds DNA. An analysis of DNA binding by BR and junction mutants suggests that an appropriate BR-DNA conformation is necessary but not sufficient for myogenesis, supporting the model that additional interactions with this region are important. The sequence specificities of E2A and Twist proteins require the corresponding BR residues. In addition, mechanisms that position the BR allow E2A to prefer distinct half-sites as a heterodimer with MyoD or Twist, indicating that the E2A BR can be directed toward different targets by dimerization with different partners. Our findings indicate that E2A and its partner bHLH proteins bind to CAN NTG sites by adopting particular preferred BR-DNA conformations, from which they derive differences in sequence recognition that can be important for functional specificity.
2999	Functional antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) trans-factors for the regulation of fatty acid synthase promoter in adipocytes.	10585876	We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA. Using the FAS gene as a model of an ADD1-regulated promoter in transiently transfected isolated rat adipocytes or mature 3T3-L1 adipocytes, a potent inhibition of the activity of the FAS-chloramphenicol acetyltransferase reporter gene was observed by overexpression of Id2 or Id3. Reciprocally, co-transfection of Id3 antisense and ADD1 expression vectors in preadipocytes potentiated the ADD1/SREBP-1c effect on the FAS promoter activity. Finally, in the non adipogenic NIH-3T3 cell line, most of the ADD1-mediated trans-activation of the FAS promoter was counteracted by co-transfection of Id2 or Id3 expression vectors. Previous studies have indicated Id gene expression to be down-regulated during adipogenesis [Moldes, Lasnier, Fève, Pairault and Djian (1997) Mol. Cell. Biol. 17, 1796-1804]. We here demonstrated that there was a dramatic rise of Id2 and Id3 mRNA levels when 3T3-L1 adipocytes or isolated rat fat cells were exposed to lipolytic and anti-lipogenic agents, forskolin and isoproterenol. Taken together, our data show that Id products are functionally involved in modulating ADD1/SREBP-1c transcriptional activity, and thus lipogenesis in adipocytes.
3000	The tyrosine kinase abl-related gene ARG is fused to ETV6 in an AML-M4Eo patient with a t(1;12)(q25;p13): molecular cloning of both reciprocal transcripts.	10590083	The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3'rapid amplification of cDNA ends-polymerase chain reaction (3'RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARG involvement in a human malignancy.
3001	Identification of GTF2IRD1, a putative transcription factor within the Williams-Beuren syndrome deletion at 7q11.23.	10575229	Williams-Beuren syndrome (WBS) is a microdeletion syndrome caused by haploinsufficiency of genes at 7q11.23. Here we describe the identification and characterization of a novel gene named GTF2IRD1, for GTF2I-repeat domain 1, within the WBS deletion region. Northern blot analysis revealed ubiquitous expression during development with two transcripts of 3.6 kb and 5.0 kb generated by alternative splicing. GTF2IRD1 encodes a protein of 944 amino acids that contains a region of high similarity to a unique motif with helix-loop-helix forming potential occurring within the transcription factor GTF2I. Analogous to TFII-I, the product of GTF2IRD1 may have the ability to interact with other HLH-proteins and function as a transcription factor or as a negative transcriptional regulator. A recent report of the identification of a muscle-specific transcription factor, MusTRD1, supports this hypothesis (O'Mahoney et al., 1998). The open reading frame described for MusTRD1 is identical to that of GTF2IRD1; however, the putative MusTRD1-protein is 486 amino acids shorter than the predicted protein encoded by GTF2IRD1. A heterozygous deletion of GTF2IRD1 may contribute to the complex WBS phenotype.
3002	Bone marrow transplantation in a child with hemophagocytic lymphohistiocytosis using a less toxic conditioning regimen.	10573638	Hemophagocytic lymphohistiocytosis (HLH) is a rare non-neoplastic, frequently fatal disease of childhood. HLA-matched bone marrow transplantation (BMT) can bring about long-term remission and an eventual cure.We report on the beneficial effect of BMT in a 2-month-old male using a less intensive conditioning regimen. The regimen included busulfan at 4 mg/kg/day (total dose 16 mg/kg), etoposide at 300 mg/m2/day (total dose 900 mg/m2), and cyclophosphamide at 50 mg/kg/day (total dose 150 mg/kg). Prophylaxis for graft-vs.-host disease included methotrexate and cyclosporine.An absolute neutrophil count of 500 microL was noticed on + day 12 (engraftment day). At present, i.e., 400 days after the procedure, the patient is asymptomatic, his physical examination is normal, and a slightly increased level of gamma-glutamyl-transpeptidase (GGT) and alkaline phosphatase are the only laboratory abnormalities.In this case, the conditioning regimen was adequate for the eradication of the disease and allowed persistent engraftment without significant toxicity. The results in our patient suggest that a less toxic regimen is feasible and permits rapid engraftment without compromising the effectiveness of chemotherapy.
3003	A transcription factor involved in skeletal muscle gene expression is deleted in patients with Williams syndrome.	10573005	Williams-Beuren syndrome (WS) is a developmental disorder caused by a hemizygous microdeletion of approximately 1.4MB at chromosomal location 7q11.23. The transcription map of the WS critical region is not yet complete. We have isolated and characterised a 3.4 kb gene, GTF3, which occupies about 140 kb of the deleted region. Northern blot analysis showed that the gene is expressed in skeletal muscle and heart, and RT-PCR analysis showed expression in a range of adult tissues with stronger expression in foetal tissues. Part of the conceptual GTF3 protein sequence is almost identical to a recently reported slow muscle-fibre enhancer binding protein MusTRD1, and shows significant homology to the 90 amino-acid putative helix-loop-helix repeat (HLH) domains of the transcription factor TFII-I (encoded for by the gene GTF2I). These genes may be members of a new family of transcription factors containing this HLH-like repeated motif. Both GTF3 and GTF2I map within the WS deleted region, with GTF2I being positioned distal to GTF3. GTF3 is deleted in patients with classic WS, but not in patients we have studied with partial deletions of the WS critical region who have only supravalvular aortic stenosis. A feature of WS is abnormal muscle fatiguability, and we suggest that haploinsufficiency of the GTF3 gene may be the cause of this.
3004	Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus.	10545951	The helix-loop-helix (HLH) protein NEUROD1 (also known as BETA2) functions as a regulatory switch for endocrine pancreatic development. In mice homozygous for a targeted disruption of Neurod, pancreatic islet morphogenesis is abnormal and overt diabetes develops due in part to inadequate expression of the insulin gene (Ins2). NEUROD1, following its heterodimerization with the ubiquitous HLH protein E47, regulates insulin gene (INS) expression by binding to a critical E-box motif on the INS promoter. Here we describe two mutations in NEUROD1, which are associated with the development of type 2 diabetes in the heterozygous state. The first, a missense mutation at Arg 111 in the DNA-binding domain, abolishes E-box binding activity of NEUROD1. The second mutation gives rise to a truncated polypeptide lacking the carboxy-terminal trans-activation domain, a region that associates with the co-activators CBP and p300 (refs 3,4). The clinical profile of patients with the truncated NEUROD1 polypeptide is more severe than that of patients with the Arg 111 mutation. Our findings suggest that deficient binding of NEUROD1 or binding of a transcriptionally inactive NEUROD1 polypeptide to target promoters in pancreatic islets leads to the development of type 2 diabetes in humans.
3005	[Luliberin and lutropin--their significance].	10544663	Human lutropin (hLH) is a heterodimeric, glycoprotein hormone released from pituitary by decapeptide luliberin (LH-RH). The both hormones plays an important role in axis hypothalamus-pituitary-gonads responsible for procreation. Many isoforms of hLH have been found and the occurrence of several genetic variants of the hormone in infertile female patients were described. Also genetic mutants of human lutropin choriogonadotropin receptor (LH/CG-R) in male young patients with precocious puberty were proved. Until now several thousands of LH-RH analogs were synthesized and some of them were applied in therapy of several gynecological, and oncological diseases or in vitro fertilization.
3006	The leukemia-associated gene TEL encodes a transcription repressor which associates with SMRT and mSin3A.	10544023	The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The TEL protein contains two functional domains that have been partially characterized: a helix-loop-helix (HLH) domain (also known as a pointed domain) at the N-terminus, which physically interacts with itself, with the SUMO-conjugating enzyme UBC9, and with FLI1; and, at the C-terminus, an ETS domain with DNA-binding properties. Little is known about the function of the central region of TEL. The HLH domain and the central region of TEL are consistently maintained in the t(12;21), which is the most frequent chromosomal translocation involving TEL. In this study, we found that the HLH domain and the central region of TEL mediate transcription repression by two distinct mechanisms. The central region involves the recruitment of a repression complex, including SMRT and mSin3A. The HLH domain represses gene transcription through a mechanism that is independent of known corepressors. Thus, TEL belongs to a growing number of transcription factors rearranged by chromosomal translocations that are associated with the corepressor complexes.
3007	Improved outcome in haemophagocytic lymphohistiocytosis after bone marrow transplantation from related and unrelated donors: a single-centre experience of 12 patients.	10520013	Haemophagocytic lymphohistiocytosis (HLH) is an autosomal recessive disease with histiocytic and lymphocytic infiltrations in multiple organs. Cure seems possible only by allogeneic bone marrow transplantation (BMT), but matched sibling donors (MSD) are restricted and high mortality rates are associated with BMT from unrelated donors (URD). We report on 12 consecutive HLH patients with an improved outcome following URD transplants. Eight patients received BMT from URD, four from MSD. Five patients had signs of active HLH at the time of BMT. The conditioning regimen consisted of 20 mg/kg busulphan, 60 mg/kg VP-16 and 120 mg/kg cyclophosphamide and, in case of URD, 90 mg/kg antithymocyte globulin. The doses of busulphan and VP-16 were reduced during the programme to 16 mg/kg and 30 mg/kg, respectively. Using a fivefold graft-versus-host disease (GVHD) prophylaxis, GVHD was absent or mild in 10, and moderate or severe in two patients undergoing unrelated transplants. One patient with URD experienced graft failure and was retransplanted on day 37. Major toxicities were hepatic veno-occlusive disease in five, capillary leak syndrome in two, pneumonia in three, sepsis in one, severe mucositis in one and seizures in two patients. All patients are alive without HLH after a median follow-up of 24.5 months. One patient has chronic GVHD, another patient has severe retardation. Three patients show slight to moderate development delay. These results indicate that in HLH, BMT from matched unrelated donors should be performed. Incomplete resolution of disease activity need not impede a successful outcome.
3008	Epitope analysis and molecular modeling reveal the topography of the C-terminal peptide of the beta-subunit of human chorionic gonadotropin.	10518802	Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common alpha-subunit and a hormone-specific beta-subunit. Among the gonadotropin beta-subunits, greater than 85% homology exists between lutropin (hLH)beta and hCGbeta in their first 114 amino acid residues. However, unlike hLHbeta, hCGbeta contains a 31-amino acid hydrophilic stretch at its carboxyl end (CTPbeta: C-terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPbeta remains unknown. In this study, we have attempted to define the topology of CTPbeta using mAb probes. We investigated three epitopes on hCGalpha, which are hidden in the hCGalphabeta dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPbeta. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPbeta. Consistent with this view, several treatments of hCG that removed CTPbeta unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPbeta on the alpha-subunit, we used an epitope-mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods 202, 173-182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPbeta. Further, we were also able to build a model of CTPbeta, completely independently of the epitope-mapping studies, using a homology-based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci. 4 (Suppl. 2), 86-97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPbeta are in contact with hCGalpha in the native hCG dimer.
3009	Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3.	10515867	Transgenic and gene targeted mice have contributed greatly to our understanding of the mechanisms underlying B-cell development. We describe here a model system that allows us to apply molecular genetic techniques to the analysis of human B-cell development. We constructed a retroviral vector with a multiple cloning site connected to a gene encoding green fluorescent protein by an internal ribosomal entry site. Human CD34(+)CD38(-) fetal liver cells, cultured overnight in a combination of stem cell factor and interleukin-7 (IL-7), could be transduced with 30% efficiency. We ligated the gene encoding the dominant negative helix loop helix (HLH) factor Id3 that inhibits many enhancing basic HLH transcription factors into this vector. CD34(+)CD38(-) FL cells were transduced with Id3-IRES-GFP and cultured with the murine stromal cell line S17. In addition, we cultured the transduced cells in a reaggregate culture system with an SV-transformed human fibroblast cell line (SV19). It was observed that overexpression of Id3 inhibited development of B cells in both culture systems. B-cell development was arrested at a stage before expression of the IL-7Ralpha. The development of CD34(+)CD38(-) cells into CD14(+) myeloid cells in the S17 system was not inhibited by overexpression of Id3. Moreover, Id3(+) cells, although inhibited in their B-cell development, were still able to develop into natural killer (NK) cells when cultured in a combination of Flt-3L, IL-7, and IL-15. These findings confirm the essential role of bHLH factors in B-cell development and demonstrate the feasibility of retrovirus-mediated gene transfer as a tool to genetically modify human B-cell development.
3010	Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.	10502414	HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation.
3011	Expression and characterization of recombinant beta-subunit hCG homodimer.	10484290	We have linked two human chorionic gonadotropin (hCG) beta-subunit cDNAs in tandem such that the expressed fusion protein consists of two mature beta-subunits joined through the carboxy terminal peptide of the first beta-subunit. A single glycine residue is inserted between the two subunits in the fusion protein. Chinese hamster ovary (CHO) cells transformed with a clone that contains the fused cDNAs express and secrete a protein that is consistent with it being a beta-hCG homodimer protein. These beta-homodimer molecules can recombine with two free alpha-subunits indicating that both beta-subunits within the homodimer are likely folded in their native conformation. Our data also suggest that the two beta-subunits fold upon each other as a globular protein and do not appear to exist as a simple fusion of two linear beta-subunits. Furthermore, the two beta-monomer subunits in the fusion protein form a stable homodimer that can bind and activate the hLH/CG receptor specifically. Recombination of the fusion protein with alpha-subunits appears to favor an arrangement where two alpha-subunits combine with a single molecule of the fusion protein. The recombined molecule consists of four subunits and is comparable to two tethered hCG moieties, which constitutes a hCG dimer. This hormone dimer can bind and activate the hLH/CG receptor with an activity approximating that of native hCG.
3012	Fusion of a novel gene, BTL, to ETV6 in acute myeloid leukemias with a t(4;12)(q11-q12;p13).	10477709	The ETV6 gene (also known as TEL) is the main target of chromosomal translocations affecting chromosome band 12p13. The rearrangements fuse ETV6 to a wide variety of partner genes in both myeloid and lymphoid malignancies. We report here 4 new cases of acute myeloid leukemia (AML) with very immature myeloblasts (French-American-British [FAB]-M0) and with a t(4;12)(q11-q12;p13). In all cases, ETV6 was found recombined to a new gene, homologous to the mouse Brx gene. The gene was named BTL (Brx-like Translocated in Leukemia). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments indicate that the expression of the BTL-ETV6 transcript, but not of the reciprocal ETV6-BTL transcript, is a common finding in these leukemias. In contrast to the majority of other ETV6 fusions, both the complete helix-loop-helix (HLH) and ETS DNA binding domains of ETV6 are present in the predicted BTL-ETV6 fusion protein, and the chimeric gene is transcribed from the BTL promoter.
3013	Head versus trunk patterning in the Drosophila embryo; collier requirement for formation of the intercalary segment.	10477305	Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. In this paper, we report the phenotypic analysis of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The parasegmental register of col activation is controlled by the combined activities of the head-gap genes buttonhead and empty spiracles and the pair-rule gene even skipped; it therefore integrates inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent. After gastrulation, positive autoregulation of col is limited to cells of anterior PS0. Conversely, heat-pulse induced ubiquitous expression of Col leads to disruption of the head skeleton. Together, these results indicate that col is required for establishment of the PS(-1)/PS0 parasegmental border and formation of the intercalary segment. Our data support neither a simple combinatorial model for segmental patterning of the head nor a direct activation of segment polarity gene expression by head-gap genes, but rather argue for the existence of parasegment-specific second order regulators acting in the head, at a level similar to that of pair-rule genes in the trunk.
3014	Luteinizing hormone measurement in polycystic ovary syndrome: a practical approach.	10474119	The biological diagnosis of polycystic ovary syndrome (PCO) remains questionable, and a single immunological hLH (ihLH) determination can be misleading. In order better to characterize these patients, we studied hLH pulsatility every 10 min for 4h using a radioimmunoassay and then compared the results with others we obtained with a biological method. Radioimmunological and biological profiles were similar in patients with PCO and in controls. We also studied pulsatility characteristics - frequency and amplitude - and calculated the area under the curve (AUC). There was no significant increase in frequency in our 10 patients with PCO but, as in other studies, increased amplitude of hLH pulses was observed. The most discriminating parameter was the AUC. For practical purposes, we propose that hLH in patients with PCO could be assessed efficiently by taking four samples every 10 min, with computerized calculation of the AUC.
3015	Isolation of human lutropin from woman urine and comparison of its properties with pituitary hormone.	10470445	Lutropin was isolated from woman preovulatory urine by immunoaffinity chromatography using a column with monoclonal antibody (mAb) anti-beta hLH(37) coupled to Sepharose CL 4B. As it was demonstrated the isolated hormone, as well as pituitary lutropin, were separated in SDS-PAGE into several well visible fractions with 30-94 kDa molecular mass and scarcely visible fractions with 14-20 kDa. All fractions reacted only with mAb anti-alpha hCG(99)-HRP but not with mAb anti-beta hLH-HRP. Pretreatment of pituitary lutropin with PN-Gase F did not affect its electrophoretic pattern. After boiling the hormone with SDS and beta ME no fractions in SDS-PAGE were observed. No substantial differences in affinity chromatography on Con A-Sepharose between pituitary and urinary lutropin were noted. Some differences between these two hormone preparations were observed in assays performed with several ELISA variants. Using two pairs of mAbs anti-beta hLH for ELISA technique no hormone was assayed in urine samples from women, collected between 12 and 16 days of menstrual cycles.
3016	Mxi1 mutations in human neurofibrosarcomas.	10470286	Mxi1 is thought to negatively regulate Myc function and may therefore be a potential tumor suppressor gene. Little effort has yet been made to find alterations involving this gene in human solid tumors. We screened 31 human gastric cancers, 7 esophageal cancers, 85 bone and soft tissue tumors of various types, including 4 neurofibrosarcomas. We also examined 29 human tumor cell lines consisting of 12 esophageal cancers, 7 glioma/glioblastomas and 10 others for Mxi1 mutations in exons 1, 2, 4 (HLH domain), 5 and 6. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and subsequent sequencing revealed three distinct polymorphisms in the intron-exon boundary upstream from exon 6. We discovered a missense mutation, GCA to GTA (Ala 54 Val), in exon 2 in a neurofibrosarcoma patient (case 1), two missense mutations, AAA to CAA (Lys 118 Gln) and GAA to GGA (Glu 154 Gly) in exon 5 of another neurofibrosarcoma patient (case 2), and 3 amino acid substitutions, GTG to GCG (Val 179 Ala), GTT to GCT (Val 181 Ala) and TTC to CTC (Phe 186 Leu), in a third neurofibrosarcoma patient (case 3). In case 3, loss of heterozygosity was also demonstrated by informative (TTC)3/(TTC)2 polymorphism. Our data demonstrate that mutations occur in the Mxi1 gene in neurofibrosarcoma. Missense mutations in the functional domain of Mxi1 in these cases may be involved in the pathogenesis of neurofibrosarcoma.
3017	A case of hemophagocytic lymphohistiocytosis with prolonged remission after syngeneic bone marrow transplantation.	10467334	We report a 7-year-old girl with hemophagocytic lymphohistiocytosis who received a syngeneic bone marrow transplant from her twin sister. She presented with high fever and cough. Laboratory findings revealed pancytopenia, elevation of liver enzymes, and hyperferritinemia. Bone marrow examination revealed histiocytic hemophagocytes and lymphoblastoid cells. Southern blot analysis of the bone marrow cells revealed a monoclonal proliferation of EBV-infected lymphocytes. Although she underwent combined chemotherapy according to the HLH-94 protocol, she developed severe pancytopenia. Following myeloablative conditioning with busulfan (16 mg/kg), cyclophosphamide (120 mg/kg), and etoposide (1.5 g/m2), she was transplanted with 6.6 x 10(8)/kg mononuclear cells from the twin sister. She remains in complete remission 23 months after transplantation.
3018	Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators.	10452845	In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell-cell signaling pathway, which is utilized in imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH) transcriptional repressors, while 4 others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Here we present strong evidence from promoter-reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes were known previously to be direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins achaete and scute. Our extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. We next show that the very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mgamma are a property of small (200-300 bp), evolutionarily conserved transcriptional enhancer elements, which can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. We also demonstrate that the characteristic inactivity of the E(spl)mgamma enhancer in the notum and margin territories of the wing disc can be overcome by elevated activity of the N receptor. We conclude that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in which these modes of regulation are operative.
3019	The notch signaling pathway is required to specify muscle progenitor cells in Drosophila.	10446272	Organization and function of the Notch signaling pathway in Drosophila are best understood with respect to its role in the process of selection of neural progenitor cells. However, there is evidence that, besides neurogenesis, the Notch signaling pathway is involved in several other developmental processes, one of which is the selection of muscle progenitor cells. Thus, the number of these cells is increased in neurogenic mutants, and it has been proposed that muscle progenitor cells are selected from clusters of equivalent cells expressing genes of the achaete-scute gene complex (AS-C). Here, I present evidence for the participation of additional elements of the Notch signaling pathway in myogenesis. Gal4 mediated expression of a Notch variant, E(spl) and Hairless shows that the selection of muscle progenitor cells obeys principles apparently identical to those acting at the selection of neural progenitor cells.
3020	Overexpression of the m4 and malpha genes of the E(spl)-complex antagonizes notch mediated lateral inhibition.	10446264	Intercellular signalling mediated by Notch proteins is crucial to many cell fate decisions in metazoans. Its profound effects on cell fate and proliferation require that a complex set of responses involving positive and negative signal transducers be orchestrated around each instance of signalling. In Drosophila the basic-helix-loop-helix (bHLH) repressor encoding genes of the E(spl) locus are induced by Notch signalling and mediate some of its effects, such as suppression of neural fate. Here we report on a novel family of Notch responsive genes, whose products appear to act as antagonists of the Notch signal in the process of adult sensory organ precursor singularization. They, too, reside in the E(spl) locus and comprise transcription units E(spl) m4 and E(spl) malpha. Overexpression of these genes causes downregulation of E(spl) bHLH expression accompanied by cell autonomous overcommitment of sensory organ precursors and tufting of bristles. Interestingly, negative regulation of the Notch pathway by overexpression of E(spl) m4 and malpha is specific to the process of sensory organ precursor singularization and does not impinge on other instances of Notch signalling.
3021	A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex.	10445028	The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development. Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery. The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown. Here, we identify a novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein that is essential for transactivation. We provide both genetic and biochemical evidence that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex.
3022	Oversecretion of IL-18 in haemophagocytic lymphohistiocytosis: a novel marker of disease activity.	10444185	We investigated the significance of interleukin (IL)-18 levels in the pathophysiology of haemophagocytic lymphohistiocytosis (HLH). IL-18 levels were significantly elevated in all nine patients with active HLH compared with those of healthy controls. Serial determination of IL-18 levels in three cases, showed a gradual decrease compared with those of IL-12, interferon (IFN)-gamma or soluble Fas ligand (sFasL) in the course of clinical improvement, and seemed to be elevated until complete disappearance of disease activity. IL-18 and IFN-gamma (CC 0.711, P = 0.018), and IFN-gamma and sFasL (CC 0.849, P = 0.0049) levels were significantly correlated. On the other hand, correlation between IL-12 and IFN-gamma, IL-18 and sFasL, or IL-18 and IL-12 was not observed. IL-18, IFN-gamma and sFasL levels significantly correlated with disease activity such as fever and alanine transaminase (ALT) levels. IL-18 mRNA expression was enhanced in spleen, but not in peripheral blood mononuclear cells (MNC), bone marrow MNC, liver from patients of active HLH, or the tumour from a patient with lymphoma-associated haemophagocytic syndrome (LAHS). These results suggest that IL-18 may play important roles in the pathogenesis of HLH, particularly through induction of Th1 cells. IL-18 measurement may be useful for the diagnosis and for the detection of smouldering disease activity.
3023	Enhancer of split [E(spl)(D)] is a gro-independent, hypermorphic mutation in Drosophila.	10440851	Enhancer of split [E(spl)] refers to a gene complex in Drosophila melanogaster, which contains a number of target genes of the Notch signaling pathway. The complex was originally identified by a dominant mutation E(spl)(D) that displays allele-specific interactions with a recessive mutation in the Notch locus called split (N(spl)). The spl phenotype is characterized by smaller eyes with irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D). This enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8, causing an increased stability of the mutant transcripts and an altered C-terminus in the mutant M8* protein. Concurrently, an insertion of a middle repetitive element in the adjacent groucho (gro) gene was observed. In this work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost the ability to enhance N(spl) completely, were analyzed at the molecular level. In each case, the structure of the mutant M8* protein was affected, suggesting a specific involvement of the aberrant protein in the enhancement of the spl phenotype. This hypothesis is supported by the finding that a perfect phenocopy of spl enhancement can be achieved with hybrid constructs, where the altered C-terminus of M8* was fused to other E(spl) bHLH proteins. Thus, the ability to interact with N(spl) is not restricted to M8* but instead can be induced by an appropriate mutation in other E(spl) bHLH genes within the context of N(spl). In a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation. However, the mutant M8* protein has lost the ability of binding to the corepressor Gro, which is an essential feature for normal E(spl) activity. Yet, other protein interactions, notably those with other bHLH proteins of either E(spl) or proneural family, are still observed. These findings suggest that the structural changes associated with the E(spl)(D) mutant protein are the primary cause for the phenotypic interactions with the recessive Notch mutation N(spl).
3024	The helix-loop-helix proteins dAP-4 and daughterless bind both in vitro and in vivo to SEBP3 sites required for transcriptional activation of the Drosophila gene Sgs-4.	10438607	The expression of Sgs genes in the salivary gland of the third instar larva of Drosophila is a spatially restricted response to signalling by the steroid hormone 20-hydroxyecdysone. For Sgs-4, we have previously demonstrated that its strictly tissue and stage-specific expression is the result of combined action of the ecdysone receptor and secretion enhancer binding proteins (SEBPs). One of these SEBPs, SEBP2, was shown to be the product of the homeotic gene fork head. Together with SEBP3, SEBP2 appears to be responsible for the spatial restriction of the hormone response of Sgs-4. Here, we show that SEBP3 is a heterogeneous binding activity that consists of different helix-loop-helix (HLH) proteins. We cloned the Drosophila homologue of human transcription factor AP-4 (dAP-4) and identified it as one of these HLH proteins. The dAP-4 protein shows great similarity to its human and Caenorhabditis counterparts within the bHLHZip domain, the second leucine zipper dimerization motif, and a third region of unknown function. The expression pattern of dAP-4 indicates that it is a ubiquitously expressed HLH protein in Drosophila. As a second component of SEBP3 we identified the Daughterless (Da) protein, which is also ubiquitously expressed and binds to SEBP3 sites independent of dAP-4. Since both dAP-4 and Da can be detected in situ at transposed Sgs-4 transcriptional control elements in polytene salivary gland chromosomes, we propose that each of the two proteins contributes to the transcriptional control of Sgs-4.
3025	Characterization of an oxygen/redox-dependent degradation domain of hypoxia-inducible factor alpha (HIF-alpha) proteins.	10403805	Hypoxia-inducible factors are heterodimeric DNA-binding complexes that control the hypoxia responses of several genes and regulate the adaptive responses to the lack of oxygen. The complex is composed of two b-HLH protein subunits, HIF-1beta (ARNT), that is constitutively expressed, and a HIF-alpha subunit, that is present only in hypoxic cells. HIF-alpha proteins are continuously synthesized, but are rapidly degraded by the ubiquitin-proteasome system under oxic conditions. Hypoxia, transition metals, iron chelators, and several antioxidants stabilize the HIF-alpha proteins, allowing the formation of the transcriptionally active HIF complex. However, the sequences and mechanisms involved in the regulated degradation of the alpha protein subunits are poorly understood. Analysis of the available cloned sequences of human and mouse members of the HIF-alpha family of proteins revealed an area of about 15 amino acids with strong sequence conservation between all the members. This area corresponds to the region encompassing amino acids 557-571 of the hHIF-1alpha subunit. Fragments of HIF-1alpha and HIF-3alpha proteins containing this conserved sequence were able to confer hypoxia regulation when expressed as fusion proteins in Hep-3B cells. Regulation was observed with all the known hypoxia "mimics," including the reducing thiol donor N-mercaptopropionylglycine (NMPG). Selective alanine substitutions of amino acids 561-568 stabilized the protein in normoxic conditions. Furthermore, transfection with an expression vector containing a fragment of hHIF-1alpha comprising amino acids 540-580 enhanced transactivation activity of the full-length hHIF-1alpha protein. These results suggest that the above-mentioned conserved sequences are likely involved in the hypoxic stabilization of HIF-alpha proteins. The mechanisms and the interacting ubiquitin-ligases involved in the selective degradation process remain unknown.
3026	Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split.	10403790	In this report we describe the initial characterization of murine, human, and Drosophila hesr-1 (for hairy and enhancer of split related-1) a novel evolutionary conserved family of hairy/enhancer of split homologs. Hesr-1 cDNAs display features typical of hairy and enhancer of split-type bHLH proteins including a N-terminal bHLH domain a conserved orange domain immediately C-terminal to the bHLH region. Despite their similarity to known hairy/enhancer of split homologs, hesr-1 cDNAs are divergent members of the hairy and enhancer of split bHLH family since the degree of sequence identity within the bHLH and their nearest homologs are relatively low. Moreover, the tetrapeptide motif, WRPW, which is found in all hairy and enhancer of split family members, is not present in hesr-1. Rather, a variant of this motif, YRPW, is found. Analysis of embryonic murine hesr-1 expression by in situ hybridization reveals strong expression in the somitic mesoderm, the central nervous system, the kidney, the heart, nasal epithelium, and limbs indicating a role for hesr-1 in the development of these tissues. Like the enhancer of split cDNAs in Drosophila, we show that hesr-1 expression depends critically on signaling through the notch pathway in murine embryos, suggesting that aspects of hesr-1 regulation and function might also be evolutionary conserved.
3027	Genetic basis of hemophagocytic lymphohistiocytosis syndrome (Review).	10402477	The group of immune disorders which leads to the occurrence of hemophagocytic lymphohistiocytosis (HLH) syndrome presents a strange paradox in that patients with these conditions associate a dramatic immune response to infection with the failure to establish an effective immune response. During the last few years, significant progress was made in the characterization and the understanding of the molecular basis involved in these inherited immune disorders. The hemophagocytic lymphohistiocytosis syndrome which characterized the evolution of the Chediak-Higashi syndrome and the Griscelli disease results from defects affecting intracellular trafficking. A defective SH2 protein interacting with T lymphocyte intracellular signaling pathways is the cause of the X-linked lymphoproliferative disease, whereas at least three distinct genetic defects can lead to the familial hemophagocytic lymphohistiocytosis. The molecular characterization of these latter defects is in progress. This review summarizes the recent advances as well as their implications in the diagnosis and the understanding of the physiopathology of these disorders.
3028	Heterotrimetric Gi/o proteins control cyclic AMP oscillations and cytoskeletal structure assembly in primary human granulosa-lutein cells.	10400315	In granulosa cells, the luteinising hormone (LH) and the follicle-stimulating hormone (FSH) receptors are coupled to the adenylyl cyclase-cAMP pathway. We identified at least eight different G proteins belonging to three families--Gs, Gq, and Gi/o--in primary human granulosa-lutein cells. By exploring the function of Gi/o by time-lapse and digital-imaging microscopy of live cells, we found that the reversible actin stress fibre-dependent cytoplasmic retraction of pre-luteinised cells in primary culture is a highly sensitive and quite rapid system allowing detection of an intracellular cAMP surge. This morphology was characterised by maintenance of connexin43-dependent cell-cell contacts and that of microtubule-directed cell processes attached to the substrate and to neighbouring cells. Inhibitors of cyclic nucleotide phosphodiesterase subfamily type 4 (PDE-4), hLH and hFSH provoked this reversible cAMP-dependent phenotype in a temporal-, spatial- and dose-dependent manner. Gi/o inhibited adenylyl cyclase in membranes, and cell treatment with islet-activating protein (IAP) caused the cAMP-dependent retracted phenotype. It is concluded that the basal intracellular cAMP level is kept within a narrow range of concentrations, below the threshold for disassembly of stress fibres, through Gs, Gi/o, adenylyl cyclases and phosphodiesterase-4. This work supports the paradigm that switching of the agonist-occupied receptors to Gs and Gi/o would control both the intracellular bursts of cAMP (through the gonadotropin-catalysed activation of Gs) and the basal cAMP (through a Gi/o-mediated braking effect).
3029	Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor, occurs through the direct interaction of importin beta with HLH-Zip.	10397761	The sterol regulatory element-binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix-leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin beta. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin beta in the absence of importin alpha. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2-importin beta complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin beta in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix-leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin beta.
3030	MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.	10393122	The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.
3031	Modulation of TEL transcription activity by interaction with the ubiquitin-conjugating enzyme UBC9.	10377438	The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The encoded protein contains two functional domains: a helix-loop-helix (HLH) domain (also known as pointed domain) located at the N terminus and a DNA-binding domain located at the C terminus. The HLH domain is involved in protein-protein interaction with itself and other members of the ETS family of transcription factors such as FLI1. TEL is a transcription factor, and we and others have shown that it is a repressor of gene expression. To understand further the role of TEL in the cell, we have used an in vivo interaction system to identify proteins that interact with TEL. We show that a protein, UBC9, interacts specifically with TEL in vitro and in vivo. UBC9 is a member of the family of ubiquitin-conjugating enzymes. These enzymes usually are involved in proteosome-mediated degradation; however, our data suggest that interaction of TEL with UBC9 does not lead to TEL degradation. Our studies show that UBC9 binds to TEL exclusively through the HLH domain of TEL. We also show that TEL expressed as fusion to the DNA-binding domain of Gal4 completely represses a Gal4-responsive promoter, but that the coexpression of UBC9 in the same system restores the activity of the promoter. Targeted point mutation of conserved amino acids in UBC9 essential for enzymatic ubiquitination of proteins does not affect interaction nor transcriptional activity. Based on our data, we conclude that UBC9 physically interacts with TEL through the HLH domain and that the interaction leads to modulation of the transcription activity of TEL.
3032	Spectrum of congenital heart defects and extracardiac malformations associated with chromosomal abnormalities: results of a seven year necropsy study.	10377306	To analyse the spectrum of congenital heart malformations, the frequency of extracardiac malformations, and the proportion of chromosome aberrations among fetuses sent for necropsy.Necropsies were performed on 815 fetuses-448 induced abortions (55%), 220 spontaneous abortions (27%), and 147 stillbirths (18%)-during a seven year period (1991-97) in the department of pathology of the Charité Medical Centre in Berlin. A congenital heart defect was identified in 129 cases (16%). For all 129 fetuses, karyotyping and an ultrasound examination had been performed.Congenital heart defects were present in 22% of induced abortions (99 cases), 9% of spontaneous abortions (20 cases), and 7% of stillbirths (10 cases). The heart malformations were classified into 13 categories. A fetus with more than one defect was included only in the category of the most serious defect. The malformations in order of frequency were: ventricular septal defect (VSD) (28%), atrioventricular septal defect (AVSD) (16%), hypoplastic left heart (HLH) (16%), double outlet right ventricle (DORV) (12%), coarctation of the aorta (CoA) (6%), transposition of the great arteries (TGA) (4%), aortic valve stenosis (AoVS) (4%), tetralogy of Fallot (TOF) (3%), truncus arteriosus communis (TAC) (3%), pulmonary valve stenosis/pulmonary valve atresia (PaVS/PaVA) (3%), tricuspid atresia (TA) (3%), single ventricle (SV) (1.5%), and atrial septal defect (ASD) (0.5%). The most common congenital heart defects were VSD, AVSD, HLH, and DORV, which made up 72% of all the cases. In 11 cases the heart defect was isolated (no other cardiovascular or extracardiac malformations present), 85 cases (66%) were associated with additional cardiac malformations, 85 cases (66%) were associated with extracardiac malformations, and chromosome anomalies were detected in 43 cases (33%).Fetal congenital heart malformations are common. These defects are often associated with other cardiovascular and extracardiac malformations, as well as with chromosome anomalies. Complex heart defects such as AVSD, HLH, and DORV are frequent in fetuses, as they often lead to spontaneous abortion or stillbirth or, after prenatal diagnosis, to deliberate termination of pregnancy.
3033	The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing.	10375526	The secreted Hedgehog (Hh) proteins have been implicated as mediators of positional information in vertebrates and invertebrates. A gradient of Hh activity contributes to antero-posterior (A/P) patterning of the fly wing. In addition to inducing localised expression of Decapentaplegic (Dpp), which in turn relays patterning cues at long range, Hh directly patterns the central region of the wing.We show that short-range, dose-dependent Hh activity is mediated by activation of the transcription factor Collier (Col). In the absence of col activity, longitudinal veins 3 and 4 (L3 and L4) are apposed and the central intervein is missing. Hh expression induces col expression in a narrow stripe of cells along the A/P boundary through a dual-input mechanism: inhibition of proteolysis of Cubitus-interruptus (Ci) and activation of the Fused (Fu) kinase. Col, in cooperation with Ci, controls the formation of the central intervein by activating the expression of blistered (bs), which encodes the Drosophila serum response factor (D-SRF), the activity of which is required for the adoption and maintenance of the intervein cell fate. Furthermore, col is allelic to knot, a gene involved in the formation of the central part of the wing. This finding completes our understanding of the sectorial organisation of the Drosophila wing.Col, the Drosophila member of the COE family (Col/Olf-1/EBF) of non-basic, helix-loop-helix (HLH)-containing transcription factors, is a mediator of the short-range organising activity of Hh in the Drosophila wing. Our results support the idea that Hh controls target gene expression in a concentration-dependent manner and highlight the importance of the Fu kinase in this differential regulation. The high degree of evolutionary conservation of the COE proteins and the diversity of developmental processes controlled by Hh signalling raises the possibility that the specific genetic interactions depicted here may also operate in vertebrates.
3034	Target specificities of Drosophila enhancer of split basic helix-loop-helix proteins.	10373509	Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5'-TGGCACGTG(C/T)(C/T)A-3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.
3035	Evidence for a novel Notch pathway required for muscle precursor selection in Drosophila.	10349619	The Notch pathway mediates cell fate choice in many species and developmental contexts. In the Drosophila mesoderm, phenotypic differences were observed when different components of the pathway were defective. To determine if these differences reflect variations in the signaling pathway or in the persistence of wild-type maternal products, we examined muscle precursors in embryos that lacked both maternally- and zygotically-derived gene products, called holonull embryos. Most holonull neurogenic embryos have the same number and arrangement of extra muscle precursors, but in Notch holonull embryos many additional cells also become muscle precursors. Thus Notch is active in cells where its known ligands and downstream effectors are not. These results indicate that Notch acts in two pathways to determine cell fates in mesoderm: the Delta-to-Notch-to-Suppressor of Hairless-to-Enhancer of split signaling pathway previously defined, and a second pathway that acts independently.
3036	Fate of the stented arterial duct.	103384531033844811094052	The technical aspects of ductal stenting have been reported, but little is known of the fate of the duct after stent implantation.Nineteen patients underwent stent implantation to maintain ductal patency. Eight had hypoplastic left heart (HLH) syndrome, 10 had pulmonary atresia, and 1 had tricuspid atresia. Median survival with HLH was 57 (12 to 907) days. Stent implantation was successful in all cases of HLH, but there were no long-term survivors. Two well-palliated infants died at transplantation. Median survival with duct-dependent pulmonary flow was 183 (0 to 1687) days, with 3 patients well at latest follow-up (56, 55, and 9 months, respectively). There were 2 operative deaths due to ductal spasm and 4 late deaths, 1 due to duct thrombosis, 1 due to chronic lung disease, and 2 of unknown cause. Stent implantation failed in 4 of the 11 cases. Assessment of endothelialization was possible in 13 cases; the stent was partially covered in 3 and fully endothelialized in all 10 cases assessed >8 weeks after implantation. In patients stented for inadequate pulmonary flow, ductal intimal hyperplasia occurred by 9 months in all 3 survivors but responded to repeated dilation.Ductal stenting cannot be recommended. In patients with HLH, it provides only short-term palliation even when combined with pulmonary artery banding. With duct-dependent pulmonary blood flow, the procedure carries high risk, and duration of palliation is poor. In patients with bilateral ducts and absent central pulmonary arteries, good palliation may be achieved, but repeated angioplasty is necessary to counteract intimal hyperplasia.
3037	Identification of Id2 as a globin regulatory protein by representational difference analysis of K562 cells induced to express gamma-globin with a fungal compound.	10330158	A fungus-derived compound (OSI-2040) which induces fetal globin expression in the absence of erythroid cell differentiation was identified in a high-throughput drug discovery program. We utilized this compound to isolate gamma-globin regulatory genes that are differentially expressed in OSI-2040-induced and uninduced cells in the human erythroleukemia cell line K562. Representational difference analysis (RDA) of cDNA revealed several genes that were significantly up- or down-regulated in OSI-2040-induced cells. One gene whose expression was markedly enhanced was the gene for the helix-loop-helix (HLH) transcription factor Id2. Southern analysis of RDA amplicons demonstrated progressive enrichment of Id2 with each successive subtraction of uninduced cDNA from induced cDNA. Northern analysis of OSI-2040-induced K562 cells confirmed that Id2 expression was directly up-regulated coordinately with gamma-globin. Analysis of other inducers of fetal globin demonstrated up-regulation of Id2 with sodium butyrate but not with hemin. Retrovirus-mediated overexpression of Id2 in K562 cells reproduced the enhancement of endogenous globin expression observed with OSI-2040 induction. Functional assays demonstrated that an E-box element in hypersensitivity site 2 is required for Id2-dependent enhancement of gamma-promoter activity. Protein binding studies suggest that alterations in E-box site occupancy by basic HLH proteins may influence this activity, thus expanding the potential role of these factors in globin gene regulation.
3038	Genetic evidence that Notch affects independently the development of machrochaeta and wing venation in Drosophila melanogaster.	10319720	Temperature sensitive experiments on the interaction of Abruptex mutations of the Notch locus and Enhancer of split indicate that the effect of temperature on the development of macrochaeta and wing venation is different. This suggest that Notch affects the development of macrochaeta and wing venation in different and independent ways being in accordance with the proposition that the signal pathway from the Notch receptor at the cell membrane to Enhancer of split complex in the nucleus is different in the case of the development of macrochaeta and wing venation. The Abruptex mutations as gain-of-function mutations of the Notch locus cannot rescue the Enhancer of split phenotype.
3039	Basic helix-loop-helix proteins can act at the E-box within the serum response element of the c-fos promoter to influence hormone-induced promoter activation in Sertoli cells.	10319327	The Sertoli cell is a terminally differentiated testicular cell in the adult required to maintain the process of spermatogenesis. Previously basic helix-loop-helix (bHLH) factors and c-fos have been shown to influence Sertoli cell-differentiated functions. The induction of Sertoli cell differentiation appears to involve the serum response element (SRE) of the c-fos promoter to activate c-fos and intermediate bHLH factor(s) that regulate down-stream Sertoli cell-differentiated genes (e.g. transferrin expression). The SRE of the c-fos promoter is influenced through the serum response factor (SRF). Interestingly, an E-box nucleotide sequence is present within the SRE. bHLH proteins act through E-box elements, and the current study investigates the possibility that bHLH proteins may directly influence the SRE of the c-fos promoter. The activation of the c-fos promoter in Sertoli cells was found to be inhibited with the overexpression of the inhibitory HLH protein Id. Analysis of major response elements within the c-fos promoter demonstrated that the expression of Id specifically inhibited the activation of SRE in Sertoli cells and no other elements tested. Mutations in the E-box of the SRE also inhibited the activation of SRE, suggesting the direct role of bHLH proteins in regulating SRE activity in Sertoli cells. In contrast, the activation of SRE containing a mutated E-box was comparable to wild-type SRE in control stromal cells. Analysis of SRE oligonucleotide gel mobility shift assays with nuclear extracts from Sertoli cells demonstrated the presence of both the SRF and the ubiquitously expressed bHLH protein E12/E47. In contrast, no E12/E47 was detected in the SRE oligonucleotide gel shift using control stromal cell nuclear extracts. Observations suggest the binding of E12/E47 to SRE may be a cell-specific event. The SRF and bHLH proteins appear to bind to the SRE and activate the c-fos promoter in Sertoli cells. Observations provide evidence that a bHLH protein can interact with the SRE of the c-fos promoter to influence hormone-induced promoter activation. Cross-talk between these nuclear transcription factors appears to be instrumental in the control of Sertoli cell-differentiated functions.
3040	Haemophagocytic lymphohistiocytosis in children.	10234636	To evaluate the clinical and diagnostic features of children presenting with haemophagocytic lymphohistiocytosis (HLH), evolution of the disease and outcomes in response to treatment.The medical records of 12 children, aged 5 weeks to 13 years at diagnosis, with HLH managed at a single institution were reviewed.Presenting features were fever, hepatosplenomegaly, pancytopenia and hypertriglyceridemia or hypofibrinogenemia. Nine patients (75%) developed central nervous system (CNS) disease. Only one child with CNS disease survived. Five children had complete responses to therapy (42%), but all relapsed at a median of 1.5 months after starting treatment (range 2 weeks to 5 months). Two of the children treated are long-term survivors (17%), both after allogeneic bone marrow transplantation. All deaths occurred in the context of active disease.Haemophagocytic lymphohistiocytosis is a disease with a poor prognosis. Central nervous system complications are common and response to treatment usually is transient. This study provides support for the use of immunomodulatory therapy for remission introduction followed by consideration of allogeneic bone marrow transplantation.
3041	The Caenorhabditis elegans genes egl-27 and egr-1 are similar to MTA1, a member of a chromatin regulatory complex, and are redundantly required for embryonic patterning.	10226007	We show here that two functionally redundant Caenorhabditis elegans genes, egl-27 and egr-1, have a fundamental role in embryonic patterning. When both are inactivated, cells in essentially all regions of the embryo fail to be properly organised. Tissue determination and differentiation are unaffected and many zygotic patterning genes are expressed normally, including HOX genes. However, hlh-8, a target of the HOX gene mab-5, is not expressed. egl-27 and egr-1 are members of a gene family that includes MTA1, a human gene with elevated expression in metastatic carcinomas. MTA1 is a component of a protein complex with histone deacetylase and nucleosome remodelling activities. We propose that EGL-27 and EGR-1 function as part of a chromatin regulatory complex required for the function of regional patterning genes.
3042	Allogeneic hematopoietic stem cell transplantation for patients with hemophagocytic syndrome (HPS) in Japan.	10217187	Seventeen cases (age at onset, 1 month to 18 years; M/F, 9/8) of hemophagocytic syndrome which received allogeneic hematopoietic stem cell transplantation (SCT) in Japan during the period 1988-1998 are reported. The patients consisted of six familial inheritance-proven erythrophagocytic lymphohistiocytosis (FEL), five familial inheritance-unknown and infective agents-unknown HLH (of which two were highly likely to have been FEL with characteristic CNS signs), and six aggressive Epstein-Barr virus (EBV)-related HLH (of which two were natural killer cell-type large granular leukemia/lymphoma-associated hemophagocytic syndrome, EBV-NK-LGLL-HPS). All cases were treated intensively with immuno-chemotherapy, or with chemotherapy before SCT. As sources of SCT, 12 cases received bone marrow cells (sibling six, father one, URD five), two cord blood, two purified CD34-positive cells, and one PBSC. SCTs were successful in all 17 cases, apart from one receiving CD34-positive SCT. Following SCT, four patients relapsed and five died with a median follow-up of 23 months. Among the relapsed cases, the two EBV-NK-LGLL-HPS previously published as successfully transplanted were included. Among the fatal cases, three patients died from relapsed active disease and the remaining two from fatal post-SCT EBV-positive T cell lymphoma and extensive chronic GVHD, respectively. As of the end of September 1998, 10 patients are alive without disease for 3.5 months to 147 months, while two post-SCT patients are still having therapy for residual/recurrent disease. The Kaplan-Meier analysis showed a 2-year event-free survival after SCT as 54.0+/-13.0%.
3043	Development of acute lymphoblastic leukemia with translocation (4;11) in a young girl with familial pericentric inversion 12.	10214360	We report a case of a 1-year-old girl with familial pericentric inv(12) who developed acute lymphoblastic leukemia (ALL) with t(4;11) 1 month after recovery from idiopathic hemophagocytic lymphohistiocytosis (HLH). The inv(12)(p13q15) was first found in bone marrow (BM) cells when she was diagnosed as having HLH, and then detected in the BM blasts together with t(4;11)(q21;q23) when she developed ALL. The inv(12) was retained in the BM cells after she achieved complete remission. Cytogenetic analysis on the PHA-stimulated peripheral lymphocytes revealed inv(12) in all of the 30 cells examined. Because the data that ALL with t(4;11) predicts an extremely poor prognosis, she received an allogeneic BM transplantation from an HLA-matched sibling at 10 months from the onset of ALL. She is now at 26 months post transplantation and maintains in a state of complete remission. Familial cytogenetic study demonstrated that 4 of 8 maternal members examined had the inv(12), but they showed no family history of a higher risk of development of hematological and other types of malignancies, suggesting that pericentric inv(12) itself might not be directly involved in the development of ALL in this case.
3044	Expression of the basic helix-loop-helix protein REBalpha in rat testicular Sertoli cells.	10208991	Sertoli cell differentiation is initiated in the embryo to promote testicular development and male sex determination. In the adult, Sertoli cells are critical for maintenance of the spermatogenic process. Previously, Sertoli cell differentiation has been shown to be regulated in part by basic helix-loop-helix (bHLH) transcription factors. This was based on the observation that promoters of a number of Sertoli cell genes contained bHLH-responsive E-box response elements and that overexpression of Id, a negatively acting HLH protein, down-regulates Sertoli cell differentiated functions. Analysis of Sertoli cell bHLH proteins demonstrated the expression of REBalpha in Sertoli cells. REBalpha and REBbeta are spliced variants of the REB gene that is implicated in cell-specific gene expression as part of dimeric bHLH complexes acting on E-box response elements. Although both the transcripts of the REB gene are widely expressed, differential expression of the REB gene transcripts REBalpha and REBbeta has been shown. In the current study, a polymerase chain reaction (PCR)-based approach demonstrated that REB gene transcripts are expressed in the testis. Characterization of the REB transcripts suggested that REBalpha is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REBalpha or REBbeta demonstrated that Sertoli cells express only REBalpha, not REBbeta. REBbeta was present in the RNA samples obtained from whole testis, suggesting expression in other testicular cell types. A Northern blot analysis of RNA from Sertoli cells treated with or without FSH or cAMP demonstrated that REBalpha is not hormone responsive. REBalpha was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REBalpha is predominantly expressed by Sertoli cells within the seminiferous tubules. The activity of REBalpha in Sertoli cells was demonstrated with an E-box gel shift with Sertoli cell nuclear extracts. The E-box gel shift was found to contain REBalpha and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REBalpha isoform of the REB gene. The results are discussed in relation to the possibility that Sertoli cells may express a cell-specific bHLH protein that can preferentially dimerize with REBalpha.
3045	Ectopic expression of individual E(spl) genes has differential effects on different cell fate decisions and underscores the biphasic requirement for notch activity in wing margin establishment in Drosophila.	10207145	A common consequence of Notch signalling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. We expressed each individual E(spl) gene using the GAL4-UAS system in order to analyse their effect in a number of cell fate decisions taking place in the wing imaginal disk. We focussed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way, namely they antagonize neural precursor and vein fates. Yet, the efficacy of this antagonism is quite distinct: E(spl)mbeta has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. We have discerned two phases within this process based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mgamma is most active in both of these processes. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns.
3046	Id gene expression as a key mediator of tumor cell biology.	10197587	Id genes encode members of the helix-loop-helix (HLH) family of transcription factors that inhibit transcription by forming inactive heterodimers with basic HLH (bHLH) proteins. There are four members of the Id gene family recognized in mammals, and the proteins they encode share homology primarily in their HLH domain. bHLH proteins typically form heterodimers with other bHLH proteins, and their basic domain binds to a DNA sequence element, the E-box, activating transcription. Products of Id genes lack the basic DNA binding domain of the bHLH transcription factors, and when they heterodimerize with bHLH proteins, the complexes are inactive. Generally, high levels of Id mRNA are detected in proliferative undifferentiated, embryonal cells and lower levels are detected in well-differentiated, mature, adult tissues. In vitro, these genes are generally expressed at lower levels in cells after the induction of differentiation. Recently, high levels of expression of Id genes have been identified in cell lines derived from a wide variety of different tumors and in tumor tissues as well. These findings suggest that not only the inappropriate proliferation of tumors but also the anaplastic characteristics that contribute to their malignant behavior may be regulated by Id gene expression.
3047	Evolutionary conservation of MyoD function and differential utilization of E proteins.	10191059	The formation of striated muscle in both vertebrates and invertebrates involves the activity of the MyoD family of basic-helix-loop-helix (bHLH) transcription factors. The high degree of evolutionary conservation of MyoD-related proteins, both in the sequence of their bHLH domains and in their general developmental expression patterns, suggests that these factors are also conserved at the level of function. We have addressed this directly using MyoD and E protein factors from vertebrates, Drosophila, and Caenorhabditis elegans. Various MyoD and E factor combinations were tested for their ability to interact in vitro and to function in vivo in the myogenic conversion of 10T12 mouse fibroblasts. We found that the ability of different homo- and heterodimers to bind DNA in vitro was an accurate measure of biological activity in vivo. A second assessment of conserved function comes from the ability of these factors to rescue a C. elegans hlh-1 (CeMyoD) null mutation. We found that both Drosophila and chicken MyoD-related factors were able to rescue a C. elegans CeMyoD loss-of-function mutation. These results demonstrate a remarkable degree of functional conservation of these myogenic factors despite differences in E-protein interactions.
3048	Notchspl is deficient for inductive processes in the eye, and E(spl)D enhances split by interfering with proneural activity.	10191054	Eye development in Drosophila involves the Notch signaling pathway at several consecutive steps. At first, Notch signaling is required for stable expression of the proneural gene atonal (ato), thereby maintaining neural potential of the cells. Second, in a process of lateral inhibition, Notch signaling is necessary to confine neural commitment to individual photoreceptor founder cells. Later on, the successive addition of cells to maturing ommatidia is under Notch control. In contrast to previous assumptions, the recessive Notch allele split (Nspl) involves specifically loss of the early proneural Notch activity in the eye, which is in agreement with bristle defects as well. As a result, fewer cells gain neural potential and fewer ommatidia are founded. Enhancement of this phenotype by the dominant mutation Enhancer of split [E(spl)D] happens within the remaining proneural cells, in which Ato expression is abolished. In line with genetic data, this process occurs primarily at the protein level due to altered protein-protein interactions between the aberrant E(spl)D and proneural proteins. Nspl is the first Notch mutation known to specifically affect Notch inductive processes during eye development.
3049	her4, a zebrafish homologue of the Drosophila neurogenic gene E(spl), is a target of NOTCH signalling.	10101116	her4 encodes a zebrafish bHLH protein of the hairy-E(spl) family. The gene is transcribed in a complex pattern in the developing nervous system and in the hypoblast. During early neurogenesis, her4 expression domains include the regions of the neural plate from which primary neurons arise, suggesting that the gene is involved in directing their development. Indeed, misexpression of specific her4 variants leads to a reduction in the number of primary neurons formed. The amino-terminal region of her4, including the basic domain, and the region between the putative helix IV and the carboxy-terminal tetrapeptide wrpw are essential for this effect, since her4 variants lacking either of these regions are non-functional. However, the carboxy-terminal wrpw itself is dispensable. We have examined the interrelationships between deltaD, deltaA, notch1, her4 and neurogenin1 by means of RNA injections. her4 is involved in a regulatory feedback loop which modulates the activity of the proneural gene neurogenin, and as a consequence, of deltaA and deltaD. Activation of notch1 leads to strong activation of her4, to suppression of neurogenin transcription and, ultimately, to a reduction in the number of primary neurons. These results suggest that her4 acts as a target of notch-mediated signals that regulate primary neurogenesis.
3050	Inhibitory effect of plasma obtained from hypophysectomized and control women on the assay of bioactive luteinizing hormone.	10099970	The purpose of this study was to determine the effect of components of female plasma on the value of bioactive luteinizing hormone (LH), especially in the presence of low immunological LH value. Using both an immunoradiometric assay (IRMA) and rat Leydig cell bioassay, immunoreactive (I) and bioactive (B) LH were assessed in plasma collected from women during a gonadotrophin releasing hormone (GnRH) test performed on day 7 of a spontaneous cycle. Two modes of response to an acute administration of GnRH were defined: normal production of gonadotrophins (group I) and excessive secretion (group II) associated with a significant difference in the B/I-LH ratio between the two groups. The B/I-LH ratio did not vary with sampling time during the test in either group. The addition of LH-free plasma collected from hypophysectomized women caused a 30% decrease in testosterone production compared to control values (in the presence or absence of hLH standard). A partial restoration of testosterone production was observed if plasma was first treated with PEG 12%. The inhibitory factor(s) was also present in plasma from ovulatory women, even after treatment by an antibody against the entire LH molecule. The effect of normal (A) or low I-LH plasma (B) on testosterone production varied strongly according to the plasma volume added to the bioassay, as well as to plasma treatments. Diethylether treatment caused a 50% decrease in testosterone secretion for plasma B (but not for A) whereas a diminution of the steroidogenesis is observed after a PEG treatment of plasma A (but not for B), suggesting that different inhibitory factors are present in plasmas A and B. Therefore the LH bioactivity measured in the rat Leydig cell assay, in terms of testosterone output, seems to represent a balance between the LH molecule and the presence of inhibitory factors in the plasma.
3051	Mutations within or upstream of the basic helix-loop-helix domain of the TWIST gene are specific to Saethre-Chotzen syndrome.	10094188	Saethre-Chotzen syndrome (ACS III) is an autosomal dominant craniosynostosis syndrome recently ascribed to mutations in the TWIST gene, a basic helix-loop-helix (b-HLH) transcription factor regulating head mesenchyme cell development during cranial neural tube formation in mouse. Studying a series of 22 unrelated ACS III patients, we have found TWIST mutations in 16/22 cases. Interestingly, these mutations consistently involved the b-HLH domain of the protein. Indeed, mutant genotypes included frameshift deletions/insertions, nonsense and missense mutations, either truncating or disrupting the b-HLH motif of the protein. This observation gives additional support to the view that most ACS III cases result from loss-of-function mutations at the TWIST locus. The P250R recurrent FGFR 3 mutation was found in 2/22 cases presenting mild clinical manifestations of the disease but 4/22 cases failed to harbour TWIST or FGFR 3 mutations. Clinical re-examination of patients carrying TWIST mutations failed to reveal correlations between the mutant genotype and severity of the phenotype. Finally, since no TWIST mutations were detected in 40 cases of isolated coronal craniosynostosis, the present study suggests that TWIST mutations are specific to Saethre-Chotzen syndrome.
3052	Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac alpha-actin promoter.	10082523	Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.
3053	Expression of the dominant-negative regulator Id4 is induced during adipocyte differentiation.	10080947	Expression of the Id4 gene was investigated during differentiation of 3T3-L1 preadipocytes into mature adipocytes. Id4 is a member of a family encoding non-DNA binding helix-loop-helix proteins proposed to inhibit the activity of basic HLH (bHLH) proteins important in many developmental processes. We show here that Id4 expression is low in confluent preadipocytes and rapidly induced by treatment with the combination of hormones which causes differentiation into mature adipocytes. Id4 expression is also induced by treatment with individual hormones, especially dexamathasone. Id4 mRNA can be detected in mouse and human adipose tissue. Genes encoding E-proteins (bHLH proteins known to interact with and be regulated by Id proteins) are expressed and regulated during differentiation in 3T3-L1 cells. These data suggest that the Id4 transcriptional regulator is playing a role in adipose cell differentiation and suggest that DNA-binding HLH proteins may also be important in regulation of differentiation of these cells.
3054	Identification of a novel transcriptional activity of mammalian Id proteins.	10076006	The Id proteins are a family of related mammalian helix-loop-helix (HLH) proteins which can interact with other HLH proteins but lack a basic region and are thus not thought to bind to DNA. Instead, they are hypothesized to act as dominant negative regulators of DNA-binding basic HLH (bHLH) proteins, by forming inactive heterodimers with these proteins. All four Id family proteins possess related HLH dimerization domains and can interact with similar bHLH proteins, although with differing affinities. The functions of the largely unrelated N- and C-terminal regions of the proteins are unknown. In this study, we have identified a novel transcriptional activity of the mammalian Id proteins. We show that when fused to the heterologous GAL4 DNA-binding domain, all four of the mammalian Id proteins can activate GAL4-dependent transcription. The HLH domain is necessary for the transactivation activity observed, suggesting that interaction with a cellular HLH protein is required. Co-transfection with exogenous Class A bHLH proteins (E-proteins) greatly potentiates the transactivation, which is abolished upon co-transfection with Class B bHLH proteins. These results are consistent with the idea that the Id proteins have a transcriptional activity when present in a DNA-binding complex.
3055	The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes.	10072784	Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless (Su(H)) mediated transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex (E(spl)-C), encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. Here we show that the E(spl)-C contains three additional Notch responsive, non-bHLH genes: m4 and ma are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide.
3056	Dual role of extramacrochaetae in cell proliferation and cell differentiation during wing morphogenesis in Drosophila.	10072780	The Extramacrochaetae (emc) gene encodes a transcription factor with an HLH domain without the basic region involved in interaction with DNA present in other proteins that have this domain. EMC forms heterodimers with bHLH proteins preventing their binding to DNA, acting as a negative regulator. The function of emc is required in many developmental processes during the development of Drosophila, including wing morphogenesis. Mitotic recombination clones of both null and gain-of-function alleles of emc, indicate that during wing morphogenesis, emc participates in cell proliferation within the intervein regions (vein patterning), as well as in vein differentiation. The study of relationships between emc and different genes involved in wing development reveal strong genetic interactions with genes of the Ras signalling pathway (torpedo, vein, veinlet and Gap), blistered, plexus and net, in both adult wing phenotypes and cell behaviour in genetic mosaics. These interactions are also analyzed as variations of emc expression patterns in mutant backgrounds for these genes. In addition, cell proliferation behaviour of emc mutant cells varies depending on the mutant background. The results show that genes of the Ras signalling pathway are co-operatively involved in the activity of emc during cell proliferation, and later antagonistically during cell differentiation, repressing EMC expression.
3057	Effective control of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis with immunochemotherapy. Histiocyte Society.	10068659	The familial form of hemophagocytic lymphohistiocytosis (HLH) is a lethal disorder. Although the prognosis for Epstein-Barr virus-associated HLH (EBV-HLH) remains uncertain, numerous reports indicate that it can also be fatal in a substantial proportion of cases. We therefore assessed the potential of immunochemotherapy with a core combination of steroids and etoposide to control EBV-HLH in 17 infants and children who met stringent diagnostic criteria for this reactive disorder of the mononuclear phagocyte system. Treatment of life-threatening emergencies was left to the discretion of participating investigators and typically included either intravenous Ig or cyclosporin A (CSA). Five patients (29%) entered complete remission during the induction phase (1 to 2 months), whereas 10 others (57%) required additional treatment to achieve this status. In 2 cases, immunochemotherapy was ineffective, prompting allogeneic bone marrow transplantation. Severe but reversible myelosuppression was a common finding; adverse late sequelae were limited to epileptic activity in one child and chronic EBV infection in 2 others. Fourteen of the 17 patients treated with immunochemotherapy have maintained their complete responses for 4+ to 39+ months (median, 15+ months), suggesting a low probability of disease recurrence. These results provide a new perspective on EBV-HLH, showing effective control (and perhaps cure) of the majority of EBV-HLH cases without bone marrow transplantation, using steroids and etoposide, with or without immunomodulatory agents.
3058	Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2.	10067894	Transcription factors with a basic helix-loop-helix (HLH) motif have been shown to be crucial for various cell differentiation processes during development of multicellular organisms. Id proteins inhibit the functions of these transcription factors in a dominant-negative manner by suppressing their heterodimerization partners through the HLH domains. Members of the Id family also promote cell proliferation, implying a role in the control of cell differentiation. Here we show that Id2 is indispensable for normal development of mice. Id2-/- mice lack lymph nodes and Peyer's patches. However, their splenic architecture is normal, exhibiting T-cell and B-cell compartments and distinct germinal centres. The cell population that produces lymphotoxins, essential factors for the development of secondary lymphoid organs, is barely detectable in the Id2-/- intestine. Furthermore, the null mutants show a greatly reduced population of natural killer (NK) cells, which is due to an intrinsic defect in NK-cell precursors. Our results indicate that Id2 has an essential role in the generation of peripheral lymphoid organs and NK cells.
3059	Encephalopathy associated with haemophagocytic lymphohistiocytosis following rotavirus infection.	10048610	A 2-year-old Japanese boy with a haemophagocytic lymphohistiocytosis (HLH) associated encephalopathy which developed after rotavirus infection is described. The neurological symptoms consisted of coma, seizures and spastic quadriplegia. On therapy with steroids, etoposide and cyclosporin A, the patient recovered without any neurological deficits. The interferon-gamma levels in serum and CSF were elevated at onset of the disease but had returned to normal at the time of clinical remission. Brain MRI revealed diffuse white matter abnormalities and parenchymal volume loss. Proton magnetic resonance spectroscopy revealed elevated lactate in the abnormal lesions observed on MRI, indicating that macrophages not exhibiting aerobic metabolism had infiltrated the CNS. At the time of clinical remission, the white matter abnormalities and brain lactate had disappeared. These findings suggested that the neurological symptoms resulted from the overproduction of cytokines by activated T-cells and macrophages. The pathophysiology of a HLH associated encephalopathy was considered to be a local immune response within the CNS, because interferon-gamma can induce the expression of major histocompatibility complex class I and II antigens on glial cells in the CNS.Haemophagocytic lymphohistiocytosis associated encephalopathy should be considered early in the differential diagnosis of cases with acute onset neuropathy.
3060	Differential biological activities of mammalian Id proteins in muscle cells.	10047451	Id proteins are helix-loop-helix (HLH) transcription factors that lack DNA-binding domains. These proteins form inactive heterodimers with basic HLH (bHLH) factors, inhibiting their DNA-binding and transcriptional activities. Consistent with a proposed role for Id proteins as inhibitors of terminal differentiation, Id1 and Id3 have been shown to negatively regulate myogenesis in cultured muscle cells. Here we have investigated the possibility that Id2 and/or Id4 can act in a similar manner. Surprisingly, while overexpression of Id2 resulted in inhibition of differentiation of Sol 8 myoblast cells, overexpression of Id4 did not. Sol 8 cells stably transfected with Id4 showed no apparent changes in expression of muscle-specific genes upon differentiation. DNA-binding activities present at the muscle creatine kinase (MCK) enhancer E-box and transcription of the MCK enhancer were not altered in Id4-overexpressing cells, compared with vector-transfected cells. Id2 is also a more potent inhibitor of protein/DNA complex formation at the MCK-R enhancer E-box than Identified in vitro. Therefore, our data support the notion that members of the Id family might be involved in the regulation of distinct developmental pathways.
3061	Frizzled regulation of Notch signalling polarizes cell fate in the Drosophila eye.	10028969	The Drosophila eye, a paradigm for epithelial organization, is highly polarized with mirror-image symmetry about the equator. The R3 and R4 photoreceptors in each ommatidium are vital in this polarity; they adopt asymmetrical positions in adult ommatidia and are the site of action for several essential genes. Two such genes are frizzled (fz) and dishevelled (dsh), the products of which are components of a signalling pathway required in R3, and which are thought to be activated by a diffusible signal. Here we show that the transmembrane receptor Notch is required downstream of dsh in R3/R4 for them to adopt distinct fates. By using an enhancer for the Notch target gene Enhancer of split mdelta, we show that Notch becomes activated specifically in R4. We propose that Fz/Dsh promotes activity of the Notch ligand Delta and inhibits Notch receptor activity in R3, creating a difference in Notch signalling capacity between R3 and R4. Subsequent feedback in the Notch pathway ensures that this difference becomes amplified. This interplay between Fz/Dsh and Notch indicates that polarity is established through local comparisons between two cells and explains how a signal from one position (for example, the equator in the eye) could be interpreted by all ommatidia in the field.
3062	The breakpoints of a constitutional inversion of chromosome 9 associated with haemophagocytic lymphohistiocytosis are not linked to the disease gene.	10027721	Haemophagocytic lymphohistiocytosis (HLH) is a severe disorder of early infancy consistent with an autosomal recessive inheritance. Neither the genetic locus nor the biochemical defect is known for the disease. A constitutional pericentric inversion of chromosome 9, with breakpoints in bands 9p23 and 9q31, has been reported in a case of HLH, suggesting a possible relationship between this chromosome abnormality and the disease. We investigated such an association, performing a genetic linkage analysis in a set of five consanguineous HLH families. 27 polymorphic markers on chromosome 9 were studied, excluding most of chromosome 9 as a putative site for the HLH gene.
3063	Three distinct domains in TEL-AML1 are required for transcriptional repression of the IL-3 promoter.	10023677	A cytogenetically cryptic (12;21) translocation is the most common molecular abnormality identified in childhood acute lymphoblastic leukemia (ALL), and it generates a chimeric TEL-AML1 protein. Fusion of the Helix-Loop-Helix (HLH) (also called the pointed) domain of TEL to AML1 has been suggested to convert AML1 from a transcriptional activator to a repressor. To define the structural features of this chimeric protein required for repression, we analysed the transcriptional activity of a series of TEL-AML1 mutants on the AML1-responsive interleukin-3 (IL-3) promoter, a potentially relevant gene target. Our results demonstrate that TEL-AML1 represses basal IL-3 promoter activity in lymphoid cells, and deletion mutant analysis identified three distinct domains of TEL-AML1 that are required for repression; the HLH (pointed) motif contained in the TEL portion of TEL-AML1, and both the runt homology domain (Rhd) and the 74 amino acids downstream of the Rhd that are present in the AML1 portion of the fusion protein. Although AML1B (and a shorter AML1 isoform, AML1A) have transcriptional activating activity on the IL-3 promoter, fusion of the AML1 gene to the TEL gene generates a repressor of IL-3 expression. Consistent with this activity, freshly isolated human ALL cells that contain TEL-AML1 do not express IL-3.
3064	Identification of DERMO-1 as a member of helix-loop-helix type transcription factors expressed in osteoblastic cells.	10022499	Several members of the basic helix-loop-helix (bHLH) type of transcription factors have now been reported, and almost every member of this class has been implicated in transcriptional regulation in cell type determination and differentiation. Previously, we reported that dominant negative HLH proteins are involved in osteoblastic phenotype expression, such as osteocalcin, and hence differentiation (Tamura and Noda [1994] J. Cell Biol. 126:773-782). In this work, we used degenerate PCR cloning in order to identify cDNA clones encoding bHLH proteins expressed in osteoblastic osteosarcoma ROS17/2.8 cells. Sequence analyses of the 47 clones revealed that 11 clones encoded products with a characteristic motif of the bHLH transcription factor family. Of these clones, sequences in the amplified region of seven clones were homologous to the mouse twist, and three clones were homologous to the mouse twist-related HLH protein, Dermo-1. To confirm Dermo-1 mRNA expression in osteoblastic cells, we performed reverse transcription polymerase chain reaction (RT-PCR) analysis using mRNA from ROS17/2.8 cells and MC3T3-E1 cells by Dermo-1 specific primers and Northern blot analysis. These analyses demonstrated that Dermo-1 mRNA was expressed in these osteoblast-like cell lines. Nucleotide sequence analysis of the partial rat Dermo-1 cDNA cloned from ROS17/2.8 library revealed that it has the highest degree of homology with the mouse Dermo-1 cDNA, and the partial amino acid sequence deduced from the obtained rat Dermo-1 was identical with the corresponding region of the mouse Dermo-1 amino acid sequence. To further examine the role of Dermo-1 in the regulation of osteoblastic differentiation, we examined mRNA levels of Dermo-1 and twist in C3H10T1/2 cells treated with recombinant human bone morphogenetic protein (rhBMP)-2. Using the RT-PCR method, the mRNA levels of Dermo-1 and twist were found to be decreased by the treatment with rhBMP-2 in C3H10T1/2 cells. We also observed that the mRNA level of Dermo-1 was decreased about fourfold by the treatment with rhBMP-2 in C3H10T1/2 cells by Northern blot analysis. Moreover, Dermo-1 mRNA was detected at lower levels in 21-day-old differentiated MC3T3-E1 cells compared with 3-day-old undifferentiated MC3T3-E1 cells. These results suggested that Dermo-1 could be involved in the osteoblastic differentiation in a negative manner.
3065	Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors.	10022839	The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. Id proteins are thought to inhibit differentiation mainly through interaction with other HLH proteins and by blocking their DNA-binding activity. Members of the ternary complex factor (TCF) subfamily of ETS-domain proteins have key functions in regulating immediate-early gene expression in response to mitogenic stimulation. TCFs form DNA-bound complexes with the serum response factor (SRF) and are direct targets of MAP kinase (MAPK) signal transduction cascades. In this study we demonstrate functional interactions between Id proteins and TCFs. Ids bind to the ETS DNA-binding domain and disrupt the formation of DNA-bound complexes between TCFs and SRF on the c-fos serum response element (SRE). Inhibition occurs by disrupting protein-DNA interactions with the TCF component of this complex. In vivo, the Id proteins cause down-regulation of the transcriptional activity mediated by the TCFs and thereby block MAPK signalling to SREs. Therefore, our results demonstrate a novel facet of Id function in the coordination of mitogenic signalling and cell cycle entry.
3066	Injury selectively down-regulates the gene encoding for the Id4 transcription factor in primary cultures of forebrain astrocytes.	9926850	Astrogliosis is an important component of the response to injury of the central nervous system (CNS). The Id family of helix-loop-helix (HLH) transcription factors has been implicated in the regulation of cellular differentiation in several different lineages and may contribute to the regulation of astrogliosis. We examined the expression of Id genes in primary cultures of mouse forebrain astrocytes under experimental conditions in which astrogliosis was elicited by mechanical injury. Astrocyte cultures expressed the four known members of the Id gene family, Id1, Id2, Id3, and Id4. After injury, at a time when astrocytes developed the characteristic phenotypic changes of astrogliosis, Id4 expression decreased dramatically. Id1, Id2, and Id3 mRNA levels did not change. These results identify Id4 as a candidate marker of astroglial activation in culture and suggest that Id4 expression plays a role in the process of astrogliosis.
3067	Ovarian responses in women to recombinant follicle-stimulating hormone and luteinizing hormone (LH): a role for LH in the final stages of follicular maturation.	9920089	During the follicular phase of the menstrual cycle, FSH stimulates follicular growth, granulosa cell aromatase activity, induction of LH receptors on the granulosa cell membrane, and estradiol secretion. As a result of negative feedback of estradiol on the pituitary, serum FSH concentrations decline. Despite the fall in FSH concentrations, the maturing follicle continues to develop to the preovulatory stage. In a prospective randomized trial, we tested the hypothesis that a key mechanism by which the dominant follicle continues to develop in the face of decreasing concentration of FSH is by acquiring LH responsiveness. In 24 women, pituitary gonadotropin secretion was down-regulated with a GnRH agonist. Follicular growth was then stimulated with recombinant human FSH (r-hFSH) until a 14-mm follicle was identified by ultrasound. The women were then randomized to 1 of 4 groups for a 2-day period: continued r-hFSH treatment, substitution of r-hFSH with saline, low dose r-hLH (150 IU, twice daily), or high dose r-hLH (375 IU, twice daily). Serum estradiol concentrations in the women receiving saline declined by the end of the 2-day randomization period. In contrast, serum estradiol concentrations continued to rise in women receiving either r-hFSH or r-hLH compared with those in the saline-treated group (P < 0.05). Pregnancies occurred in each of the gonadotropin treatment groups. These findings indicate that once FSH initiates follicular growth, either FSH or LH is capable of sustaining follicular estradiol production. Extrapolating these findings to the normal menstrual cycle suggests that the maturing follicle may continue to develop in the presence of diminishing FSH concentrations by acquiring the capacity to respond to LH.
3068	Characterization of monoclonal antibodies recognizing alpha and beta subunits of human chorionic gonadotropin hormone.	9876951	Human chorionic gonadotropin (hCG) hormone is required for maintenance of early pregnancy and is a potential marker in the diagnosis and prognosis of both pregnancy and trophoblastic diseases. Murine hybridomas were generated against purified hCG. Seven hybrid clones secreting antibodies against hCG molecule with IgG1/kappa subclass were established. The indirect ELISA result demonstrated that six MAbs (BEL-1 to BEL-6) recognized hCG in both holo and free beta subunit form with negligible cross-reactivity against a closely related hormone, human luteinizing hormone (hLH). In this fusion, only one MAb (ALC-1) showed a cross-reaction with hLH, which designated an alpha subunit specific. The outcome of Western blot ascertained that ALC-1 recognized the conformational epitope on alpha subunit of hCG at Mr 23 kDa band in nonreducing condition (NR). In contrast, epitopes belonging to all MAbs recognized beta subunit of hCG were in linear peptide structure at Mr 34 kDa band (NR) and Mr 26 kDa band (R). These six MAbs were further characterized by using two beta subunit carboxy-terminal synthetic peptides (beta109-119 and beta109-145). Three of them (BEL-1, BEL-3, and BEL-4) recognized only epitope harboring in beta109-145 fragment, the others recognized both types of the synthetic peptide. In order to select the most suitable MAbs specific to beta subunit of hCG for exploiting with ALC-1 in the sandwich-type immunometric assay, competitive ELISA was employed. Six individual MAbs specific to beta subunit of hCG were used to compete with biotinylated ALC-1 to evaluate the proximity of their epitopes on the holo form of hCG molecule. Of all six MAbs, BEL-5 depicted the lowest percent inhibition result, which indicated the bottom-most steric hindrance effect. Consequently, MAb BEL-5 will be the most appropriate antibody to utilize in concert with ALC-1 in place of devising a sandwich-type immunometric assay for measuring holo-hCG level.
3069	Mxi1 is a repressor of the c-Myc promoter and reverses activation by USF.	9872993	The basic region/helix-loop-helix/leucine zipper (B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and forms heterodimers with Max protein that bind to E-box sites in DNA and stimulate genes required for proliferation. A second B-HLH-LZ protein, Mxi1, is induced during terminal differentiation, and forms heterodimers with Max that also bind E-boxes but tether the mSin3 transcriptional repressor protein along with histone deacetylase thereby antagonizing Myc-dependent activation. We show that Mxi1 also antagonizes Myc by a second pathway, repression of transcription from the major c-myc promoter, P2. Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ and COOH-terminal sequences, including putative casein kinase II phosphorylation sites. Repression targeted elements of the myc P2 promoter core (-35/+10), where it reversed transactivation by the constitutive transcription factor, USF. We show that Zn2+ induction of a stably transfected, metallothionein promoter-regulated mxi1 gene blocked the ability of serum to induce transcription of the endogenous c-myc gene and cell entry into S phase. Thus, induction of Mxi1 in terminally differentiating cells may block Myc function by repressing the c-myc gene P2 promoter, as well as by antagonizing Myc-dependent transactivation through E-boxes.
3070	Time-course effects of human recombinant luteinizing hormone on porcine Leydig cell specific differentiated functions.	9863627	Since recombinant hormones are considered as safer and more reliable in their bioactivity than extractive hormones, the recently available human recombinant luteinizing hormone (r-hLH), will probably replace hCG in the near future, for clinical purposes. This prompted us to investigate whether or not, and by which mechanisms, r-hLH can induce a desensitization of signal transduction and/or an up-regulation of steroidogenic capacity in Leydig cells. The effects of a 30 min to 24 h exposure to r-hLH (10(-9) M) on the differentiated functions of cultured immature porcine Leydig cells were studied by measuring the following parameters: LH/hCG receptor number and mRNA, hCG-, cholera toxin- and forskolin-induced cAMP production, G protein alphas subunit content of the membrane, hCG-, cholera toxin-, forskolin-, 8Br-cAMP-, 22R-OH-cholesterol-, progesterone-, 170H-progesterone-, DHEA-, delta4-androstenedione-induced testosterone secretion and StAR, 3beta-HSD, cytochrome P-450scc and P-450c17 mRNAs. hCG binding sites and LH/hCG receptor mRNA were slowly down regulated by r-hLH, reaching 47+/-1 and 18+/-7% of control at 24 h, respectively. Down-regulation of both hCG- and cholera toxin-induced cAMP production occurred earlier and was more marked, and at 24 h represented only 2.7+/-0.5 and 12.5+/-3.6% of control. Due to the synergistic effect of r-hLH and forskolin on cAMP production, the forskolin-induced cAMP was higher in r-hLH treated than in control cells, but this response also declines with time and was, at 24 h, only 32% of that observed at 30 min. This decreased cAMP production was associated with a less marked decline in the amount of membrane content of Galphas protein. The testosterone production in response to hCG, cholera toxin, forskolin and 8Br-cAMP declined to reach a nadir at 6 h but increased thereafter and at 24 h was significantly higher than in control cells. In contrast, the conversion of several precursors into testosterone remained stable or increased slightly during the first hours of r-hLH treatment and significantly increased at 24 h and this was associated with an increase of StAR, 3beta-HSD, P-450scc and P-450c17 mRNAs. Taken together, the present results indicate that, despite the marked down-regulation of transmembrane signaling, r-hLH increased the steroidogenic capacity of Leydig cells by increasing the expression of several genes encoding the proteins involved in testosterone synthesis.
3071	Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family.	9856573	Waardenburg syndrome (WS) is an autosomal-dominant neural crest cell disorder phenotypically characterized by hearing impairment and disturbance of pigmentation. A presence of dystopia canthorum is indicative of WS type 1, caused by loss of function mutation in the PAX3 gene. In contrast, type 2 WS (WS2) is characterized by normally placed medial canthi and is genetically heterogeneous; mutations in MITF (microphthalmia associated transcription factor) associated with WS2 have been identified in some but not all affected families. Here, we report on a three-generation Indian family with a point mutation in the MITF gene causing WS2. This mutation, initially reported in a Northern European family, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking the HLH-Zip or Zip structure necessary for normal interaction with its target DNA motif. Comparison of the phenotype between the two families demonstrates a significant difference in pigmentary disturbance of the eye. This family, with the first documented case of two unrelated WS2 families harboring identical mutations, provides additional evidence for the importance of genetic background on the clinical phenotype.
3072	Gonadotrophin secretion patterns in testicular cancer patients with greatly increased human chorionic gonadotrophin serum concentrations.	9834462	Despite the fact that a number of alterations of the hypothalamic-pituitary-gonadal hormone axis have been identified in patients with testicular cancer, little is known about the gonadotrophin secretion pattern in such patients who have greatly increased human chorionic gonadotrophin (hCG) serum concentrations. The aim of this study was to assess this issue in detail using a longitudinal study design and a panel of highly sensitive and specific immunoassays. Eleven patients with non-seminomatous (n=11), and one with seminomatous testicular cancer with pretreatment hCG serum concentrations exceeding 10(5) pg/ml (>1000 mIU/ml) were selected and followed for a mean of 166 days (mean of 14 serum samples/patient) after initial diagnosis. Serum concentrations of hCG, its free alpha- (hCGalpha) and beta- (hCGbeta) subunits, human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) were determined by highly sensitive and specific enzymometric immunoassays based on a panel of monoclonal antibodies (MCA) established in our laboratory. A potential FSH-like activity (FSA) of hCG in the respective sera was determined by radioreceptor assays (RRA) for LH/CG and FSH. Specificity of FSA at the level of the receptor was assessed by MCA-based immunoabsorption studies. At diagnosis, hCG (9.8x10(7)+/-4.84x10(7) pg/ml; range 1.1x10(5)-5x10(8) pg/ml) was greatly increased and serum hFSH was undetectable (<9 pg/ml) in 11 patients, and one patient had very low, albeit detectable (approximately 30 pg/ml) hFSH concentrations. hLH was below the limit of detection (<2 pg/ml) in five individuals. During successful chemotherapy, hCG rapidly declined to physiological concentrations and hFSH/hLH returned to normal or even reached supraphysiological values. There was a highly significant negative correlation between hCG and hFSH (P=0.0001) and, to a lesser extent, hLH (P=0.0265). The ability of serum hCG to block the binding of [125I]rFSH (rat FSH) to its receptor was found to be 0.01-0.1% compared with the FSH standard; this could be reversed by an anti-hCG MCA. Addition of a specific MCA against hFSH blocked 3 microg/ml of the hFSH standard, but had no effect on the FSA of serum hCG in the FSH RRA. As observed during pregnancy, secretion of gonadotrophin -- particularly that of FSH -- is substantially or completely suppressed in patients with testicular cancer when serum hCG concentrations exceed 10(5)-10(6) pg/ml (approximately 10(3)-10(4) mIU/ml). As determined by RRA, the intrinsic FSA of tumour-derived hCG is most probably responsible for the suppression of hFSH in this group of patients with testicular cancer.
3073	The mouse Id2 and Id4 genes: structural organization and chromosomal localization.	9831657	The Id proteins belong to a family of nuclear HLH proteins lacking a basic region and thought to function as dominant-negative regulators of bHLH proteins during cell growth and differentiation. In this paper, we report the genomic organization of the mouse Id2 and Id4 genes. These genes each span approximately 3 kb of the mouse genome and are each organized as three exons with recognizable splice donor and acceptor consensus sequences. Their genomic organization is very similar, consistent with their having evolved from a common, ancestral Id-like gene. Using FISH analysis, we have localized the mouse Id2 and Id4 genes to mouse chromosome 12 and 13, respectively.
3074	Cumulation of the tendency to segregate auditory streams: resetting by changes in location and loudness.	9821783	In four experiments, the accumulation, over time, of a tendency to hear separate high and low streams in a sequence of high (H) and low (L) tones, presented in a galloping rhythm (HLH-HLH-...), was studied. Each trial was composed of two parts, an induction sequence, then a test sequence, with no break between them. The test sequence was always heard at the far left. When the induction sequence and the test sequence were identical, the presence of the induction sequence increased the tendency for the test sequence to split into two streams. However, when the sequences differed in location (cued by differences in interaural timing or intensity over headphones and by loudspeaker placement in a free field) or when they differed in loudness, the accumulation of the segregative tendency was reset, and the test sequence sounded more integrated. When the induction sequence changed in location or loudness in gradual steps toward the value of the test sequence, resetting was much less. It appears that the accumulation of information about streams in different frequency regions is sensitive to sudden changes in parameters, even when they affect the frequency regions equally. This prevents the system from accumulating data across unrelated events.
3075	Overexpression of Mos(rat) proto-oncogene product enhances the positive autoregulatory loop of MyoD.	9804168	The myogenic b-HLH transcription factor MyoD activates expression of muscle-specific genes and autoregulates positively its own expression. Various factors such as growth factors and oncogene products repress transcriptional activity of MyoD. The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate myogenic differentiation by specific phosphorylation of MyoD which favors heterodimerization of MyoD and E12 proteins. Here we show that overexpression of Mos enhances the expression level of MyoD protein in myoblasts although phosphorylation of MyoD by Mos does not modify its stability but promotes transcriptional transactivation of the MyoD promoter linked to the luciferase reporter gene. Moreover, co-expression of MyoD with Mos(wt) but not with the kinase-inactive Mos(KM) greatly enhances expression of endogenous MyoD protein and the DNA binding activity of MyoD/E12 heterodimers in 10T1/2 cells. Our data suggest that Mos increases the ability of MyoD to transactivate both muscle-specific genes and its own promoter and could therefore participate in the positive autoregulation loop of MyoD and muscle differentiation.
3076	Helix-loop-helix (E2-5, HEB, TAL1 and Id1) protein interaction with the TCRalphadelta enhancers.	9796921	In order to dissect the correlation between aberrant TAL1 basic-helix-loop-helix (b-HLH) expression and the exclusive development of T cell acute lymphoblastic leukemias (T-ALL) of the TCRalphabeta lineage, we have assessed the ability of class A b-HLH proteins to regulate the TCRalpha and delta enhancers. We demonstrate that E47S binds to TCRalpha but not to TCRdelta E-boxes in vitro. Despite this, neither E2-5 nor HEB transactivate the TCRalpha enhancer in NIH 3T3, nor did Id1 modify endogenously driven TCRalpha [alphaE1-4] activity in a TCRalphabeta cell line. We also demonstrate that TAL1 inhibits both binding of E47S to aE3 and aE4 and endogenous transactivation of the TCRalpha enhancer. Comparison of the activity of the minimal [alphaE1-2] fragment, which contains no E-boxes, with the accessory [aE3-4] fragment, which contains two, suggested some contribution from the latter to TCRalpha enhancer activity in HPB-ALL. TCR [alphaE1-2] activity was partially (40%) inhibited by TAL1 but not at all by Id1. In contrast, [alphaE3-4] activity was almost completely inhibited by TAL1 (80%) and slightly reduced by Id1 (15%). These data demonstrate that class A b-HLH regulation of the TCRalpha enhancer E-boxes differs from their B lymphoid Igmicro counterparts and suggest a novel mechanism of transcriptional inhibition by TAL1, which may be, at least partly, independent of E-box-mediated activation, as we currently recognize it. They also clearly demonstrate that the restriction of TAL1 deregulation to T-ALL of the TCRalphabeta lineage is not due to induction of TCRalpha enhancer activity by the TAL1 protein.
3077	Molecular cloning of Zcoe2, the zebrafish homolog of Xenopus Xcoe2 and mouse EBF-2, and its expression during primary neurogenesis.	9784615	Xcoe2 is a recently identified HLH transcription factor of the Xenopus primary neurogenesis pathway, which is necessary downstream of Neurogenin to stabilize neuroblast determination (Dubois, L. et al., 1998. Curr. Biol. 8, 199-209). We report here the embryonic expression pattern of Zcoe2, its zebrafish homolog. As observed for Xcoe2, Zcoe2 is strongly expressed in a subset of the neurogenin1- (ngn1-) positive primary neuroblasts of the spinal cord. In the anterior neural plate, in contrast, Zcoe2 is expressed earlier and more widely than ngn1. This pattern is strongly maintained in the presumptive mesencephalon and rhombomeres 1-4 until the 2-3-somite stage. This expression of Zcoe2 in the brain anlage calls for a re-analysis in zebrafish of the functional relationship demonstrated in Xenopus between Coe2 and Neurogenin factors. At later stages, Zcoe2 is expressed in early forming neurons of the anterior brain and is a marker of the olfactory placodes.
3078	Polymerase chain reaction amplification of archival material for Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, and parvovirus B19 in children with bone marrow hemophagocytosis.	9781644	Bone marrow hemophagocytosis may occur as an incidental finding, or it may be a manifestation of a systemic and potentially lethal disorder. When systemic, the proliferation is termed hemophagocytic lymphohistiocytosis (HLH), a clinicopathologic entity characterized by a widespread proliferation of benign hemophagocytic histiocytes, fever, pancytopenia, deranged liver function, and frequently coagulopathy and hepatosplenomegaly. A variety of infectious agents, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), and parvovirus B19 (PVB19), have been associated with HLH, but the relative frequency of each using one technique has not been evaluated. In addition, infectious causes of incidental bone marrow hemophagocytosis, not occurring in the setting of HLH, have not been evaluated. Review of bone marrow reports from bone marrow examinations done between December 1986 and June 1997 showed that 20 children aged 2 months to 15 years had bone marrow examinations that indicated hemophagocytosis. Archival materials from 19 patients were successfully retrieved, and DNA was extracted from archived unstained coverslips with subsequent polymerase chain reaction for EBV, CMV, HHV6, and PVB19 genomic DNA. DNA extracted from 16 bone marrow specimens of age-matched children was used as negative controls. Eleven of the 19 patients fulfilled the clinical and pathological criteria for HLH; the remaining eight patients had isolated hemophagocytosis without a systemic presentation. Viral DNA was detected in 8 of 11 patients with HLH but in none of eight patients with isolated hemophagocytosis. EBV was present in five of the bone marrows, followed in frequency by HHV6, CMV, and PVB19. Infection with more than one agent was present in three patients. Only one control patient was positive for HHV6 DNA; the remaining control patients were negative for all viruses. Viral infection, detected by PCR analysis of bone marrow, is a common finding in patients with HLH but not in patients with isolated bone marrow hemophagocytosis. This technique may provide another marker to aid in the diagnosis of HLH and suggests a different cause of hemophagocytosis occurring in patients with and without HLH.
3079	A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer.	9780002	The human embryonal carcinoma cells NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambdaZAP II NEC14 cell library with the 32P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N- and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but different from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.
3080	Eve and ftz regulate a wide array of genes in blastoderm embryos: the selector homeoproteins directly or indirectly regulate most genes in Drosophila.	9778506	The selector homeoproteins are a highly conserved group of transcription factors found throughout the Eumetazoa. Previously, the Drosophila selector homeoproteins Eve and Ftz were shown to bind with similar specificities to all genes tested, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Here, we demonstrate that the expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve. Suspecting that the selector homeoproteins may affect many more genes than previously thought, we have characterized the expression of randomly selected genes at different stages of embryogenesis. At cellular blastoderm, 25-50% of genes whose transcription can be monitored are regulated by both Eve and Ftz. In late embryogenesis, 87% of genes are directly or indirectly controlled by most or all selector homeoproteins. We argue that this broad control of gene expression is essential to coordinate morphogenesis. Our results raise the possibility that each selector homeoprotein may directly regulate the expression of most genes.
3081	Defective natural killer cell function in patients with hemophagocytic lymphohistiocytosis and in first degree relatives.	9773832	Hemophagocytic lymphohistiocytosis (HLH), also referred to as familial erythrophagocytic lymphohistiocytosis, is a rare disorder of infancy associated with proliferation of activated histiocytes and T cells, anemia, thrombocytopenia, and fevers. This disorder appears to be due to the uncontrolled activation of T cells producing IL-2, tumor necrosis factor-alpha, and interferon-gamma. Untreated, the disorder is universally fatal. Various deficits in immune function have been described during acute disease activity including impaired T cell function, impaired monocyte-mediated antibody-dependent cytotoxicity, impaired natural killer cell function, and impaired IL-1 production. We examined natural killer cell function in familial HLH patients to determine whether this finding was consistently associated with the disease. We also examined natural killer cell function in asymptomatic parents and siblings of patients. Impaired natural killer cell function was identified in all patients and in some family members, including obligate carrier parents. This implies that one potential genetic defect in HLH may result in depressed natural killer function, but that this may not be sufficient to reliably predict eventual progression to disease.
3082	[Treatment of hemophagocytic lymphohistiocytosis, HLH, with bone marrow transplantation].	9743950	Hemophagocytic lymphohistiocytosis (HLH) is a rare disease of infancy and young childhood. The clinical presentation includes recurrent unexplained fever with hepatosplenomegaly. Cytopenia, hypofibrinogenemia and/or hypertriglyceridemia and hemophagocytosis in bone marrow, spleen and lymphnode confirm the diagnosis. Hemophagocytosis may not be present at the beginning. In these cases, diagnosis is facilitated by a positive family history, a relapsing course of the disease, the frequent involvement of the central nervous system and positive findings on immunological work-up. Treatment by chemotherapy and immunosuppressants can achieve sustained remissions in most patients and reinduction of remission after relapse is possible. Most children however, eventually die from progressive disease. At present, allogeneic bone marrow transplantation is the only curative therapeutic option. Between August 1992 and May 1997 eleven consecutive patients with HLH received bone marrow from unrelated (n = 7) or matched sibling donors (n = 4). The conditioning regimen consisted of busulfan, VP-16 and cyclophosphamide. Patients engrafted after a median time of 16 days (13-43). Only one patient developed grade III acute GVHD, another patient, grade II acute GVHD. Although regimen-related toxicity was extensive, all patients have survived without signs of HLH after a median follow up of 20 months (8-63). One patient suffers from chronic GVHD, three patients reveal psychomotoric retardation and one patient has severe impairment with spastic tetraparesis, amaurosis and seizures. Our experience shows that HLH can be successfully treated by allogeneic BMT from unrelated donors.
3083	A distal regulatory region of the insulin-like growth factor binding protein-2 (IGFBP-2) gene interacts with the basic helix-loop-helix transcription factor, AP-4.	9741833	Insulin-like growth factor binding protein-2 (IGFBP-2), the predominant IGFBP in the fetal circulation and an induced protein during several types of malignancies, belongs to a family of structurally related proteins that bind the mitogens, IGF-1 and IGF-2. The present study focused on functional analysis of the 5 '-flanking region (approximately 1.3 kb) of the IGFBP-2 gene to identify nuclear factors that mediate hepatic transcription of this gene. Luciferase (LUC) reporter constructs containing progressive deletions of 5'-flanking DNA and the intact promoter of the porcine IGFBP-2 gene were examined for functional activity by transient transfection of human HepG2 liver cells. LUC activity of the transfected reporter gene driven by the IGFBP-2 promoter and flanking sequences to -1397 (numbering relative to initiation codon at +1) was 22-fold higher than that of promoterless parent LUC vector. This activity was decreased by 60% with deletion of sequences to -874 bp, and dropped to basal levels with further truncation to -764 bp. The region between -874 and -765 bp (110 bp) functioned as a potent stimulator of heterologous SV40 promoter activity (110 bp/SV40-LUC construct) and was found to contain two noncontiguous basic helix-loop-helix (bHLH) transcription factor binding motifs (E-boxes [CAN NTG]: CACCTG and CAAATG). In electrophoretic mobility shift assays, nuclear proteins prepared from HepG2 cells formed two complexes (C1, C2) with double-stranded oligonucleotides containing either HLH sequence, mutations of which resulted in loss of complex formation. Southwestern blot analysis identified an HepG2 nuclear protein with molecular mass of 48 kDa, similar to that of the bHLH transcription factor AP-4, which bound the CACCTG motif. Cotransfection of HepG2 cells with the 110-bp/SV40-LUC construct and an expression vector encoding human AP-4 increased IGFBP-2 fragment-dependent SV40 promoter activity by 16-fold. This AP-4-mediated stimulation was lost following block mutation of both bHLH motifs within the IGFBP-2 110-bp fragment. Results demonstrate the functional importance of sequences upstream of the promoter in IGFBP-2 gene transcription and identify a novel mechanism by which bHLH proteins potentially may affect cell proliferation and differentiation via induction of IGFBP-2 synthesis.
3084	Forced expression of Id-1 in the adult mouse small intestinal epithelium is associated with development of adenomas.	9737997	Ids are dominant-negative helix-loop-helix (HLH) proteins that play overlapping yet distinct roles in antagonizing basic HLH transcription factors. Although Ids affect myogenesis, neurogenesis, and B-cell development, little is known about their in vivo functions in epithelia. We have examined the effects of forced expression of Id-1 in the small intestinal epithelium of adult chimeric mice. 129/Sv embryonic stem cells, transfected with DNA containing Id-1 under the control of transcriptional regulatory elements that function in all intestinal epithelial cell lineages, were introduced into C57Bl/6 (B6) blastocysts heterozygous for the ROSA26 marker. The B6 ROSA26/+ intestinal epithelium of the resulting adult chimeras produces Escherichia coli beta-galactosidase, allowing identification of this internal control cell population. Chimeras produced from nontransfected embryonic stem cells served as additional controls. Immunohistochemical studies of the control chimeras indicated that the small intestinal epithelium supports a complex pattern of endogenous Id expression. Id-1 is restricted to the cytoplasm; levels do not decrease as descendants of multipotent intestinal stem cells differentiate. Id-2 and Id-3 are only detectable in nuclei; levels increase markedly as epithelial cells differentiate. Forced expression of Id-1 in the 129/Sv epithelium results in a decline in Id-2 and Id-3 to below the limits of immunodetection. A subset of chimeric-transgenic mice lacked growth factor- and defensin-producing Paneth cells in their 129/Sv epithelium and also developed intestinal adenomas. These changes were not present in normal control chimeras. Adenomas were composed of proliferating beta-Gal-positive and -negative epithelial cells, suggesting that they arose through cooperative interactions between 129/Sv(Id-1) and B6 ROSA26/+ cells. These chimeras provide a model for studying how perturbations in Id expression affect tumorigenesis.
3085	The K box, a conserved 3' UTR sequence motif, negatively regulates accumulation of enhancer of split complex transcripts.	9735368	Cell-cell interactions mediated by the Notch receptor play an essential role in the development of the Drosophila adult peripheral nervous system (PNS). Transcriptional activation of multiple genes of the Enhancer of split Complex [E(spl)-C] is a key intracellular response to Notch receptor activity. Here we report that most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3' untranslated regions (3' UTRs). We present three lines of evidence that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes. First, K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species. Second, the wild-type m8 3' UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences. Finally, m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, we find that K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, we present sequence analyses that implicate genes of the iroquois Complex (Iro-C) and engrailed as additional targets of K box-mediated regulation.
3086	The helix-loop-helix protein Id2 is overexpressed in human pancreatic cancer.	9731481	Id2 belongs to the Id family of helix-loop-helix (HLH) proteins, which upon heterodimerization with basic HLH proteins prevent basic HLH proteins from DNA binding. Proteins of the Id family act as negative regulatory transcriptional factors, and their expression correlates with cell proliferation and arrested differentiation in many cell lineages. In this study, we characterized the expression of Id2 in normal and cancerous pancreatic tissues. Pancreatic cancers markedly overexpressed Id2 mRNA in comparison to the normal pancreas. Furthermore, there was abundant Id2 immunoreactivity in the cancer cells within the pancreatic tumor mass. In PANC-1 human pancreatic cancer cells, steady-state Id2 mRNA levels increased upon serum addition and decreased after induction of differentiation with either sodium butyrate or 12-O-tetradecanoylphorbol-13-acetate. Inhibition of Id2 expression with Id2 antisense oligonucleotides inhibited the growth of these cells, whereas random and sense oligonucleotides were without effect. These findings suggest that Id2 may have a role in human pancreatic cancer.
3087	Biomarker and morphological characteristics of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis.	9722893	Viruses may induce primary as well as secondary hemophagocytic lymphohistiocytosis (HLH), but it may not be possible to discriminate between these two in patients with a negative family history. Among these HLH cases, fulminant and fatal virus-associated hemophagocytic syndrome (VAHS) occurs mostly in relation to Epstein-Barr virus (EBV) infection. Although the immunological characteristics of EB-VAHS were previously reported, data on non-EB-VAHS were sporadic and fragmentary. This study has compared the clearly distinguishable groups of EBV-positive vs. EBV-negative HLH cases.Among 26 patients with EBV-related HLH and 12 patients with non-EBV HLH, peripheral blood mononuclear cell (PBMC) subsets and serum concentrations of cytokines at the active phase of the disease were compared. Blood and bone marrow smears were also compared.The frequency of the CD3+HLADR+ subset in PBMC (median 34.3% vs. 4.8%), of serum concentrations of interferon (IFN)-gamma (median 105 U/ml vs. 2.4 U/ml), and of soluble interleukin-2-receptor (sIL-2R) (median 14,700 U/ml vs. 3,412 U/ml) were significantly different between these two groups. Morphological characteristics were noted for EBV-related HLH cases. Mortality also differed between these two groups, 9/26 vs. 0/12 (P = 0.05). Data indicate pronounced immunological imbalance and poor prognosis in EBV-related HLH cases. These parameters could be useful for determining an EBV involvement as well as risk factors in the early care and treatment of HLH patients.
3088	Allogeneic peripheral blood stem cell transplantation for active hemophagocytic lymphohistiocytosis.	9720748	Hemophagocytic lymphohistiocytosis (HLH) is a lethal disorder of immune dysregulation. Successful allogeneic BMT is the only therapy that has produced long-term disease-free survival. However, for patients with active HLH the outcome of BMT remains poor when compared with those transplanted in remission. We describe a patient with active, recurrent HLH treated with high-dose chemotherapy and related allogeneic peripheral blood stem cell (PBSC) transplantation. Hematologic recovery was rapid and the post-transplant course was uncomplicated. The patient is alive and well 2 1/2 years after transplant. Allogeneic PBSC transplantation may deserve further evaluation as an alternative source of stem cells for transplantation in patients with active HLH.
3089	Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning.	9716413	Mesodermal development is a multistep process in which cells become increasingly specialized to form specific tissue types. In Drosophila and mammals, proper segregation and patterning of the mesoderm involves the bHLH factor Twist. We investigated the activity of a Twist-related factor, CeTwist, during Caenorhabditis elegans mesoderm development. Embryonic mesoderm in C. elegans derives from a number of distinct founder cells that are specified during the early lineages; in contrast, a single blast cell (M) is responsible for all nongonadal mesoderm formation during postembryonic development. Using immunofluorescence and reporter fusions, we determined the activity pattern of the gene encoding CeTwist. No activity was observed during specification of mesodermal lineages in the early embryo; instead, the gene was active within the M lineage and in a number of mesodermal cells with nonstriated muscle fates. A role for CeTwist in postembryonic mesodermal cell fate specification was indicated by ectopic expression and genetic interference assays. These experiments showed that CeTwist was responsible for activating two target genes normally expressed in specific subsets of nonstriated muscles derived from the M lineage. In vitro and in vivo assays suggested that CeTwist cooperates with the C. elegans E/Daughterless homolog in directly activating these targets. The two target genes that we have studied, ceh-24 and egl-15, encode an NK-2 class homeodomain and an FGF receptor (FGFR) homolog, respectively. Twist activates FGFR and NK-homeodomain target genes during mesodermal patterning of Drosophila and similar target interactions have been proposed to modulate mesenchymal growth during closure of the vertebrate skull. These results suggest the possibility that a conserved pathway may be used for diverse functions in mesodermal specification.
3090	Id2 promotes apoptosis by a novel mechanism independent of dimerization to basic helix-loop-helix factors.	9710627	Members of the helix-loop-helix (HLH) family of Id proteins have demonstrated roles in the regulation of differentiation and cell proliferation. Id proteins inhibit differentiation by HLH-mediated heterodimerization with basic HLH transcription factors. This blocks their sequence-specific binding to DNA and activation of target genes that are often expressed in a tissue-specific manner. Id proteins can also act as positive regulators of cell proliferation. The different mechanisms proposed for Id-mediated promotion of entry into S phase also involve HLH-mediated interactions affecting regulators of the G1/S transition. We have found that Id2 augments apoptosis in both interleukin-3 (IL-3)-dependent 32D.3 myeloid progenitors and U2OS osteosarcoma cells. We could not detect a similar activity for Id3. In contrast to the effects of Id2 on differentiation and cell proliferation, Id2-mediated apoptosis is independent of HLH-mediated dimerization. The ability of Id2 to promote cell death resides in its N-terminal region and is associated with the enhanced expression of a known component of the programmed cell death pathway, the proapoptotic gene BAX.
3091	Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis.	9692544	The results reported here indicate that retinoic acid (RA) induces growth arrest and differentiation only in MyoD-expressing muscle cells. Transient transfection assays reveal a functional interaction between MyoD, a key myogenic regulator and RA-receptors, principal mediators of RA actions. Interestingly, we demonstrate that RXR-MyoD-containing complexes are recruited at specific MyoD DNA-binding sites in muscle cells. Furthermore, we also demonstrate that RA-receptors and the muscle basic helix-loop-helix (b-HLH) proteins interact physically. Mutational analysis suggests that this interaction occurs via the basic region of muscle b-HLH proteins and the DNA-binding domain of RA-receptors and is important for functional interactions between these two families of transcription factors. In conclusion, these results highlight novel interactions between two distinct groups of regulatory proteins that influence cell growth and differentiation.
3092	The development of an enzyme immunometric assay for LH and the effects of the methods on the immunoreactivity of the conjugates.	9682127	Using an affinity purified sheep anti-human luteinizing hormone IgG-horseradish peroxidase conjugate (sheep anti-hLH IgG-HRP) and rabbit anti-human luteinizing hormone antiserum (rabbit anti-hLH) directed against different antigenic determinants, a solid-phase "sandwich" enzyme immunometric assay (EIMA) for human luteinizing hormone (hLH) was developed. The assay was validated and compared with a liquid phase "two site" immunoradiometric assay (IRMA) for hLH which uses same two antibodies. The sheep anti-hLH IgG, which had been affinity purified by eluting at pH 3.5 from a hLH-sepharose 4 B column, was labelled with 125I. The IRMA is based on simultaneous addition of two antibodies to standards and samples. After overnight incubation, separation was achieved by addition of Sheep anti-Rabbit Fc (SARFc) antiserum. In EIMA; partially denaturated (at pH 2.5) Sheep anti-Rabbit FcIgG (SARFcIgG) coated polystyrene tubes or microtitre plates were employed as solid-phase second antibody. The substrate was N,N'-o-phenylene diamine (2 mg/ml) and H2O2 (O.02%). Two methods, modified NaIO4 and 4-(N-maleimido methyl)-cyclohexane-1-carboxylic acid N-hydroxy succinimide ester (SMCC), were employed in the preparation of sheep anti-hLH IgG-HRP conjugate. The immunoreactivity and peroxidase activity of conjugate prepared with NaIO4 method was impared to various extends. Both EIMA and IRMA had good specificity, were not susceptible to interference from serum components and exhibited very low non-specific binding. The values determined by EIMA were independent of the serum volume employed. Standard added to serum samples was accurately determined and the results obtained from the analysis of serum samples correlated closely with those obtained by IRMA.
3093	Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper.	9680483	The oncoprotein c-Myc (a member of the helix-loop-helix-leucine zipper (b-HLH-LZ) family of transcription factors) must heterodimerize with the b-HLH-LZ Max protein to bind DNA and activate transcription. It has been shown that the LZ domains of the c-Myc and Max proteins specifically form a heterodimeric LZ at 20 degreesC and neutral pH. This suggests that the LZ domains of the c-Myc and Max proteins are playing an important role in the heterodimerization of the corresponding gene products in vivo. Initially, to gain an insight into the energetics of heterodimerization, we studied the stability of N-terminal disulfide-linked versions of the c-Myc and Max homodimeric LZs and c-Myc-Max heterodimeric LZ by fitting the temperature-induced denaturation curves monitored by circular dichroism spectroscopy. The c-Myc LZ does not homodimerize (as previously reported) and the c-Myc-Max heterodimeric LZ is more stable than the Max homodimeric LZ at 20 degreesC and pH 7.0. In order to determine the critical interhelical interactions responsible for the molecular recognition between the c-Myc and Max LZs, the solution structure of the disulfide-linked c-Myc-Max heterodimeric LZ was solved by two-dimensional 1H-NMR techniques at 25 degreesC and pH 4.7. Both LZs are alpha-helical and the tertiary structure depicts the typical left-handed super-helical twist of a two-stranded parallel alpha-helical coiled-coil. A buried salt bridge involving a histidine on the Max LZ and two glutamate residues on the c-Myc LZ is observed at the interface of the heterodimeric LZ. A buried H-bond between an asparagine side-chain and a backbone carbonyl is also observed. Moreover, evidence for e-g interhelical salt bridges is reported. These specific interactions give insights into the preferential heterodimerization process of the two LZs. The low stabilities of the Max homodimeric LZ and the c-Myc-Max heterodimeric LZ as well as the specific interactions observed are discussed with regard to regulation of transcription in this family of transcription factors.
3094	Familial hemophagocytic lymphohistiocytosis: improved neurodevelopmental outcome after bone marrow transplantation.	9672524	A child with familial hemophagocytic lymphohistiocytosis (HLH) underwent allogeneic bone marrow transplantation (BMT) at age 5 months. At that time he showed delayed psychomotor development, and computed tomography revealed diffuse calcification. After BMT, a gradual neurodevelopmental normalization was observed. The potential ability of BMT to reverse neurodevelopmental deterioration in HLH should be considered.
3095	Hypoxia-inducible factor 1alpha (HIF-1alpha) is a non-heme iron protein. Implications for oxygen sensing.	966075610375303	The hypoxia-inducible factor 1 complex (HIF-1) is involved in the transcriptional activation of several genes, like erythropoietin and vascular endothelial growth factor, that are responsive to the lack of oxygen. The HIF-1 complex is composed of two b-HLH proteins: HIF-1beta that is constitutively expressed, and HIF-1alpha, that is present only in hypoxic cells. The HIF-1alpha subunit is continuously synthesized and degraded by the ubiquitin-proteasome under oxic conditions. Hypoxia, transition metals, iron chelators, and several antioxidants stabilize the HIF-1alpha protein, allowing the formation of the transcriptionally active HIF-1 complex. The mechanisms of oxygen sensing and the pathways leading to HIF-1alpha stabilization are unclear. Because the involvement of a heme protein oxygen sensor has been postulated, we tested the heme sensor hypothesis by using a luciferase-expressing cell line (B-1), that is highly responsive to hypoxia. Exposure of B-1 cells to carbon monoxide and heme synthesis inhibitors failed to show any effect on the hypoxia responsiveness of these cells, suggesting that heme proteins are not involved in hypoxia sensing. Measurement of iron in recombinantly expressed HIF-1alpha protein revealed that this protein binds iron in vivo. Iron binding was localized to a 129-amino acid peptide between sequences 529 and 658 of the HIF-1alpha protein. Although the exact structure of the iron center has not been yet defined, a 2:1 metal/protein molar ratio suggests a di-iron center, probably similar to the one found in hemerythrin. This finding is compatible with a model where redox reaction may occur directly in the iron center of the HIF-1alpha subunit, affecting its survival in oxic conditions.
3096	A subset of notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex.	9655811	The Notch signalling pathway is involved in many processes where cell fate is decided. Previous work showed that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signalling represses atonal expression during lateral specification. In this paper we investigate which known components of the Notch pathway are involved in each signalling process. Using clonal analysis we show that a ligand of Notch, Delta, is required along with Notch for both proneural enhancement and lateral specification, while the downstream components Suppressor-of-Hairless and Enhancer-of-Split are involved only in lateral specification. Our data point to a distinct signal transduction pathway during proneural enhancement by Notch. Using misexpression experiments we also show that particular Enhancer-of-split bHLH genes can differ greatly in their contribution to lateral specification.
3097	Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes.	9584154	Proteins of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including neurogenesis, lymphopoiesis, myogenesis, and sex determination. Using a yeast two-hybrid screen, we have identified a new bHLH transcription factor, ABF-1, from a human B-cell cDNA library. Within the bHLH region, ABF-1 shows a remarkable conservation with other HLH proteins, including tal-1, NeuroD, and paraxis. Its expression pattern is restricted to a subset of lymphoid tissues, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and activated human B cells. ABF-1 is capable of binding an E-box element either as a homodimer or as a heterodimer with E2A. Furthermore, a heterodimeric complex containing ABF-1 and E2A can be detected in EBV-immortalized lymphoblastoid cell lines. ABF-1 contains a transcriptional repression domain and is capable of inhibiting the transactivation capability of E47 in mammalian cells. ABF-1 represents the first example of a B-cell-restricted bHLH protein, and its expression pattern suggests that ABF-1 may play a role in regulating antigen-dependent B-cell differentiation.
3098	Lack of transcriptional repression by max homodimers.	9632139	Max, a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) protein, plays a central role in the transcriptional regulation of myc oncoprotein-responsive genes. Myc-max heterodimers bind to consensus E-box motifs near or within the promoters of these genes and activate gene expression, whereas heterodimers between max and members of the mad family of bHLH-ZIP proteins promote transcriptional repression. In contrast to all other members of the myc network, max readily homodimerizes and binds to identical E-box sites in vitro. However, the role for max homodimers in transcriptional repression in vivo is unclear. Upstream stimulatory factor (USF) is a bHLH-ZIP protein which does not interact with members of the myc-max-mad family. By replacing the HLH-ZIP domain of max with that from USF, we created a chimeric protein, max(USF), which was indistinguishable from max with respect to its ability to homodimerize and bind DNA. As expected, however, max(USF) was unable to heterodimerize with any of the tested max partner proteins and was incapable of suppressing c-myc target genes. Thus, transcriptional repression is an exclusive property of max-mad heterodimers and cannot be achieved by max homodimers alone.
3099	Unrelated donor marrow transplantation for inborn errors.	9630323	From December 1989 to December 1997 40 children aged 1 year to 19 years with inborn errors other than severe combined immunodeficiencies underwent unrelated donor (UD) bone marrow transplantation (BMT) in one of 10 institutions of the Italian Bone Marrow Transplant Group participating in this program. The diseases leading to BMT included Fanconi Anemia (10), Thalassemia (8), Wiskott Aldrich syndrome (5), haemophagocytic lymphohystiocytosis (6), osteopetrosis (3), storage diseases (6), Chediak Higashi syndrome (1), Schwachman syndrome (1). Thirty-three pairs were A, B, DRB1 matched. Three pairs were one antigen mismatched and one pair was two antigens mismatched. The remaining three pairs lacked information on molecular biology. Twelve children underwent a preparative regimen including radiotherapy. The remaining 28 children were conditioned with a chemotherapy regimen which included Busulfan. GvHD disease prophylaxis included CSA and MTX alone (9) or associated with ALG (17) or in vivo Campath 1G (12). The remaining two children received CSA alone. Thirty-five children showed donor engraftment; three children with thalassemia and one with osteopetrosis failed to engraft. Five children developed secondary graft failure. Actuarial 5 year disease-free survival was 62%; grade III-IV acute GvHD developed in seven of 38 evaluable children (18%); chronic GvHD developed in seven of 27 evaluable children (26%). We confirm that Wiskott Aldrich syndrome, HLH, and osteopetrosis represent an absolute indication for UD-BMT. Prognosis of UD-BMT for FA could improve in children grafted in an early phase, but a better preparative regimen has to be identified. UD-BMT in thalassemia is acceptable only in a restricted subset of patients selected for poor compliance to therapy.
3100	Heterogeneity of immune markers in hemophagocytic lymphohistiocytosis: comparative study of 9 familial and 14 familial inheritance-unproved cases.	9628431	Although immune dysfunction is suspected in patients with hemophagocytic lymphohistiocytosis (HLH), the difference between immune dysfunction in patients with familial erythrophagocytic lymphohistiocytosis (FEL) and familial inheritance-unproved lymphohistiocytosis (FIU) remains unknown. The aim of this study was to determine useful markers to distinguish patients with FEL from those with FIU.Clinical features and laboratory findings, especially natural killer (NK) cell activity and the relative frequencies of peripheral blood mononuclear cell (PBMC) subsets, and serum levels of interferon-gamma and soluble interleukin-2 receptor were compared in 9 patients with FEL and 14 age-matched patients with FIU. Twenty-seven healthy infants served as controls. The treatment and outcome were also compared for patients with FEL and FIU.Comparison between patients with FEL and FIU revealed significantly lower NK activity in those with FEL (p = 0.03) but failed to show any significant differences in PBMC subsets, except that the percentage of CD3+ T cells was higher in patients with FEL (p = 0.02). CD4- and CD8-dominant phenotypes were characteristic findings in both groups of patients, although increased CD19+ B cells were restricted to patients with FIU. NK activity was deficient (< 5%) in four of the seven patients with FEL tested but in only one of eight patients with FIU. By comparison to values for age-matched controls, the percentages of CD3+, CD3+DR+ and CD45RO+ PBMCs in patients with FEL were significantly high (p < 0.05) and those of CD19+ and CD45RA+ subsets were lower than normal. Among patients with FIU, PBMC subsets included significantly reduced CD3+, CD4+, CD45RA+, and CD4+CD45RA+. In this small series, the outcome of patients with FEL and FIU treated with chemotherapy was not significantly different at the time of evaluation.These results indicate considerable immune heterogeneity among patients with HLH younger than 2 years. Although NK activity was useful but not diagnostic, determination of PBMC subsets and patterns of cytokine expression was not helpful in distinguishing patients with FEL from those with FIU, suggesting that the immune responses characteristic of these diseases may reflect different triggering factors, including viruses. The impact of this immune heterogeneity on patients' outcome remains to be determined.
3101	The function of hairy-related bHLH repressor proteins in cell fate decisions.	9619101	Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.
3102	Immunohistochemical localization of FSH and LH in the pituitary of male ruin lizards (Podarcis sicula campestris De Betta).	9615194	The pars distalis from the pituitary gland of adult male ruin lizards (Podarcis sicula campestris De Betta), captured during the reproductive period (May and June), was studied immunohistochemically using specific antibodies against hFSH beta, hLH beta and oLH beta with the avidin-biotin-peroxidase complex (ABC) procedure to determine the localization of both gonadotropins. The immunostaining with anti-hFSH beta and anti-hLH beta allowed identification of morphologically distinct FSH containing cells and LH containing cells, whereas anti-oLH beta serum showed cross-reactivity with cells immunostained with the anti-hFSH beta and anti-hLH beta sera. The gonadotropic cells took up approximatively 10.5% of the area of the pars distalis: 10% was positive for FSH, whereas only 0.5% was positive for LH. The FSH cells were distributed throughout the pars distalis, whereas the LH cells were only located in the rostral region. Double-immunostaining procedure did not reveal cells containing both gonadotropic hormones. The findings indicate that FSH and LH are produced in separate pituitary cells.
3103	Autopsy findings in 27 children with haemophagocytic lymphohistiocytosis.	9602326	Primary haemophagocytic lymphohistiocytosis (HLH) is a fatal childhood disorder. The diagnosis is difficult to establish, clinically as well as histopathologically, and it is markedly underdiagnosed. Because of these difficulties, we wanted to elucidate the histopathological findings in population-based patient material.The post-mortem findings in 27 children with primary HLH diagnosed in Sweden between 1971 and 1986 was reviewed. Twelve of these patients had an affected sibling and three additional children had parental consanguinity. Some of the children showed generalized disease, whereas in others only one or a few organs were affected. The major histological alteration was an accumulation of primarily lymphocytes, but also of histiocytes, some of which exhibited evidence of haemophagocytosis. The haemophagocytic activity may be difficult to detect if there are pronounced post-mortem changes, particularly in the spleen, and it is therefore preferable to perform the autopsy as soon as possible after death in order to minimize autolysis. Haemophagocytosis was most commonly observed in the spleen (17/24), the lymph nodes (17/23) and the bone marrow (9/23), indicating that a negative bone marrow examination does not rule out this diagnosis. Three additional patients had discrete signs of haemophagocytosis in the bone marrow. In the spleen, the lymph nodes and the bone marrow, lymphocytic depletion, pronounced in some cases, could be observed, even without prior treatment with steroids or cytostatics. In the liver, most of the patients demonstrated an infiltration of lymphocytes into the portal tracts similar to that seen in chronic persistent hepatitis (22/27), a finding which is uncommon in infancy and therefore suggestive of the diagnosis HLH. Other organs involved included the thymus, lungs intestine, pancreas, kidney, heart and striated muscle.The diagnosis of HLH must be based on clinical, histological and additional laboratory findings. A negative bone marrow examination is common. Previous treatment with steroids and/or cytostatic drugs may attenuate or even eliminate the typical histological findings. Liver findings similar to those in chronic persistent hepatitis are common.
3104	Calcium regulation of basic helix-loop-helix transcription factors.	9601609	The basic helix-loop-helix (bHLH) family of transcription factors is essential for numerous developmental and growth control processes. The regulation of bHLH proteins occurs at many levels, including tissue specific expression, differential oligomerization and DNA binding specificities, interaction with negatively acting HLH proteins and post-translational modifications. This review focuses on what is emerging as another level of bHLH protein regulation, calcium regulation through interaction with Ca2+ loaded calmodulin and S-100 proteins. The mechanism and implications of these Ca2+ regulated interactions are discussed.
3105	Nonmyogenic factors bind nicotinic acetylcholine receptor promoter elements required for response to denervation.	9582305	Nicotinic acetylcholine receptors (AChRs) belong to a class of muscle proteins whose expression is regulated by muscle electrical activity. In innervated muscle fiber, AChR genes are transcriptionally repressed outside of the synapse, while after denervation they become reexpressed throughout the fiber. The myogenic determination factors (MDFs) of the MyoD family have been shown to play a central role in this innervation-dependent regulation. In the chicken AChR alpha-subunit gene promoter, two E-boxes that bind MDFs are necessary to achieve the enhancement of transcription following muscle denervation. However, the deletion of promoter sequences located upstream to these E-boxes greatly impairs the response to denervation (Bessereau, J. L., Stratford- Perricaudet, L. D., Piette, J., Le Poupon, C. and Changeux, J. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1304-1308). Here we identified two additional cis-regulatory elements of the alpha-subunit gene promoter that cooperate with the E-boxes in the denervation response. One region binds the Sp1 and Sp3 zinc finger transcription factors. The second region binds at least three distinct factors, among which we identified an upstream stimulatory factor, a b-ZIP-HLH transcription factor. We propose that among MDF-responsive muscle promoters, a specific combination between myogenic and nonmyogenic factors specify innervation-dependent versus innervation-independent promoters.
3106	Elevation of the serum Fas ligand in patients with hemophagocytic syndrome and Diamond-Blackfan anemia.	9531589	Fas ligand (FasL) is a membrane protein that is expressed in activated T cells and natural killer cells. FasL binds to Fas on target cells and induces apoptosis. There exists a soluble form of FasL (sFasL), and sFasL also induces apoptosis of Fas-bearing cells. The serum sFasL concentrations were reported to be elevated in patients with large granular lymphocytic leukemia and natural killer cell lymphoma. In this study, we have measured serum sFasL concentrations in other hematological disorders, including severe aplastic anemia (SAA), hemophagocytic lymphohistiocytosis (HLH), and Diamond-Blackfan anemia (DBA). The serum sFasL concentration of age-matched healthy controls was 0.16 +/- 0.11 ng/mL (mean +/- SD, n = 22). The serum sFasL levels in the patients with HLH and DBA were 3.75 +/- 3.82 (n = 19; P < .0001, HLH v control) and 2.76 +/- 2.43 ng/mL (n = 6; P = .012, DBA v control), respectively. Serum interferon-gamma concentration was elevated in the patients with HLH (1.61 +/- 2.62 ng/mL) but not in those with DBA (below the detectable level). These results suggest that the Fas-FasL system plays a role, at least in part, in the pathophysiology of HLH and DBA.
3107	Induction of basic helix-loop-helix protein-containing complexes during erythroid differentiation.	9572395	The involvement of basic helix-loop-helix (bHLH) transcription factors in erythroid differentiation and development has been established by forced expression of the proteins TAL1 and Id1 in cultured cell lines and by targeted disruption of the mouse TAL1 gene. To better understand the mechanism by which bHLH proteins regulate erythropoiesis, we have investigated HLH protein-DNA interactions in mouse erythroleukemia (MEL) cells before and during chemically induced differentiation. Three bHLH (E-box) binding activities were found to be induced in nuclei from differentiating MEL cells. Using specific antisera, we have demonstrated that these complexes are dimers of TAL1 and ubiquitous E proteins. Similar complexes were detected in nuclear extracts from a human erythroid cell line, K562, and from mouse fetal liver. All three bHLH complexes were disrupted in vitro by Id1, a dominant-negative HLH protein that we and others have previously shown to antagonize MEL cell differentiation. During differentiation of an Id1-overexpressing MEL cell line, induction of a complex containing TAL1 and E2A was not only blocked but reduced below the levels seen in undifferentiating cells. These observations are consistent with the idea that TAL1 and Id1 have opposing effects on erythroid differentiation and that the level of TAL1/E2A heterodimer and/or another E protein-containing complex may influence the decision of a cell to terminally differentiate.
3108	Structure, chromosomal locus, and promoter of mouse Hes2 gene, a homologue of Drosophila hairy and Enhancer of split.	9570950	Hes2 encodes a mammalian basic helix-loop-helix transcriptional repressor homologous to the products of Drosophila hairy and Enhancer of split. Here, we isolated and characterized the mouse Hes2 gene. This gene consists of four exons, and all the introns are located within the protein-coding region at positions homologous to those of other Hes genes. On the inter-specific backcross analyses, mouse Hes2 is mapped to the distal region of Chromosome 4 near the Hes3 and Hes5 loci, which are different from the Hes1 locus on Chromosome 16. Upstream of the transcription initiation site, there are GC-rich regions, but a typical TATA box is not present. Transient transfection analyses demonstrated that, while Hes1 and Hes5 promoter activities are significantly upregulated by the active form of Notch, a key regulator of cellular differentiation, Hes2 and Hes3 promoter activities are not. These results suggest that Hes genes are functionally classified into two groups: those that are regulated by Notch and those that are not.
3109	Familial hemophagocytic lymphohistiocytosis. Primary hemophagocytic lymphohistiocytosis.	9561910	Hemophagocytic lymphohistiocytosis represents a spectrum of pathogenetically different diseases including the rapidly fatal autosomal recessive disease of familial hemophagocytic lymphohistiocytosis (FHL). The onset is usually during the first years of life with fever, cytopenia, and hepatosplenomegaly. Neurologic symptoms may supervene. Similar symptoms may occur in the infection-(virus-)associated or malignancy-associated hemophagocytic syndromes (IAHS/MAHS). Triggering infections can be found in all these diseases and do not allow for reliable differentiation. An international treatment protocol (HLH-94) has been developed for FHL, but immunomodulatory treatment may be justified in IAHS and MAHS as well, since they also have a high fatality rate.
3110	A role for Sp and helix-loop-helix transcription factors in the regulation of the human Id4 gene promoter activity.	9516472	Id family helix-loop-helix (HLH) proteins are involved in the regulation of proliferation and differentiation of several cell types. To identify cis- and trans-acting factors that regulate Id4 gene expression, we have analyzed the promoter regulatory sequences of the human Id4 gene in transient transfections and gel mobility shift assays. We have identified two functional elements, both located downstream from the TATA motif, that control Id4 promoter activity. One element contains a consensus E-box, and we demonstrated that the protein complex binding to the E-box contains the bHLH-zip upstream stimulatory factor (USF) transcription factor. Enforced expression of USF1 leads to E-box-mediated stimulation of promoter activity. The E-box also mediated stimulatory effects of several bHLH transcription factors, and co-expression of Id4 blocked the stimulatory effect mediated by the bHLH factors. A second element is a GA motif, located downstream from the transcriptional start sites, mutation of which resulted in a 20-fold increase in transcriptional activity. Gel-shift analysis and transfections into Drosophila Schneider SL2 cells showed that the repressor element is recognized by both Sp1 and Sp3 factors. These data suggest that Id4 transcription control is highly complex, involving both negative and positive regulatory elements, including a novel inhibitory function exerted by Sp1 and Sp3 transcription factors.
3111	Mmip1: a novel leucine zipper protein that reverses the suppressive effects of Mad family members on c-myc.	9528857	C-myc, a member of the basic helix-loop-helix-leucine zipper (bHLH-ZIP) protein family activates target genes in heterodimeric association with another bHLH-ZIP protein, Max. Max readily homodimerizes, competes with C-myc-Max heterodimers, and represses transcription. Four additional bHLH-ZIP proteins, Mad1, Mxi1, Mad3 and Mad4, heterodimerize with Max and also repress transcription of c-myc-responsive genes. We employed a yeast two-hybid approach to identify proteins which interact with Mxi. We identified a novel ZIP-containing protein, Mmip1 (Mad member-interacting protein 1) that strongly dimerizes with all four Mad members, but not with c-myc, Max, or with unrelated HLH proteins. The Mmip1-Mxi association is mediated by the ZIP domain of each polypeptide and is as strong or stronger than the associations between c-myc and Max or Max and Mxi1. In vitro, Mmip1 can inhibit DNA binding by Max-Mad heterodimers and, in vivo, can reverse the suppressive effects of Mad proteins on c-myc functions. Mmipl is found in a variety of cells types, is induced by serum stimulation, and can be co-immunoprecipitated from fibroblasts in association with Mxi1. By interfering with the dimerization between Max and Mad family member proteins, Mmip1 can indirectly up-regulate the transcriptional activity of c-myc and suppress the antiproliferative actions of Mad proteins.
3112	Age-resolving osteopetrosis: a rat model implicating microphthalmia and the related transcription factor TFE3.	9480987	Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.
3113	XCoe2, a transcription factor of the Col/Olf-1/EBF family involved in the specification of primary neurons in Xenopus.	9501982	Primary neurogenesis in Xenopus is a model for studying the control of neural cell fate decisions. The specification of primary neurons appears to be driven by transcription factors containing a basic region and a helix-loop-helix (HLH) motif: expression of Xenopus neurogenin-related-1 (X-ngnr-1) defines the three prospective domains of primary neurogenesis, and expression of XNeuroD coincides with neuronal differentiation. The transition between neuronal competence and stable commitment to a neuronal fate remains poorly characterised, however.Drosophila Collier and rodent early B-cell factor/olfactory-1 define a family of HLH transcription factors containing a previously unknown type of DNA-binding domain. We isolated an orthologous gene from Xenopus, Xcoe2, which is expressed in precursors of primary neurons. Xcoe2 is transcribed after X-ngnr-1 and before XNeuroD. Overexpression of a dominant-negative mutant of XCoe2 prevented neuronal differentiation. Conversely, overexpressed wild-type Xcoe2 could promote ectopic differentiation of neurons, in both the neural plate and the epidermis. In contrast to studies with X-ngnr-1 or XNeuroD, the supernumerary neurons induced by Xcoe2 appeared in a 'salt-and-pepper' pattern, resulting from the activation of X-Delta1 expression and feedback regulation by lateral inhibition.XCoe2 may play a pivotal role in the transcriptional cascade that specifies primary neurons in Xenopus embryos: by maintaining Delta-Notch signalling, XCoe2 stabilises the higher neural potential of selected progenitor cells that express X-ngnr-1, ensuring the transition between neural competence and irreversible commitment to a neural fate; and it promotes neuronal differentiation by activating XNeuroD expression, directly or indirectly.
3114	Mitfmi-enu122 is a missense mutation in the HLH dimerization domain.	9501313	NaN
3115	Detection of the Der (21)t(12;21) chromosome forming the TEL-AML1 fusion gene in childhood acute lymphoblastic leukemia.	9498702	The t(12;21) (p13;q22) is observed in approximately 20-25% of childhood B-lineage acute lymphoblastic leukemia (ALL) cases in both Asian and Caucasian populations. This translocation results in the fusion of TEL, a recently described ETS-like gene on 12p13, and AML1, which was shown to be involved in the formation of fusion genes with ETO and EVI1 in myeloid leukemias. Fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis are useful in detecting this translocation which is not readily identified with routine cytogenetic techniques. The t(12;21) is associated with a distinct subgroup of patients characterized by an age between 1 and 10 years, an early B immunophenotype, and a good prognosis. A high incidence of the deletion of non-translocated TEL is another characteristic of leukemic cells with this translocation. TEL-AML1 hybrid protein thought to be critical in leukemogenesis possesses the HLH domain of TEL fused to almost the entire AML1 protein, although the detailed mechanisms of leukemogenesis remain obscure. RT-PCR combined with FISH analysis of posttreatment samples appears to be useful in detecting early relapse or minimal residual disease and thus, is expected to optimize the treatment strategy for patients with t(12;21).
3116	Fluorescence in situ hybridization characterization of new translocations involving TEL (ETV6) in a wide spectrum of hematologic malignancies.	9454771	The ETV6 (also known as TEL) gene on chromosome 12p13 is the target of a number of translocations associated with various hematologic malignancies. The contribution of ETV6 to leukemogenesis occurs through different mechanisms that involve either its helix-loop-helix dimerization domain or its E26 transformation-specific (ETS) DNA-binding domain. Using fluorescence in situ hybridization we characterized seven new ETV6 rearrangements in chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma. These aberrations, not always discernible at the cytogenetic level, include a t(5;12)(q31;p13), t(6;12;17)(p21;p13;q25), t(7;12)(p15;p13), t(7;12)(p12;p13), t(7;12)(q36;p13), t(12;13)(p13;q12), and a not completely defined t(12;?)(p13;?). Loss or disruption of the second ETV6 allele by a del(12)(p12p13) or by an intragenic ETV6 deletion was detected in two cases. In six cases the 12p13 breakpoint occurred in the 5' end of ETV6, upstream to exons encoding the HLH domain, whereas the remaining case had a breakpoint between the exons coding for the HLH domain and the exons coding for the ETS domain of ETV6. These observations provide further evidence for the multiple contributions of ETV6 in the pathogenesis of a wide range of hematologic malignancies.
3117	A basic-helix-loop-helix protein expressed in precursors of Drosophila longitudinal visceral muscles.	9486535	Basic-helix-loop-helix (bHLH) transcription factors are involved in the control of many developmental processes in vertebrates and invertebrates. The HLH domain mediates formation of homo- or heterodimers. We have taken advantage of these dimerisation properties to identify a novel Drosophila HLH protein using the yeast two-hybrid system. Expression of bHLH54F at the blastoderm stage is restricted to a small subpopulation of mesodermal cells near the posterior pole. During germ band retraction these cells spread along the future midgut region. Later bHLH54F-expressing cells make up the longitudinal portion of the visceral musculature. Characterisation of this expression pattern demonstrates that precursors of the outer, longitudinal muscles of the midgut are distinct in origin and morphology from precursors of the inner, circular muscles.
3118	Identification of a highly conserved module in E proteins required for in vivo helix-loop-helix dimerization.	9446597	Basic helix-loop-helix (bHLH) transcription factors often function as heterodimeric complexes consisting of a tissue-specific factor such as SCL/tal or MyoD bound to a broadly expressed E protein. bHLH dimerization therefore appears to represent a key regulatory step in cell lineage determination and oncogenesis. Previous functional and structural studies have indicated that the well defined HLH domain is both necessary and sufficient for dimerization. Most of these studies, however, have employed in vitro systems for analysis of HLH dimerization, and their implications for the requirements for in vivo dimerization remain unclear. Using multiple approaches, we have analyzed bHLH dimerization in intact, living cells and have identified a novel domain in E proteins, domain C, which is required for in vivo dimerization. Domain C, which lies just carboxyl-terminal to helix 2 of the HLH domain, represents the most highly conserved region within E proteins and appears to influence the in vivo conformation of the adjacent HLH domain. These results suggest that HLH dimerization in vivo may represent a complex, regulated process that is distinct from HLH dimerization in vitro.
3119	A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma.	9462753	Congenital (or infantile) fibrosarcoma (CFS) is a malignant tumour of fibroblasts that occurs in patients aged two years or younger. CFS is unique among human sarcomas in that it has an excellent prognosis and very low metastatic rate. CFS is histologically identical to adult-type fibrosarcoma (ATFS); however, ATFS is an aggressive malignancy of adults and older children that has a poor prognosis. We report a novel recurrent t(12;15)(p13;q25) rearrangement in CFS that may underlie the distinctive biological properties of this tumour. By cloning the chromosome breakpoints, we show that the rearrangement fuses the ETV6 (also known as TEL) gene from 12p13 with the 15q25 NTRK3 neurotrophin-3 receptor gene (also known as TRKC). Analysis of mRNA revealed the expression of ETV6-NTRK3 chimaeric transcripts in all three CFS tumours analysed. These were not detected in ATFS or infantile fibromatosis (IFB), a histologically similar but benign fibroblastic proliferation occurring in the same age-group as CFS. ETV6-NTRK3 fusion transcripts encode the helix-loop-helix (HLH) protein dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. Our studies indicate that a chimaeric PTK is expressed in CFS and this may contribute to oncogenesis by dysregulation of NTRK3 signal transduction pathways. Moreover, ETV6-NTRK3 gene fusions provide a potential diagnostic marker for CFS.
3120	The MXI1 tumor suppressor gene is not mutated in primary prostate cancer.	9458379	For prostate cancer, allelic deletions from the long arm of chromosome 10 (#10q23-25), the locus of the putative tumor suppressor gene MXI1 (#10q24-25), have been identified as a frequently occurring genetic event. During the development of several human malignancies, the c-myc proto-oncogene has been identified to enhance cellular transformation, mitogenesis and cell proliferation. The MXI1 gene, belonging to the helix-loop-helix (bHLH) gene family, was demonstrated to display tumor suppressor function by antagonizing c-myc induced transcriptional activities. Due to the detection of point mutations in the retained alleles of four primary adenocarcinomas of the prostate, MXI1 gene alterations have been suggested to be involved in the development and/or the progression of prostate cancer. To evaluate the role of MXI1 gene alterations for the development of adenocarcinoma of the prostate, 42 primary prostate cancers of different stage (T1-4) and histological grade (G1-3) were investigated for alterations within exons 4 and 5 of the MXI1 gene (spanning 6 exons in total), encoding for the functional HLH-Zip domain, by RNA-SSCP analysis and direct PCR-DNA-sequencing following the microscopically guided tumor cell dissection from 5 microm fresh-frozen buffer-soaked tissue sections. Even by application of this highly elaborated technical approach, MXI1 gene alterations could not be deleted in any of the tumor specimens investigated. Therefore, a substantial involvement of MXI1 gene alterations in the development of prostate cancer appears unlikely. The newly identified putative tumor suppressor gene PTEN, located at #10q23, might be responsible for the frequently observed allelic deletions from #10q23-25 in prostate cancer.
3121	Characterization of human lutropin carboxyl-terminus isoforms.	9449621	Human lutropin (hLH) exhibits both carbohydrate and peptidic heterogeneities that affect its biological potency and the duration of its activity in vivo. Peptidic changes within the hLH beta-subunit are characterized as intrachain proteolytic nicking and carboxyl terminus heterogeneity. To date, the carboxyl terminus of hLHbeta appears to end at either position Gln114 or Gly117, as determined by sequencing of purified subunit. Furthermore, the complementary DNA for hLHbeta predicts a protein containing an additional peptidic stretch, which would make the beta-subunit 121 residues long. This extension may be responsible for the particular intracellular behavior of hLHbeta. To investigate the carboxyl terminus polymorphism of natural hLHbeta, monoclonal antipeptide antibodies were raised against a synthetic peptide mimicking the 104-119 portion of hLHbeta. One antibody, designated LHP09, was found to specifically react with the recombinant hLHbeta ending at position hLHbeta[Leu119] but not with other recombinant forms ending at [Ser116], [Phe120] or [Leu121]. Immunochemical analysis of hLH, either pituitary or urinary in origin, indicated that only pituitary hLH contains a Leu119-ending form of hLHbeta. Finally, immunohistochemical detection was performed using LHP09 and showed specific staining of a normal adult pituitary gland. These observations support the in vivo existence of intrapituitary molecular forms of hLHbeta ending at various positions between Gln114 and Leu121.
3122	Gonadotropic control of secretion of dimeric inhibins and activin A by human granulosa-luteal cells in vitro.	9447456	It is well established that human granulosa cells and luteal cells express inhibin/activin subunit protein and secrete immunoreactive inhibin. The gonadotropic control of secretion of different molecular forms of inhibin and activin A by granulosa-luteal cells (G-LCs) was investigated using recently developed specific enzyme immunoassays (EIAs).Granulosa-luteal cells obtained at IVF egg pickup were cultured in a serum-free medium at 37 degrees C in a water-saturated incubator with 5% CO2 for up to 5 days. Experiments with varying concentrations of human FSH, hLH, and hCG were carried out.FSH raised the secretion of inhibin A and pro-alpha C-containing inhibins after 24 and 48 hr in culture. Inhibin B was raised after 24 hr and activin A was raised after 48 hr of FSH treatment. LH treatment for 24 hr stimulated inhibin A, inhibin B, pro-alpha C, and activin A. hCG stimulated G-LC secretion of inhibin A after 48 hr and pro-alpha C after 24 hr. Paradoxically, inhibin B secretion was inhibited by 1 and 10 ng/ml hCG after 48 hr. Activin A was stimulated by hCG after 24 and 48 hr of incubation. G-LC secretion of estradiol and progesterone was also stimulated significantly by LH and hCG.Secretion of dimeric inhibins and activin A is controlled differentially by gonadotropins.
3123	The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression.	9428421	During the development of the Drosophila adult peripheral nervous system (PNS), inhibitory cell-cell interactions mediated by the Notch receptor are essential for proper specification of sensory organ cell fates. We have reported previously (M. W. Leviten, E. C. Lai and J. W. Posakony (1997) Development 124, 4039-4051) that the 3' untranslated regions (UTRs) of many genes involved in Notch signalling, including Bearded (Brd) and the genes of the Enhancer of split Complex (E(spl)-C), contain (often in multiple copies) two novel heptanucleotide sequence motifs, the Brd box (AGCTTTA) and the GY box (GTCTTCC). Moreover, the molecular lesion associated with a strong gain-of-function mutant of Brd suggested that the loss of these sequence elements from its 3' UTR might be responsible for the hyperactivity of the mutant gene. We show here that the wild-type Brd 3' UTR confers negative regulatory activity on heterologous transcripts in vivo and that this activity requires its three Brd box elements and, to a lesser extent, its GY box. We find that Brd box-mediated regulation decreases both transcript and protein levels, and our results suggest that deadenylation or inhibition of polyadenylation is a component of this regulation. Though Brd and the E(spl)-C genes are expressed in spatially restricted patterns in both embryos and imaginal discs, we find that the regulatory activity that functions through the Brd box is both temporally and spatially general. A Brd genomic DNA transgene with specific mutations in its Brd and GY boxes exhibits hypermorphic activity that results in characteristic defects in PNS development, demonstrating that Brd is normally regulated by these motifs. Finally, we show that Brd boxes and GY boxes in the E(spl)m4 gene are specifically conserved between two distantly related Drosophila species, strongly suggesting that E(spl)-C genes are regulated by these elements as well.
3124	The metalloprotease-disintegrin Kuzbanian participates in Notch activation during growth and patterning of Drosophila imaginal discs.	9428413	The Notch transmembrane protein is the receptor of an evolutionary conserved pathway that mediates intercellular signalling leading to the specification of different cell types during development. In this pathway, many aspects of the signal transduction mechanism remain poorly understood, especially the role of proteolytic processing of Notch. We present genetic evidence indicating that the metalloprotease-disintegrin kuzbanian (J. Rooke, D. Pan, T. Xu and G. M. Rubin (1996) Science 273, 1227-1231) is a new component of the Notch signalling pathway and is involved in Notch activation. kuzbanian genetic mosaics demonstrate that, during neurogenesis, wing margin formation and vein width specification kuzbanian is autonomously required in the cell where Notch is activated. Genetic interactions between kuzbanian and different genes of the Notch pathway indicate that kuzbanian is required upstream of Suppressor of Hairless. Moreover, the requirement of kuzbanian for signalling by a ligand-dependent Abruptex receptor, but not by a constitutively activated form of Notch, suggests that kuzbanian is involved in the generation of a Notch functional receptor and/or in its activation. However, differences in the phenotypes of loss-of-function Notch and kuzbanian mutations suggest the existence of alternative Kuzbanian-independent mechanisms that generate Notch functional receptors.
3125	Role of E boxes in the repression of E-cadherin expression.	9425291	Decreased expression of the intercellular adhesion molecule E-cadherin correlates with tumor aggressiveness and invasion capacity of cell lines. The decrease of E-cadherin expression results primarily from reduced mRNA expression. We show here that the activity of a human E-cadherin promoter construct is cell specific and correlates with E-cadherin mRNA expression. This spectrum of activity is conserved by a region as short as 81 bp obtained after 5' deletion. Mutation analysis revealed that two E box elements of this minimal promoter are involved in the silencing of E-cadherin promoter activity occurring in cancer cells. E boxes are mainly known as target sequences for bHLH transcription factors that are involved in the control of tissue differentiation and are antagonised by HLH proteins. However, mutation data and cotransfection experiments using HLH protein expression vectors indicate that bHLH transcription factors are not significantly involved in the E box mediated silencing of this E-cadherin promoter fragment.
3126	Helix-loop-helix proteins in Schwann cells: a study of regulation and subcellular localization of Ids, REB, and E12/47 during embryonic and postnatal development.	9418957	Although basic helix-loop-helix (bHLH) proteins play an important role in transcriptional control in many cell types, the role of HLH proteins in Schwann cells has yet to be assessed. In this study, we have analyzed the expression of the dominant negative HLH genes, Id1 to Id4 and the class A gene REB, during Schwann cell development. We found that mRNA derived from these genes was present in the Schwann cell lineage throughout development including embryonic precursors and mature cells. The mRNA levels were not significantly regulated during development. Nevertheless, by using antibodies against the four different Id proteins, we found clear regulation of some of these genes at the protein level, in particular Id 2, 4, and REB, both in amount and nuclear/cytoplasmic localization. All these proteins are found in the nuclei of Schwann cell precursors but are not seen in nuclei of Schwann cells of newborn nerves. We observed extensive overlap in Id expression, especially in Schwann cell precursors that co-expressed all four Id proteins and REB. We also showed that Id 1 and 2 were up-regulated as Schwann cells progressed through the cell cycle. These data indicate that HLH transcription factors act as regulators of Schwann cell development and point to the existence of as yet unidentified cell type-specific bHLH proteins in these cells.
3127	Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation.	9372912	The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E. Hara, M. Hall, and G. Peters, EMBO J. 16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation.
3128	Use of a two-hybrid system to identify mutations in Max that confer increased affinity for Myc.	9407000	A yeast two-hybrid system was used to identify mutants of Max that exhibit an increased affinity for Myc. Truncated forms of the Max helix-loop-helix/leucine zipper motif (HLH/Zip) were first expressed in a two- hybrid system in which the bait protein was the HLH/Zip motif of Myc. Deletion of amino acids both amino-terminal and carboxy-terminal to the leucine zipper of Max reduced Myc/Max heterodimer formation as evidenced by a 160-fold reduction in the expression of the lacZ gene. A library of partially randomized sequences encoding this minimal leucine zipper of Max was then screened using the two-hybrid system. Mutant forms of the Max leucine zipper were identified whose affinities for Myc, as measured by beta-galactosidase activity in yeast lysates, were from 8- to 200-fold greater than the wild-type Max zipper. These Max mutants were shown to interact specifically with Myc and not with wild-type Max. Of 29 mutants analyzed, all had a unique amino acid sequence. This result illustrates the value of a genetic screen in the identification of a collection of mutant forms of the Max leucine zipper whose structures would not have been predicted based on principles of structure-based design.
3129	Co-purification of a ribonuclease and human chorionic gonadotrophin beta-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases.	9406851	An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) beta-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG beta-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (< 0.001 microg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 microg/ml RNase). The co-purification of hCG beta-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.
3130	Dimerization and DNA binding facilitate alpha-helix formation of Max in solution.	9399572	Max is a basic region/helix-loop-helix/leucine zipper (b/HLH/Z) protein that forms a hetero-complex with the Myc family proteins Myc, Mad, and Mxi1, and a homo-complex with itself. These complexes specifically bind to double-stranded DNA containing CACGTG sequences. Here, we report on the structural properties in aqueous solution of a 109-amino-acid protein, Max110, corresponding to the N-terminal domain of Max containing the b/HLH/Z motif (residues 2-110), as characterized by combined use of circular dichroism (CD) and sedimentation equilibrium experiments. The results showed that the alpha-helical content of Max110 increases with increasing protein concentration. The sedimentation equilibrium data indicated that Max110 exists as a monomer at low protein concentration, and forms a dimer at high protein concentration. Further increases in the alpha-helical content of Max110 occur upon addition of DNA with the CACGTG recognition sequence. Thus, dimerization and binding to DNA of Max both favor an increase of the alpha-helical content.
3131	A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system.	9371806	Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.
3132	Helix-loop-helix factors in growth and differentiation of the vertebrate nervous system.	9388783	Neural development involves the initial growth phase of dividing precursor cells and the subsequent differentiation phase of postmitotic cells. Recent studies indicate that the transition from the former phase to the latter is controlled antagonistically by multiple helix-loop-helix (HLH) genes. Cascades of neuronal HLH genes promote differentiation whereas anti-neuronal HLH genes repress them under the control of Notch and keep cells at the precursor stage. This antagonistic regulation may be essential for generation of the proper number of neurons and for morphogenesis of the nervous system.
3133	The Drosophila gene Bearded encodes a novel small protein and shares 3' UTR sequence motifs with multiple Enhancer of split complex genes.	9374401	Gain-of-function alleles of the Drosophila gene Bearded (Brd) cause sensory organ multiplication and loss phenotypes indistinguishable at the cellular level from those caused by loss-of-function mutations in the genes of the Notch pathway (Leviten, M. W. and Posakony, J. W. (1996). Dev. Biol. 176, 264-283). We have carried out a molecular analysis of the structure and expression of both wild-type and mutant Brd transcription units. We find that the Brd transcript is truncated and accumulates to substantially higher levels in the gain-of-function mutants, due to the insertion of a transposable element of the blood family in the Brd 3' untranslated region (UTR). The wild-type Brd 3' UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that we refer to as the 'Brd box'. Moreover, the 3' UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif we refer to as the 'GY box' (GTCTTCC). We find that both the Brd box and the GY box are also present in the 3' UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and two of the Brd boxes present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them. Finally, we find that Brd encodes a novel small protein of only 81 amino acids that is predicted to include a basic amphipathic alpha-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic alpha-helix, suggesting that the two proteins have related biochemical functions.
3134	Helix-loop-helix motif in GnRH associated peptide is critical for negative regulation of prolactin secretion.	9368654	The GnRH associated prolactin inhibiting factor (GAP) reveals the signature sequence associated with the helix-loop-helix structural motif. A number of different peptide fragments of GAP were designed, synthesized and analysed by circular dichroism and by an in vivo assay for prolactin secretion inhibiting activity. Peptides corresponding to the two individual alpha-helices and a 44-residue peptide comprising the entire helix-loop-helix motif show significant helical propensity in circular dichroism spectra. However, a peptide corresponding to the loop sequence shows no helical propensity. Albeit, the peptide corresponding to helix-loop-helix motif was found to inhibit prolactin secretion and augment circulating levels of gonadotropins in the in vivo assay; other shorter peptides did not show such activity. The activity profile of the 44-residue peptide was biphasic and very similar to that of the recombinant GAP. Thus, the prolactin inhibiting activity of this factor is defined by its helix-loop-helix motif as in the case of the transcription factors of developmental genes. The structural features of a homology-based model of GAP in complex with E47, a ubiquitous HLH-type developmental gene regulator, are consistent with the structural requirements of the negative regulation of transcription by helix-loop-helix proteins.
3135	Transcriptional regulation of the mBMP-4 gene through an E-box in the 5'-flanking promoter region involving USF.	9367898	We analyzed the promoter activity of murine bone morphogenetic protein-4 (mBMP-4) and determined that the 5'-flanking region of exon I plays a critical role for transcription and position -265 to -246 of 5'-flanking region of mBMP-4 gene acts as a cis-element important for the regulation of BMP4 transcription in MC3T3E1 cells. By use of site-directed mutagenesis, we have established that the E-box, CACGTG, located within this short region of promoter, is essential for transcriptional activation of the mBMP-4 gene. Upstream stimulatory factor (USF), a member of the helix-loop-helix (HLH) group of regulatory proteins, was found to bind to this E-box using supershift gel mobility analysis. It is proposed that the HLH transcription regulatory proteins play an important role in mBMP-4 gene transcription in this osteoblastic cell line.
3136	A leech homolog of twist: evidence for its inheritance as a maternal mRNA.	9358036	In the development of leeches such as Helobdella robusta, mesodermal and ectodermal fates segregate to cells DM and DNOPQ, respectively, at fourth cleavage. As one step in identifying genes that may act in mesoderm determination, we have cloned the H. robusta homolog to the Drosophila gene twist. This homolog, designated Hro-twi, exhibits high (> 90%) amino acid identity with other twist-class genes within its basic-helix loop-helix (b-HLH) DNA binding motif and dimerization domain. Like twist, Hro-twi contains CAX-rich stretches: three stretches 5' to the b-HLH and one located 3' of the b-HLH motif. RT-PCR analysis suggests that Hro-twi is present throughout development, beginning as a maternal transcript in the oocyte.
3137	A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding.	9356439	We describe a method to design dominant-negative proteins (D-N) to the basic helix-loop-helix-leucine zipper (B-HLHZip) family of sequence-specific DNA binding transcription factors. The D-Ns specifically heterodimerize with the B-HLHZip dimerization domain of the transcription factors and abolish DNA binding in an equimolar competition. Thermal denaturation studies indicate that a heterodimer between a Myc B-HLHZip domain and a D-N consisting of a 12-amino acid sequence appended onto the Max dimerization domain (A-Max) is -6.3 kcal.mol-1 more stable than the Myc:Max heterodimer. One molar equivalent of A-Max can totally abolish the DNA binding activity of a Myc:Max heterodimer. This acidic extension also has been appended onto the dimerization domain of the B-HLHZip protein Mitf, a member of the transcription factor enhancer binding subfamily, to produce A-Mitf. The heterodimer between A-Mitf and the B-HLHZip domain of Mitf is -3.7 kcal.mol-1 more stable than the Mitf homodimer. Cell culture studies show that A-Mitf can inhibit Mitf-dependent transactivation both in acidic extension and in a dimerization-dependent manner. A-Max can inhibit Myc-dependent foci formation twice as well as the Max dimerization domain (HLHZip). This strategy of producing D-Ns may be applicable to other B-HLHZip or B-HLH proteins because it provides a method to inhibit the DNA binding of these transcription factors in a dimerization-specific manner.
3138	Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.	9348318	Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.
3139	Characterization of a putative helix-loop-helix motif in nucleotide excision repair endonuclease, XPG.	9346928	Complementation group G of xeroderma pigmentosum (XPG) is one of the most rare and pathophysiologically heterogeneous forms of this inherited disease. XPG patients exhibit varying phenotypes, from having a very mild defect in DNA repair to being severely affected, and a few cases are also associated with the neurological degeneracy and growth retardation of Cockayne's syndrome. The XPG gene encodes a 134-kDa nuclear protein that is essential for the incision steps of nucleotide excision repair. XPG protein contains a putative helix-loop-helix (HLH) motif in the region that is most conserved among the members of structure-specific endonuclease family. To establish the functional significance of the HLH motif, we used several approaches, including theoretical modeling, functional complementation assay, structure-specific endonuclease assay, and DNA binding assay. A secondary structure of the motif was predicted by energy minimization and the Monte Carlo simulation and empirically proven using the circular dichroism to contain a high content of alpha-helix. When an XPG mutant lacking the HLH was overexpressed in UV135 cells, which have defects in the hamster homolog of XPG, the mutant gene failed to confer to the hamster cells the resistance to UV light. A recombinant XPG protein lacking the HLH motif was purified from insect cells and tested for a structure-specific endonuclease activity. The mutant protein failed to cleave the flap strand. A recombinant peptide containing the HLH (amino acids 758-871) was expressed in and purified from bacteria, tested for DNA binding activity, and found to bind to a DNA substrate with the flap structure. These results suggest that the HLH motif is required for the catalytic and DNA binding activities of XPG.
3140	Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia.	9326218	Translocations in hematologic disease of myeloid or lymphoid origin with breakpoints at chromosome band 12p13 frequently result in rearrangements of the Ets variant gene 6 (ETV6). As a consequence either the ETS DNA-binding domain or the Helix-Loop-Helix (HLH) oligomerization domain of ETV6 is fused to different partner genes. We show here that a t(9;12)(p24;p13) in a case of early pre-B acute lymphoid leukemia and a t(9;15;12)(p24;q15;p13) in atypical chronic myelogenous leukemia in transformation involve the ETV6 gene at 12p13 and the JAK2 gene at 9p24. In each case different fusion mRNAs were found, with only one resulting in an open reading frame for a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the protein tyrosine kinase (PTK) domain of JAK2. The cloning of the complete human JAK2 coding and genomic sequences and of the genomic junction fragments of the translocations allowed a characterization of the different splice events leading to the various mRNAs. JAK2 plays a central role in non-protein tyrosine kinase receptor signaling pathways, which could explain its involvement in malignancies of different hematologic lineages. Besides hop in Drosophila no member of the JAK family has yet been implicated in tumorigenesis.
3141	The basic domain of myogenic basic helix-loop-helix (bHLH) proteins is the novel target for direct inhibition by another bHLH protein, Twist.	9343420	In vertebrates, the basic helix-loop-helix (bHLH) protein Twist may be involved in the negative regulation of cellular determination and in the differentiation of several lineages, including myogenesis, osteogenesis, and neurogenesis. Although it has been shown that mouse twist (M-Twist) (i) sequesters E proteins, thus preventing formation of myogenic E protein-MyoD complexes and (ii) inhibits the MEF2 transcription factor, a cofactor of myogenic bHLH proteins, overexpression of E proteins and MEF2 failed to rescue the inhibitory effects of M-Twist on MyoD. We report here that M-Twist physically interacts with the myogenic bHLH proteins in vitro and in vivo and that this interaction is required for the inhibition of MyoD by M-Twist. In contrast to the conventional HLH-HLH domain interaction formed in the MyoD/E12 heterodimer, this novel type of interaction uses the basic domains of the two proteins. While the MyoD HLH domain without the basic domain failed to interact with M-Twist, a MyoD peptide containing only the basic and helix 1 regions was sufficient to interact with M-Twist, suggesting that the basic domain contacts M-Twist. The replacement of three arginine residues by alanines in the M-Twist basic domain was sufficient to abolish both the binding and inhibition of MyoD by M-Twist, while the domain retained other M-Twist functions such as heterodimerization with an E protein and inhibition of MEF2 transactivation. These findings demonstrate that M-Twist interacts with MyoD through the basic domains, thereby inhibiting MyoD.
3142	Misleading results with Opus Magnum hLH assay.	9342035	NaN
3143	Synergy between suppressor of Hairless and Notch in regulation of Enhancer of split m gamma and m delta expression.	9271437	The Notch signaling pathway is known to regulate cell fate decisions in a variety of organisms from worms to humans. Although several components of the pathway have been characterized, the actual mechanism and molecular results of signaling remain elusive. We have examined the role of the Notch signaling pathway in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes, whose gene products have been shown to be downstream players in the pathway. Using a reporter assay system in Drosophila tissue culture cells, we have observed a significant induction of E(spl) m gamma and m delta expression after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. We show that Notch-inducible expression of E(spl) m gamma and m delta both in cultured cells and in vivo is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter.
3144	The murine Sim-2 gene product inhibits transcription by active repression and functional interference.	9271372	The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription.
3145	Cloning and developmental expression of Xenopus cDNAs encoding the Enhancer of split groucho and related proteins.	9300818	The two full-length cDNAs encoding ESG1 (Enhancer of split groucho) and related AES (Amino Enhancer of split) proteins of 767 and 197 amino acids, respectively, were cloned and sequenced from the African frog Xenopus laevis. The amino acid sequence of Xenopus ESG1 protein had 61% identity to the full-length Drosophila groucho. Xenopus AES protein exhibited 91%, 58% and 48% identity to the mouse AES, amino-terminal regions of Xenopus ESG1 and Drosophila groucho, respectively. Northern blot analysis showed that widespread RNA expression of ESG1 of 2.8 kb, ESG2 of 3.6 kb and AES of 2.2 kb transcripts were seen in adult tissues, whereas ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed in the developing embryos. The overall structural relationships of ESG and AES proteins among human, mouse, rat, Xenopus and Drosophila were analysed.
3146	Identification in a fish species of two Id (inhibitor of DNA binding/differentiation)-related helix-loop-helix factors expressed in the slow oxidative muscle fibers.	9288909	Helix-loop-helix (HLH) proteins related to the inhibitor of DNA binding/differentiation (Id) serve as general antagonists of cell differentiation. They lack a basic DNA-binding domain and are thought to function in a dominant negative manner by sequestering basic HLH (bHLH) transcription factors that are involved in cell determination and differentiation. Four Id-encoding genes have been shown in mammals, they have a distinct pattern of expression suggesting different functions for each member in different cell lineage. In this study we describe the identification and cloning of two trout cDNAs which encode helix-loop-helix proteins showing a high degree of similarity with mammalian Id family members. One cDNA encodes a trout putative Id1 protein (TId1) that is 63% identical to the human Id1 protein over the entire length and 78% identical within the HLH region. The other cDNA encodes a trout putative Id2 protein (TId2) that shows 82% identity to the human Id2 protein and only one change that is conservative over the HLH region. In the 3' untranslated region, TId2 mRNA exhibits 16 nucleotides upstream from the AATAAA site, a palindromic sequence similar to the cytoplasmic polyadenylation element (CPE) which is also present in Id2 and Id3 mRNAs from mammals and in XIdx/XIdI mRNA from Xenopus. In the fish, TId1 and TId2 are expressed in a tissue-specific manner, with slightly different patterns. During myogenesis, TId1 and TId2 are highly expressed in the myotomal musculature of fish embryos and of early alevins but are down-regulated in that of late alevins. In muscle from juveniles and adults, TId1 and TId2 transcripts are abundant in the slow oxidative fibers while they are absent in the fast glycolytic fibers. This expression pattern suggests that Id genes play a role in the regulation of muscle fiber phenotype in addition to controlling early myogenesis. On the whole, the identification of two HLH-Id encoding genes in a major taxonomic group like teleosts, suggests an early divergence of Id genes in vertebrate evolution. The observation that Id transcripts are present selectively in the slow muscle reveals that their expression is more complicated than previously appreciated.
3147	Differential diagnosis of hemophagocytic syndrome: underlying disorders and selection of the most effective treatment.	9277044	Hemophagocytic syndrome consists of primary and secondary HLH. Efficacy of therapeutic measures and prognosis depend on degree of hypercytokinemia-associated organ failure at disease onset and underlying disorders. The underlying diseases related to hemophagocytosis and informative markers useful for differential diagnoses to select the most effective treatment are discussed. Differential diagnosis is difficult between confirmed FEL and familiality-unknown infantile VAHS (or sporadic FEL cases) in primary HLH and also among IAHS, benign EB-VAHS and EBV-related LAHS in secondary HLH. For primary HLH, assay of NK activity and for secondary HLH, studies on the serum cytokine pattern, EBV genomes and clonality determination might prove useful.
3148	TEL is one of the targets for deletion on 12p in many cases of childhood B-lineage acute lymphoblastic leukemia.	9264373	Abnormalities of the short arm of chromosome 12 including loss of heterozygosity (LOH) and TEL/AML-1 fusion resulting from a t(12;21)(p13;q22) translocation are frequently observed in childhood acute lymphoblastic leukemia (ALL). We investigated 21 DNA samples of childhood ALL which had LOH at 12p13. Rearrangement of TEL was observed in eight cases and another case showed a homozygous deletion of TEL. Two informative samples with TEL rearrangement had a deletion localized to the 5' region of this gene. The deletion in these two cases includes the helix-loop-helix (HLH) domain. This is consistent with the hypothesis that the normal tel can heterodimerize with the TEL/AML-1 gene product and inhibit the transforming capacity of the chimeric protein. Presumably, loss of the HLH of the normal remaining TEL allele abrogates this tumor suppressor-like function. The case with homozygous deletion of TEL is also consistent with this gene having qualities of a tumor suppressor. One unusual case had T-ALL rather than B-lineage ALL and the leukemic cells had rearrangement of TEL, but they did not have an alteration of the remaining TEL allele suggesting that the etiology of this disease may be different. This analysis further emphasizes the importance of loss of the normal TEL allele in childhood precursor B-lineage ALL.
3149	The role of lin-22, a hairy/enhancer of split homolog, in patterning the peripheral nervous system of C. elegans.	9247331	In C. elegans, six lateral epidermal stem cells, the seam cells V1-V6, are located in a row along the anterior-posterior (A/P) body axis. Anterior seam cells (V1-V4) undergo a fairly simple sequence of stem cell divisions and generate only epidermal cells. Posterior seam cells (V5 and V6) undergo a more complicated sequence of cell divisions that include additional rounds of stem cell proliferation and the production of neural as well as epidermal cells. In the wild type, activity of the gene lin-22 allows V1-V4 to generate their normal epidermal lineages rather than V5-like lineages. lin-22 activity is also required to prevent additional neurons from being produced by one branch of the V5 lineage. We find that the lin-22 gene exhibits homology to the Drosophila gene hairy, and that lin-22 activity represses neural development within the V5 lineage by blocking expression of the posterior-specific Hox gene mab-5 in specific cells. In addition, in order to prevent anterior V cells from generating V5-like lineages, wild-type lin-22 gene activity must inhibit (directly or indirectly) at least five downstream regulatory gene activities. In anterior body regions, lin-22(+) inhibits expression of the Hox gene mab-5. It also inhibits the activity of the achaete-scute homolog lin-32 and an unidentified gene that we postulate regulates stem cell division. Each of these three genes is required for the expression of a different piece of the ectopic V5-like lineages generated in lin-22 mutants. In addition, lin-22 activity prevents two other Hox genes, lin-39 and egl-5, from acquiring new activities within their normal domains of function along the A/P body axis. Some, but not all, of the patterning activities of lin-22 in C. elegans resemble those of hairy in Drosophila.
3150	A chimeric enhancer-of-split transcriptional activator drives neural development and achaete-scute expression.	9234693	Drosophila melanogaster neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Here, we test if E(spl) proteins function as promoter-bound repressors by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. Our results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway.
3151	Binding of upstream stimulatory factor and a cell-specific activator to the calcitonin/calcitonin gene-related peptide enhancer.	9218472	The calcitonin/calcitonin gene-related peptide (CT/CGRP) gene is selectively transcribed in thyroid C cells and neurons. We have previously shown that the rat CT/CGRP cell-specific enhancer is synergistically regulated by a helix-loop-helix (HLH) protein and the OB2 octamer-binding protein. In this report, we show that the HLH-OB2 enhancer is required for full promoter activity, even in the context of other HLH elements. Since this enhancer appears to be a major controlling element, we have characterized the HLH and OB2 DNA binding proteins. We have identified the major HLH complex as a heterodimer of the ubiquitous upstream stimulatory factor (USF)-1 and USF-2 proteins. USF bound the enhancer with a reasonably high affinity (KD 1.6 nM), comparable to other genes. Characterization of a series of mutations revealed that a portion of the HLH motif is also recognized by OB2 and confirmed that HLH activity requires OB2. We have shown that OB2 is a single DNA binding protein based on UV cross-linking studies. The 68-kDa protein-DNA complex was detected only in C cell lines, including a human C cell line that has robust HLH-OB2 enhancer activity. These results suggest that the calcitonin/CGRP gene is controlled by the combinatorial activity of a ubiquitous USF HLH heterodimer and an associated cell-specific activator.
3152	Structure/calcium affinity relationships of site III of calmodulin: testing the acid pair hypothesis using calmodulin mutants.	9214312	Calmodulin mutants in which the calcium binding affinity of site IV was greatly reduced by a D133E mutation were prepared using site-specific, cassette-mediated mutagenesis as a multisite calcium binding protein model to examine structure/calcium affinity relationships in site III of calmodulin. Tryptophan was introduced in position 92 of the calmodulin mutants as a fluorescent label to monitor the calcium-induced structural changes in the C-terminal domain of calmodulin. The five calmodulin mutants, 3xCaM, 3zCaM, 4xCaM, 4zCaM, and 4xzCaM, were designed so that there were three or four acidic amino acid residues in chelating positions of site III with acid pairs on either the X and/or Z coordinating axes. The calcium dissociation constant of site III, KIII, of the five calmodulin mutants changes in a descending order from 3xCaM (237 microM), 3zCaM (140 microM), 4xCaM (5.8 microM), 4zCaM (3 microM), to 4xzCaM (2 microM), and these KIII values are significantly lower than that of F92W/D133E calmodulin (335 microM) in which three acidic residues with no acid pairs were present in site III [Wu, X., & Reid, R. E. (1997) Biochemistry 36, 3608-3616]. These results indicate that the calcium affinity of site III increases when the number of the acidic chelating residues increases from three to four, when the number of acid pairs increases from zero to one and further to two, and when the location of the acid pair is changed from the X axis to the Z axis. This study provides the first evidence that the acid pair hypothesis which correlates the nature of the chelating residues with the calcium affinity of the hlh motif is applicable to a multisite calcium binding protein model. The Hill coefficients indicate that reversal of the sequence of filling of the calcium binding sites in the C-terminal domain from IV --> III to III --> IV also changes the site cooperativity from positive to negative. The cooperativity returns to positive when the proteins are titrated in the presence of a calmodulin-binding peptide. Data from the present study also demonstrate that calmodulin mutants with a decreased calcium affinity have a reduced efficiency in phosphodiesterase regulation at low calcium concentrations (50 microM). However, high calcium concentrations (15 mM) restore the phosphodiesterase regulatory activity of the calmodulin mutants to a level obtained with F92W calmodulin, indicating that the mutations alter calcium regulation of calmodulin-mediated phosphodiesterase activity without affecting the interaction between calmodulin and the enzyme.
3153	Translocation breakpoint maps 5 kb 3' from TWIST in a patient affected with Saethre-Chotzen syndrome.	9215678	Saethre-Chotzen syndrome, a common autosomal dominant craniosynostosis in humans, is characterized by brachydactyly, soft tissue syndactyly and facial dysmorphism including ptosis, facial asymmetry, and prominent ear crura. Previously, we identified a yeast artificial chromosome that encompassed the breakpoint of an apparently balanced t(6;7) (q16.2;p15.3) translocation associated with a mild form of Saethre-Chotzen syndrome. We now describe, at the DNA sequence level, the region on chromosome 7 affected by this translocation event. The rearrangement occurred approximately 5 kb 3' of the human TWIST locus and deleted 518 bp of chromosome 7. The TWIST gene codes for a transcription factor containing a basic helix-loop-helix (b-HLH) motif and has recently been described as a candidate gene for Saethre-Chotzen syndrome, based on the detection of mutations within the coding region. Potential exon sequences flanking the chromosome 7 translocation breakpoint did not hit known genes in database searches. The chromosome rearrangement downstream of TWIST is compatible with the notion that this is a Saethre-Chotzen syndrome gene and implies loss of function of one allele by a positional effect as a possible mechanism of mutation to evoke the syndrome.
3154	Uncoupling of p21 induction and MyoD activation results in the failure of irreversible cell cycle arrest in doxorubicin-treated myocytes.	9215525	Doxorubicin (Dox, Adriamicin), a potent broad spectrum anthracycline anticancer drug, selectively inhibits muscle specific gene expression in cardiac cells in vivo and prevents terminal differentiation of skeletal muscle cells in vitro. By inducing the expression of the helix-loop-helix (HLH) transcriptional inhibitor ld2, Dox represses the myogenic function of the MyoD family of muscle regulatory factors (MRFs). In many cell types, terminal differentiation is coupled to an irreversible exit from the cell cycle and MyoD plays a critical role in the permanent cell cycle arrest of differentiating myocytes by upregulating the cyclin dependent kinase inhibitor (cdki) p21. Here, we correlate Dox effects on cell cycle with changes of E2F/DP complexes and activity in differentiating C2C12 myocytes. In Dox-treated quiescent myoblasts, which fail to differentiate into myotubes under permissive culture conditions, serum re-stimulation induces cyclin/cdk re-association on the E2F/DP complexes and this correlates with an evident increase in E2F/DP driven transcription and re-entry of myoblasts into the cell cycle. Despite Dox ability to activate the DNA-damage dependent p53/p21 pathway, when induced in the absence of MyoD or other MRFs, p21 fails to maintain the postmitotic state in Dox-treated myocytes induced to differentiate. Thus, uncoupling p21 induction and MyoD activity results in a serum-reversible cell cycle arrest, indicating that MRF specific activation of cdki(s) is required for permanent cell cycle arrest in differentiating muscle cells.
3155	Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles.	9215284	Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 +/- 471 vs. 111 +/- 26 pg/mL, mean +/- SEM; P < 0.05). In vitro treatment with hCG increased (P < 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P < 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.
3156	Luteinizing hormone resistance syndromes.	9238276	Luteinizing hormone (LH) plays its effects on ovarian and testicular cells through binding to a specific cell surface receptor. We recently described two kindreds with LH resistance due to abnormalities of the LH receptor (LH-R) gene. Affected XY members presented with severe or mild fetal undermasculinization (female external genitalia or micropenis) and primary hypogonadism, while an XX affected member showed normal pubertal development, increased plasma concentrations of LH, and amenorrhea. The first kindred included three XY phenotypic female siblings with Leydig cell hypoplasia and primary hypogonadism and a fully developed XX sister with elevated plasma concentrations of LH and amenorrhea. PCR amplification of genomic DNA and direct sequencing of the entire exon 11 of the LH-R revealed that all four affected individuals had a homozygous mutation (Arg554-->Stop codon) in the third cytosolic loop of the LH-R, which resulted in a truncated LH-R unable to transduce the hormonal signal. The second kindred included a 6-year-old XY boy with a micropenis and bilaterally descended testes, who demonstrated no response to exogenous human chorionic gonadotrophin postnatally. This patient had a nonconservative homozygous amino acid substitution (Ser616-->Tyr616) in the seventh transmembrane domain of his LH-R gene that was inherited from his heterozygous parents. The mutant receptor expressed in heterologous cells in vitro demonstrated no appreciable binding of 125I-labeled hLH, nor did it confer cAMP responsiveness to LH. Homozygous inactivating mutations of the LH-R cause complete or mild testicular failure in genetic males, resulting in female external genitalia or micropenis and primary hypogonadism. Similar mutations in genetic females may cause failure of ovulation and corpus luteum formation, resulting in amenorrhea. Follicular growth and development are apparently sufficient to allow feminization at puberty.
3157	Functional dissection of the Drosophila enhancer of split protein, a suppressor of neurogenesis.	9177203	The Enhancer of split [E(spl)] gene complex of Drosophila comprises seven related genes encoding a special type of basic helix-loop-helix proteins, the function of which is to suppress the neural developmental fate. One of these proteins is E(spl) itself. To gain insight into the structural requirements for E(spl) function, we have expressed a large number of deletion variants in transgenic flies. Three protein domains were identified as essential for suppression of bristle development: the carboxyl-terminal tetrapeptide WRPW, the region comprising the putative helix III and helix IV, and the region between helix IV and the WRPW motif. Lack of the basic helix-loop-helix domain, helix III or IV, only partially inhibits the suppressor activity of the protein. Truncated variants that lack all the regions carboxyl-terminal to helix IV elicit the development of additional neural progenitors, and thus act as dominant-negative variants. All these results suggest that E(spl) suppresses neural development by direct interaction with other proteins, such as groucho and the proneural proteins.
3158	Non-familial haemophagocytic lymphohistiocytosis in children.	10968072	Haemophagocytic Lymphohistiocytosis (HLH) is a rare clinical illness with a high mortality. There are reported effective treatment and a favourable outcome if diagnosed early. Five cases of childhood non-familial HLH seen over a 3 year period in our hospital are presented. The diagnosis was not suspected in the referring hospitals even after a bone marrow biopsy examination in two cases. Delay in referral was between 2 weeks to 2 months. A viral trigger was detected in only two cases. There were two deaths. Cause of death in both cases were cytomegalovirus pneumonitis and disseminated intravascular coagulopathy. Respond to treatment was better if started earlier. One case spontaneously resolved. Earlier diagnosis will lead to prompt treatment and a better outcome.
3159	Expression and functional role of the Id HLH family in cultured astrocytes.	9191087	The Id family of helix-loop-helix factors (Id1, Id2, and Id3) expressed in many types of cells has been reported to negatively regulate myoblast differentiation and is required for G1/S progression of arrested fibroblasts. Our previous studies have indicated that Id1, Id2, and Id3 mRNA expression appear in the subventricular zone of 1-day-old rat brains. At later ages, Id3 mRNA was only expressed in astrocytes. We now report that Id1 and Id3 mRNA expression increased in astrocytes during the first hour of serum stimulation. Subsequently, the Id1 and Id3 mRNA levels gradually declined to basal level as observed in cultures without serum stimulation. However, there was no significant difference in Id2 mRNA expression between serum-treated and control astrocyte cultures within 1 h of serum induction. In addition, a strong nuclear immunostaining for Id2 and Id3 proteins was observed 24 h after serum stimulation. This observation is consistent with our results that show an increase in Id2 and Id3 protein levels following 24 h serum induction. Furthermore, DNA synthesis in FCS-stimulated astrocytes was blocked by antisense oligonucleotides against Id3 mRNA. The addition of Id3 antisense oligonucleotides caused approximately 50% reduction in Id3 mRNA and protein levels when compared to that in sense-treated cultures. The results indicate that the inhibition of DNA synthesis in FCS-stimulated astrocytes is due to a decrease in Id3 levels by the antisense. These observations suggest that Id3 may play an important role in the regulation of astrocyte proliferation.
3160	A C. elegans E/Daughterless bHLH protein marks neuronal but not striated muscle development.	9187144	The E proteins of mammals, and the related Daughterless (DA) protein of Drosophila, are ubiquitously expressed helix-loop-helix (HLH) transcription factors that play a role in many developmental processes. We report here the characterization of a related C. elegans protein, CeE/DA, which has a dynamic and restricted distribution during development. CeE/DA is present embryonically in neuronal precursors, some of which are marked by promoter activity of a newly described Achaete-scute-like gene hlh-3. In contrast, we have been unable to detect CeE/DA in CeMyoD-positive striated muscle cells. In vitro gel mobility shift analysis detects dimerization of CeE/DA with HLH-3 while efficient interaction of CeE/DA with CeMyoD is not seen. These studies suggest multiple roles for CeE/DA in C. elegans development and provide evidence that both common and alternative strategies have evolved for the use of related HLH proteins in controlling cell fates in different species.
3161	Maid: a maternally transcribed novel gene encoding a potential negative regulator of bHLH proteins in the mouse egg and zygote.	9186056	We isolated an abundant novel cDNA SSEC-8 from a subtraction cDNA library enriched for maternal transcripts that are still present in the mouse 2 cell stage embryo. This gene is evolutionarily conserved and maps to the distal region of mouse chromosome 2. The deduced polypeptide sequence of the encoded protein contains a conserved helix-loop-helix (HLH) motif without a basic DNA binding domain, suggesting that it functions as a negative regulator of basic (b) HLH transcription factors. Gel mobility shift assays show that in vitro translated protein prevents the E12/MyoD bHLH dimer from binding to DNA. Also, transient overexpression of this protein in C2C12 cells reduced the transcription of a CAT-reporter regulated by an E12/MyoD driven enhancer. The 3'-UTR contains consensus sequences of cytoplasmic polyadenylation elements (CPE's), and the length of its poly (A) tail changes during oocyte maturation, indicating that its expression is controlled by timely activation of translation. This new gene, Maid, models the translational and transcriptional regulation of gene expression during the transition from gamete to embryo.
3162	Cytokine production regulating Th1 and Th2 cytokines in hemophagocytic lymphohistiocytosis.	9166851	Hemophagocytic lymphohistiocytosis (HLH) is caused by the hyperactivation of T cells and macrophages. The clinical characteristics associated with this disease result from overproduction of Th1 cytokines including interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-alpha). In this study, we analyzed the production of IL-12 and IL-4, which determine Th1 and Th2 response, respectively, and IL-10, which antagonizes Th1 cytokines, in 11 patients with HLH. IL-12 was detected in plasma in all patients (mean peak value, 30.0 +/- 5.0 pg/mL), while IFN-gamma was massively produced in nine patients (mean peak value, 79.2 +/- 112.0 U/mL). IL-4 was not detected in any of the patients. Plasma IL-10 levels were elevated in all patients (mean peak value, 2,698.0 +/- 3,535.0 pg/mL). There was a positive correlation between the levels of IFN-gamma and IL-10 (P < .01). The plasma concentrations of these cytokines were initially high, before decreasing after the acute phase. However, the decrease in IL-10 levels was slower than that of IFN-gamma. Although the concentration of IL-12 was high at the acute phase, in some patients, a peak in the level was delayed until the chronic phase. Thus, in HLH, production of cytokines that promote development of Th1 cells appears to be predominant over that for Th2 cell development. Overproduction of IL-10 was also observed indicating that a mechanism suppressing hyperactivation of Th1 cells and monocytes/macrophages functions in patients with this disease.
3163	The characterization of the Olf-1/EBF-like HLH transcription factor family: implications in olfactory gene regulation and neuronal development.	9151732	The Olf-1/EBF helix-loop-helix (HLH) transcription factor has been implicated in olfactory gene regulation and in B-cell development. Using homology screening methods, we identified two additional Olf-1/EBF-like cDNAs from a mouse embryonic cDNA library. The Olf-1/EBF-like (O/E) proteins O/E-1, O/E-2, and O/E-3 define a family of transcription factors that share structural similarities and biochemical activities. Although these O/E genes are expressed within olfactory epithelium in an identical pattern, they exhibit different patterns of expression in the developing nervous system. Although O/E-1 mRNA is present in several tissues in addition to olfactory neurons and developing B-cells, O/E-2 and O/E-3 are expressed at high levels only in olfactory tissue. In O/E-1 knock-out animals, the presence of two additional O/E family members in olfactory neurons may provide redundancy and allow normal olfactory neurodevelopment. Further, the identification of the O/E family of HLH transcription factors and their embryonic expression patterns suggest that the O/E proteins may have a more general function in neuronal development.
3164	Transcriptional activation of human LIM-HOX gene hLH-2 in chronic myelogenous leukemia is due to a cis-acting effect of Bcr-Abl.	9175786	DNA methylation plays an important role in gene regulation. A human LIM-HOX gene, namely hLH-2, was highly expressed in chronic myelogenous leukemia (CML) and located on chromosome 9q33-34.1, in the same region as the reciprocal translocation that creates the Bcr-Abl chimera of Philadelphia chromosome [Wu et al. (1996) Oncogene 12, 1205]. To elucidate the mechanism of hLH-2 transcriptional activation, we studied the methylation status of hLH-2 in normal bone marrow and CML cells. When blots containing genomic DNA digested with Hpa II or Msp I were hybridized with full-length cDNA probe, it was discovered that hLH-2 was methylated in normal bone marrow cells in which hLH-2 was not expressed; in contrast, both alleles of hLH-2 locus in CML cells were heavily hypomethylated. Furthermore, using the sensitive RT-PCR technique, we examined the expression of LH-2 in mouse x human hybrids and a wide array of mouse cell lines containing Abl or Bcr-Abl and failed to identify a consistent expression pattern in the cell lines tested. These results suggest that the transcriptional activation of hLH-2 in CML is likely due to a cis-acting effect, but not a trans-acting effect of the Bcr-Abl fusion protein. Because hypomethylated genes generally are transcribed more efficiently than hypermethylated genes, the high level of hLH-2 mRNA in CML cells probably is a consequence of the low level of methylation of the gene in the leukemic cells.
3165	The polymyositis-scleroderma autoantigen interacts with the helix-loop-helix proteins E12 and E47.	9148967	The basic helix-loop-helix (bHLH) transcription factors E12 and E47 regulate cellular differentiation and proliferation in diverse cell types. While looking for proteins that bind to E12 and E47 by the yeast interaction trap, we isolated the rat (r) homologue of the human (h) polymyositis-scleroderma autoantigen (PM-Scl), which has been localized to the granular layer of the nucleolus and to distinct nucleocytoplasmic foci. The rPM-Scl and hPM-Scl homologues are 96% similar and 91% identical. We found that rPM-Scl mRNA expression was regulated by growth factor stimulation in cultured rat aortic smooth muscle cells. rPM-Scl bound to E12 and E47 but not to Id3, Gax, Myb, OCT-1, or Max. The C terminus of rPM-Scl (amino acids 283-353) interacted specifically with a 54-amino acid domain in E12 that is distinct from the bHLH domain. Finally, cotransfection of rPM-Scl and E47 specifically increased the promoter activity of a luciferase reporter construct containing an E box and did not affect the basal activity of the reporter construct. rPM-Scl appears to be a novel non-HLH-interacting partner of E12/E47 that regulates E2A protein transcription.
3166	Successful correction of hemophagocytic lymphohistiocytosis with related or unrelated bone marrow transplantation.	9160694	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation leading to widespread lymphocytic and hemophagocytic infiltration of vital organs. Apparent cure has only been achieved with allogeneic bone marrow transplantation (BMT). This report describes 20 consecutive patients, who underwent either matched sibling donor (n = 4) or unrelated donor (URD; n = 16) BMT. Age at the time of BMT was 0.4 to 5.3 years (median, 0.8 years). Central nervous system disease was present at diagnosis in 13 patients. At BMT, 14 patients were in a clinical remission, whereas 6 patients had active HLH. All patients were engrafted after cytoreduction with busulfan, cyclophosphamide, and etoposide. The probability of grade II-III acute graft-versus-host disease (GVHD) for all patients was 57% (95% confidence limit [CL], 0.28, 0.86), and 73% (95% CL, 0.44, 1.0) in URD patients. The overall probability of survival at 3 years was 45% (95% CL, 0.23, 0.67) and 44% (95% CL, 0.19, 0.68) when URD BMT was evaluated separately. Favorable BMT outcome was associated with clinical remission status at the time of BMT. The preparative regimen was well tolerated, and in the 9 surviving patients it provided durable engraftment and was effective at eradicating the underlying disease.
3167	Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing.	9169839	The veins in the Drosophila wing have a characteristic width, which is regulated by the activity of the Notch pathway. The expression of the Notch-ligand Delta is restricted to the developing veins, and coincides with places where Notch transcription is lower. We find that this asymmetrical distribution of ligand and receptor leads to activation of Notch on both sides of each vein within a territory of Delta-expressing cells, and to the establishment of boundary cells that separate the vein from adjacent interveins. In these cells, the expression of the Enhancer of split gene m beta is activated and the transcription of the vein-promoting gene veinlet is repressed, thus restricting vein differentiation. We propose that the establishment of vein thickness utilises a combination of mechanisms that include: (1) independent regulation of Notch and Delta expression in intervein and vein territories, (2) Notch activation by Delta in cells where Notch and Delta expression overlaps, (3) positive feedback on Notch transcription in cells where Notch has been activated and (4) repression of veinlet transcription by E(spl)m beta and maintenance of Delta expression by veinlet/torpedo activity.
3168	Repression of muscle-specific gene activation by the murine Twist protein.	9168805	Inhibition of myogenic differentiation can be achieved by various mechanisms. The murine bHLH protein Twist has been shown to inhibit muscle differentiation in mammalian cells. Here we demonstrate that this inhibition is cell autonomous and does not alter cell proliferation. By overexpression of E12, we can distinguish the inhibitory mechanisms of Twist and the dominant negative HLH factor Id. A difference is seen both for the native muscle-specific enhancers of myogenin and myosin light chain 1/3 and for an enhancer consisting only of four E-boxes. Mutagenesis experiments revealed that both the basic region and an evolutionarily conserved carboxy-terminal domain are required for the Twist-specific type of inhibition. Loss of either of these regions renders Twist less efficient and more similar to Id. Gel mobility shift assays demonstrate that Twist can bind to the muscle creatine kinase E-box and inhibit DNA binding of heterodimers of E12 with myogenic bHLH transcription factors like MyoD. However, a fourfold excess of Twist compared to MyoD is required for both effects. Our results suggest that Twist inhibits muscle-specific gene activation by formation of actively inhibitory complexes rather than by sequestering E-proteins.
3169	Expression of the Id family helix-loop-helix regulators during growth and development in the hematopoietic system.	9129018	To better understand the molecular mechanism(s) by which growth and differentiation of the primitive hematopoietic stem cell is initiated, as well as the means by which the maturing cell can commit to development along a specific cell lineage, we elected to study the Id family of helix-loop-helix (HLH) transcriptional regulators. Some members of the HLH family are expressed in a stage-specific manner during hematopoietic development and can regulate the ability of immature hematopoietic cells to terminally differentiate. None of the four Id family genes were detected in the most primitive progenitors. Id-1 was widely expressed in proliferating bi- and unipotential progenitors, but its expression was downregulated in cells of increasing maturity; conversely, Id-2 and, to a limited extent, Id-3 gene expression increased as cells matured and lost proliferative capacity. Id-2 expression ran counter to that of Id-1 not only during maturation, but during periods of cell growth and arrest as well. This is quite distinct from the nonhematopoietic tissues, in which these two factors are coordinately expressed and suggests that Id-1 and Id-2 might be regulating very different events during hematopoiesis than they regulate in other cell types.
3170	HLH-94: a treatment protocol for hemophagocytic lymphohistiocytosis. HLH study Group of the Histiocyte Society.	9121398	NaN
3171	Transcriptional activation of human LIM-HOX gene, hLH-2, in chronic myelogenous leukemia is due to a cis-acting effect of Bcr-Abl.	9168938	DNA methylation plays an important role in gene regulation. A human LIM-HOX gene, namely hLH-2, was highly expressed in chronic myelogenous leukemia (CML) and located on chromosome 9q33-34.1, in the same region as the reciprocal translocation that creates the Bcr-Abl chimera of Philadelphia chromosome (H.-K. Wu et al., 1996, Oncogene 12, 1205-1212). To elucidate the mechanism of hLH-2 transcriptional activation, we studied the methylation status of hLH-2 in normal bone marrow and CML cells. When blots containing genomic DNA digested with Hpa II or Msp I were hybridized with full-length cDNA probe, it was discovered that hLH-2 was methylated in normal bone marrow cells in which hLH-2 was not expressed; in contrast, both alleles of the hLH-2 locus in CML cells were heavily hypomethylated. Furthermore, using a sensitive RT-PCR technique, we examined the expression of LH-2 in mouse x human hybrids and a wide array of mouse cell lines containing Abl or Bcr-Abl, and we failed to identify a consistent expression pattern in the cell lines tested. These results suggest that the transcriptional activation of hLH-2 in CML is likely due to a cis-acting effect, but not a trans-acting effect, of the Bcr-Abl fusion protein. Because hypomethylated genes generally are transcribed more efficiently than hypermethylated genes, the high level of hLH-2 mRNA in CML cells probably is a consequence of the low level of methylation of the gene in the leukemic cells.
3172	Target-dependent effect of phosphorylation on the DNA binding activity of the TAL1/SCL oncoprotein.	9111058	Activation of the TAL1 (or SCL) gene, initially identified through its involvement by a recurrent chromosomal translocation, is the most frequent gain-of-function mutation recognized in T-cell acute lymphoblastic leukemia. The translational products of this gene contain the basic domain helix-loop-helix motif characteristic of a family of transcription factors that bind to a consensus nucleotide sequence termed the E-box. Previous work established that the TAL1 proteins are phosphorylated exclusively on serine and identified Ser122 as a substrate for the mitogen-activated protein kinase ERK-1. We provide evidence that an additional serine residue, Ser172, located in a conserved region proximal to the DNA binding domain and sharing homology with a similarly positioned sequence in the HLH oncoprotein LYL1, can be phosphorylated in vitro and in vivo by the catalytic subunit of cAMP-dependent protein kinase. Phosphorylation was found to alter TAL1 DNA binding activity in a target-dependent manner that was influenced by both the specific CANNTG E-box core motif and its flanking sequences. In contrast, the ability of TAL1 to interact with the E2A gene product E12 and its subcellular localization in transfected COS cells were unaffected by Ser172 phosphorylation. These results suggest this serine residue has a regulatory function and indicate a mechanism by which phosphorylation could affect DNA binding site discrimination.
3173	The crystal structure of an intact human Max-DNA complex: new insights into mechanisms of transcriptional control.	9115440	Max belongs to the basic helix-loop-helix leucine zipper (bHLHZ) family of transcription factors. Max is able to form homodimers and heterodimers with other members of this family, which include Mad, Mxi1 and Myc; Myc is an oncoprotein implicated in cell proliferation, differentiation and apoptosis. The homodimers and heterodimers compete for a common DNA target site (the E box) and rearrangement amongst these dimer forms provides a complex system of transcriptional regulation. Max is also regulated by phosphorylation at a site preceding the basic region. We report here the first crystal structure of an intact bHLHZ protein bound to its target site.The X-ray crystal structure of the intact human Max protein homodimer in complex with a 13-mer DNA duplex was determined to 2.8 A resolution and refined to an R factor of 0.213. The C-terminal domains in both chains of the Max dimer are disordered. In contrast to the DNA observed in complex with other bHLH and bHLHZ proteins, the DNA in the Max complex is bent by about 25 degrees, directed towards the protein. Intimate contacts with interdigitating sidechains give rise to the formation of tetramers in the crystal.The structure confirms the importance of the HLH and leucine zipper motifs in dimerization as well as the mode of E box recognition which was previously analyzed by X-ray crystallography of shortened constructs. The disorder observed in the C-terminal domain suggests that contacts with additional protein components of the transcription machinery are necessary for ordering the secondary structure. The tetramers seen in the crystal are consistent with the tendency of Max and other bHLHZ and HLH proteins to form higher order oligomers in solution and may play a role in DNA looping. The location of the two phosphorylation sites at Ser1 and Ser10 (the latter is the N-cap of the basic helix) suggests how phosphorylation could disrupt DNA binding.
3174	Highly conserved asparagine in the basic domain of Myc is dispensable for DNA binding, transformation, and apoptosis.	9169089	The basic region of c-Myc and other basic helix-loop-helix (b-HLH)-containing proteins bind to the palindromic DNA sequences CANNTG. For the myogenic factor MyoD, a member of the b-HLH family, mutation of several basic region residues abrogates muscle differentiation, but not DNA binding. One of the amino acid positions displaying this behavior in MyoD aligns with a highly conserved asparagine in Myc. This conserved asparagine displays complete tolerance for alanine substitution as measured by DNA binding. To test the possibility of whether the basic region of Myc encodes a second biological function, the conserved asparagine in c-Myc (N360) was mutated to alanine and tested for the Myc-dependent functions of cellular transformation and apoptosis. In contrast to the deleterious effects of such mutations in MyoD, the alanine mutant functions normally for both Myc-dependent cellular transformation and apoptosis induction. Therefore, a basic region function distinct from DNA binding may not be a general feature of HLH transcription factors.
3175	HLH transcription factor activity in osteogenic cells.	9138075	To examine possible mechanisms underlying osteoblast differentiation from mesenchymal stem cells, we investigated bHLH functional activity in cell lines representing different stages of osteoblast maturation. Interaction of nuclear proteins with oligonucleotides corresponding to various bHLH binding sequences (known as E-boxes) was determined in mobility shift assays. Both ADD-1 oligonucleotide, a binding site for transcription factor ADD-1, and OCE-1, an E-box from osteocalcin promoter, produced retarded bands after incubation with nuclear extracts from osteogenic cells. Cells at different stages of osteogenic maturation demonstrated similar patterns and intensity of binding, as did cells treated with different osteogenic inducers. Binding to ADD-1 and OCE-1 was not tissue-specific as it was also observed in fibroblastic 10T1/2 cells. MEF-1 oligonucleotide, the E-box sequence from the muscle creatine kinase enhancer, demonstrated no changes in binding with nuclear extracts from moderately differentiated (W-20) or relatively mature (ROS 17/2.8) cells under any conditions tested. However, in poorly differentiated RI-2J cells, which do not express osteogenic markers unless treated with dexamethasone, induction of differentiation was reflected in transient inhibition of binding to MEF-1. Inhibition of binding was not seen under differentiation-restrictive conditions. Promoter-reporter studies also demonstrated inhibition of MEF-1 driven CAT expression by dexamethasone under differentiation-permissive conditions in RI-2J cells. These data suggest that bHLH gene expression is not required for the early steps of osteogenesis; moreover, inhibition of bHLH protein binding to a MEF1-type E box might be an integral part of osteogenic commitment.
3176	Purification and characterization of the Streptomyces lividans initiator protein DnaA.	9079932	The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains. The larger size of the S. lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II. The S. lividans protein was overproduced as a His-tagged fusion protein. The purified protein (isoelectric point, 5.7) has a weak ATPase activity. By DNase I footprinting studies, each of the 17 DnaA boxes (consensus sequence, TTGTCCACA) in the S. lividans oriC region was found to be protected by the DnaA fusion protein. Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif or with amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes. A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S. lividans mutant DnaA proteins with little or no capacity for binding to DnaA boxes. Thus, like in E. coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA. A mutant protein lacking a stretch of acidic amino acids corresponding to domain II is not affected in its DNA binding capacity. Whether the acidic domain II interacts with accessory proteins remains to be elucidated.
3177	Functional characterization of ETV6 and ETV6/CBFA2 in the regulation of the MCSFR proximal promoter.	9050885	The ETV6/CBFA2 (TEL/AML1) fusion gene occurs as a result of the chromosome translocation t(12;21)(p13;q22) in up to 30% of children diagnosed with B cell precursor (cd10+, cd19+) acute lymphoblastic leukemia. Leukemic cells that have acquired the t(12;21) usually demonstrate loss of the remaining normal ETV6 (TEL) allele. Using reporter gene assays we have functionally characterized both the normal ETV6 and ETV6/CBFA2 fusion proteins in the regulation of the MCSFR proximal promoter. Neither ETV6 or ETV6/CBFA2 has any significant, detectable effect on the promoter by itself. However, both ETV6 and ETV6/CBFA2 inhibit the activation of the promoter by CBFA2B(AML1B) and C/EBPa. We have shown that a 29-bp region of the MCSFR promoter containing the binding sites for CBFA2B and C/EBPa is sufficient for the inhibition by ETV6 and ETV6/CBFA2. Mutational analysis of the MCSFR promoter revealed that binding of both CBFA2B and C/EBPa to their respective sites is necessary for the inhibition by ETV6 and ETV6/CBFA2. Deletion of the helix-loop-helix (HLH) region from the cDNAs of ETV6 and ETV6/CBFA2 decreased but did not completely abrogate the ability of either construct to inhibit promoter activation. We also found that the ETS DNA binding region of ETV6 is necessary for inhibition of the promoter. Addition of ETS1 and FLI1, two ETS family members that have homology in the 5' HLH region, but not Spi1, an ETS family member without the 5' HLH region, also inhibited reporter gene expression. Our data show that the inhibition mediated by ETV6 and ETV6/CBFA2, in the context of the MCSFR promoter, depend on interactions with other proteins, not just CBFA2B. Our results also indicate that the transactivation characteristics of ETV6/CBFA2 are a combination of positive and negative regulatory properties.
3178	Prolonged resolution of hemophagocytic lymphohistiocytosis following myeloablative chemotherapy and subsequent autologous peripheral blood stem cell transplantation.	9085745	A 30-month-old boy with hemophagocytic lymphohistiocytosis (HLH) received an autologous peripheral blood stem cell transplant (PBSCT) following high-dose chemotherapy. He presented with hemophagocytic syndrome (HPS) at 6 months of age, but relapsed despite the repeated administration of prednisolone, VP-16, cyclosporin A (CsA), and other cytotoxic agents. PBSC were obtained using combination chemotherapy with etoposide (VP16, 450 mg/m2), doxorubicin (70 mg/m2), vincristine (2 mg/m2) and cyclophosphamide (CY, 1200 mg/m2). 2.7 x 10(5)/kg CFU-GM PBSC were transplanted after similar high-dose VP16 preconditioning used for allogeneic BMT for HLH. The boy continues to remain in complete remission 30 months after PBSCT while receiving low-dose PSL/CsA therapy. High-dose chemotherapy followed by PBSCT may be an optional therapeutic approach for patients with HLH.
3179	[Measurement of biological activity of hLH. Multicenter study].	9180965	This report describes the results of a multicentric study of biological methods for luteinizing hormone (hLH). The production of testosterone by animal Leydig cells is used by five laboratories with different methodologies including rat, mouse and pig cells. Dose-response curves of testosterone production, quality criteria and results on a physiological population of men and women, are reported and discussed. It is concluded that the bioactive determination of hLH must be considered as a reference method used in discordances between clinic and immunological methods.
3180	Haemophagocytic lymphohistiocytosis in association with granular lymphocyte proliferative disorders in early childhood: characteristic bone marrow morphology.	9074410	Five paediatric cases of haemophagocytic lymphohistiocytosis (HLH) which showed proliferation of granular atypical lymphocytoid cells in bone marrow are reported. All cases were girls aged 8 months to 4 years who had marked hepatosplenomegaly. Marker analysis on peripheral blood mononuclear cells revealed an increase in the CD3+HLADR+ subset in three cases and the CD3- CD56+ subset in one case. An Epstein-Barr virus genome was detected in three cases, and monoclonality was confirmed in two cases. A characteristic morphology of large granular lymphocytes (LGL) was identified, with elongated bizarre features that resembled horsetail-, tadpole-, cucumber- or shooting star-type configurations on the bone marrow smear. Serum concentrations of soluble interleukin-2 receptor and interferon-gamma were elevated in all cases. All five cases required multi-agent chemotherapy which resulted in two complete remission, two partial remissions and one no response. Refinement of treatment is required for these paediatric GLPD cases which probably comprise a specific high-risk subgroup among secondary HLH patients which had previously escaped notice.
3181	Neuropathologic findings and neurologic symptoms in twenty-three children with hemophagocytic lymphohistiocytosis.	90634099063402	Primary hemophagocytic lymphohistiocytosis (HLH) is an autosomal recessive disorder with very high mortality rates, mainly affecting infants and young children, which is characterized by fever, hepatosplenomegaly, and cytopenias. Of great clinical importance are the neurologic symptoms, which are common and may even dominate the clinical picture and precede the systemic presentation. These symptoms are extremely variable, ranging from irritability, bulging fontanelle, and neck stiffness, to convulsions, cranial nerve palsies, ataxia, hemiplegia/tetraplegia, and unconsciousness.To elucidate this neurologic involvement further, we reviewed the neuropathologic postmortem findings from 23 children and their neurologic symptoms.Macroscopically, edema was present in many cases, and in some with advanced disease, softening and destruction of the tissue were conspicuous. The microscopic picture was exceedingly variable, ranging from almost normal to very advanced changes. In the mildest form of HLH, only the meninges were involved with infiltration of lymphocytes and macrophages (stage I), whereas more advanced cases in addition also showed perivascular infiltrates (stage II). In even more advanced disease there was also a diffuse infiltration in the tissue (stage III), as well as a multifocal necrosis. A prominent astrogliosis was present in such cases. Hemophagocytosis was seen in most patients, most commonly in the leptomeninges.HLH affecting the central nervous system imitates several neurologic disorders and may be misdiagnosed. A staging system for the neuropathologic findings is presented. In children with obscure central nervous systems symptoms and a progressive encephalopathy, the diagnosis of HLH may be considered, in particular because treatments are available.
3182	Hemophagocytic lymphohistiocytosis in infancy and childhood.	90634089063402	The current status of hemophagocytic lymphohistiocytosis (HLH) in infants and children has been studied.Eighty-two cases of pediatric HLH, for which there was no confirmed familial inheritance, were comparatively studied between 36 patients less than 2 years of age and 46 patients more than 2 years of age.In all cases, persistent fever, cytopenia, liver dysfunction, and hepatosplenomegaly were the most frequently noted symptoms. Hyperferritinemia (> 1000 micrograms/L) and elevated blood levels of lactate dehydrogenase (> 1000 IU/L) were observed in 90% and 89.7%, respectively. These figures were considerably higher than for either hypertriglyceridemia (> 2 mmol/L) (50%) or hypofibrinogenemia (< 1.5 gm/L) (57.4%), indicating that increased serum ferritin and lactate dehydrogenase concentrations might be good diagnostic parameters for HLH. These parameters are nonspecific but are of follow-up and prognostic value in these HLH cases. No differences were found in clinical signs and symptoms or other laboratory findings for the two age groups. Immunochemotherapy was administered in the similar regimens to patients in both groups. Of the 82 patients, 13 (15.9%) succumbed to a fatal course within 2 months after diagnosis, and Kaplan-Meir analysis for all cases predicted the overall disease-free patient survival at 4 years from the onset of disease to be 57.2% (95% confidence interval (CI), 45.1% to 69.3%). There was a slightly, but not statistically significant, poorer prognosis for the younger patients: 44.2% (95% CI, 26.0% to 62.4%) survival for the infant group versus 67.2% (95% CI, 51.8% to 82.6%) survival for the older group (p = 0.0569).Refinement of the treatment is mandatory to improve the outcome of HLH in both infants and older pediatric patients.
3183	Successful engraftment of haploidentical stem cell transplant for familial haemophagocytic lymphohistiocytosis using both bone marrow and peripheral blood stem cells.	9054677	Familial haemophagocytic lymphohistiocytosis (HLH) is a disease with a very poor prognosis unless patients receive a bone marrow transplant. It is often difficult to find an HLA-matched donor and haploidentical familial donors may be considered. The main complication of this type of transplant is graft rejection. We describe a patient with familial HLH who received a haploidentical transplant using both mobilized peripheral blood and bone marrow stem cells in an attempt to overcome graft rejection by increasing the stem cell dose. The peripheral blood stem cell inoculum was CD34 enriched using a Cellpro column and T-cell depleted by Campath-1M, the patient received conditioning for a matched sibling donor transplant with the addition of Campath 1G. There was rapid and full engraftment and the patient remains disease free at 5 months. This technique may be applicable for other fatal inborn errors in the absence of an HLA-matched donor.
3184	Filamentous phage displaying the extracellular domain of the hLH/CG receptor bind hCG specifically.	9048631	We have expressed the extracellular binding domain of the human LH/CG receptor as a fusion with the cpIII filamentous phage coat protein. These fusion phage are able to specifically bind ELISA plates coated with hCG. Preincubation of the phage with hCG before exposure to the coated plates inhibited binding of phage, whereas human FSH had no effect. Furthermore, addition of anti-hLH/CG receptor antisera inhibited binding of phage to the plates. We have also tested the effect of monoclonal antibodies to hCG on phage binding. Monoclonal antibodies that can bind hCG when it is bound to native receptor do not inhibit phage binding. These include B105 and A109 Alternatively, B107 and B109 have an inhibitory effect on phage binding. They cannot bind hCG when it is bound to receptor and are likely directed against epitopes at or near the receptor binding interface. Therefore, these fusion phage exhibit the same binding specificity as the wild-type receptor and likely display the extracellular domain of the hLH/CG receptor on their surface in the native conformation. Phage display is a potentially useful tool for studying the hLH/CG receptor structure and in screening for synthetic compounds that compete with hormone for receptor binding.
3185	USF2/FIP associates with the b-Zip transcription factor, c-Maf, via its bHLH domain and inhibits c-Maf DNA binding activity.	9070273	In screening for proteins that interact with the basic zipper (bZip) transcription factor, c-Maf, we isolated USF2/FIP. USF2 is a member of the bHLH-Zip protein family, possessing a basic (b) DNA binding region, a helix-loop-helix (HLH) motif, and a leucine zipper (Zip) structure. Mutants of USF2 that lacked a Zip formed heterodimers with c-Maf, but did not homodimerize. Deletion of the USF2 basic region or mutation of its helices abrogated its binding to c-Maf, but had no effect on homodimerization. A functional c-Maf bZip motif was necessary for both homodimerization and heterodimerization with USF2. These data suggest a tetrameric configuration for Maf-USF2 complexes. In the presence of USF2, the DNA binding activity of c-Maf was markedly reduced. Therefore, USF2 and c-Maf may interact to regulate gene expression.
3186	Expression of luteinizing hormone/human chorionic gonadotrophin (LH/HCG) receptor mRNA in the human ovary.	9239715	The gonadotrophins follicle stimulating hormone (FSH) and luteinizing hormone (LH) are key hormones in the regulation of ovarian function. In the present study, the expression of LH/human chorionic gonadotrophin (HCG) receptor mRNAs in the human ovary was examined. Northern blot analysis was used to measure relative amounts of LH/HCG receptor mRNA, and in-situ hybridization was used to localize LH/HCG receptor transcripts. Northern blot analysis of human ovaries detected three transcripts (5.4, 3.6 and 2.4 kb) for the LH/HCG receptor. LH/HCG receptor mRNA concentrations increased from preovulatory follicles to the corpus luteum of the midluteal phase, and decreased at the late luteal phase. Using in-situ hybridization, LH/HCG receptor mRNA was located predominantly in granulosa cells in the same follicle. Cloning of the human LH/HCG receptor cDNA previously revealed the existence of two alternative forms of the receptor differing by the presence (HLH-Ra) and absence (HLH-Rb) of 62 amino acids by exon 9. We have studied the functional significance of these receptor isoforms and have confirmed that they are generated by alternative splicing. A reverse transcription-polymerase chain reaction amplification was used to detect different isoforms of LH receptor mRNAs in ovary and placenta. The expression of the two mRNA forms of LH/HCG receptor were detected in ovary, and at very low concentrations in placenta. Treatment with HCG caused a dose-dependent increase in cAMP production with an initial response evident at approximately 1 ng/ml HCG in COS-7 cells expressing HLH-Ra. However, a complete loss of signal transduction was found in cells transfected with the truncated HLH-Rb.
3187	Identification of a CG/LH binding site in two strains of Mycobacterium vaccae.	9187538	We have previously reported the presence of a chorionic gonadotropin-like (CG-like) protein in the bacterium Xanthomonas maltophilia (X. maltophilia). We have also shown that X. maltophilia possesses a unique binding site for the native ligand and hCG, but not for human luteinizing hormone (hLH), and that binding of the native ligand or hCG to the receptor causes changes in the growth rates of culturing X. maltophilia. In this study we have characterized a CG/LH binding site in two strains of Mycobacterium vaccae. The binding site is specific for hCG and hLH, and Scatchard analysis reveals a biphasic, high affinity binding pattern. This is the second identified bacterial species to possess a CG-specific binding site.
3188	Evidence to suggest that expression of MITF induces melanocyte differentiation and haploinsufficiency of MITF causes Waardenburg syndrome type 2A.	9170159	MITF (microphthalmia-associated transcription factor) encodes a transcription factor with a basic-helix-loop-helix-leucine zipper (bHLH-Zip) motif. Ectopic expression of MITF is found to convert NIH/3T3 fibroblasts into cells with characteristics of melanocytes. MITF transfectants formed foci, which superficially resembled those induced by oncogenes, but did not exhibit malignant phenotypes. Instead, they contained dendritic cells that express melanogenic marker proteins such as tyrosinase and tyrosinase-related protein 1. Such properties were not observed in cells transfected with the closely related gene, TFE3. These findings indicated that MITF is involved in melanocyte differentiation. Two mutations (C760-->T and C895-->T) in MITF are found to be associated with individuals with Waardenburg syndrome type 2 (WS2). These mutations create stop codons in exon 7 and 8, respectively, and probably result in truncated proteins lacking HLH-Zip or Zip structure. To understand how these MITF mutations cause WS2 in heterozygotes, mutant MITF proteins were generated and used for DNA-binding and luciferase reporter assays. The mutated MITF proteins lose their DNA-binding activity and fail to transactivate the promoter of the tyrosinase gene. However, these mutated proteins do not appear to interfere with the activity of wild-type MITF protein in these assays, indicating that they do not show a dominant-negative effect. These findings suggest that the phenotypes of the two WS2 families are caused by loss-of-function mutations in one of the two MITF alleles, resulting in haploinsufficiency of the MITF protein, the transcription factor necessary for normal melanocyte differentiation.
3189	The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis.	9043084	The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, we isolated the Xenopus homolog of Suppressor of Hairless (X-Su(H)), a component of the Notch signaling pathway in Drosophila. Using animal cap assays, we show that X-Su(H) induces ESR-1 expression, perhaps directly, when modified by the addition of ankyrin repeats. Using a DNA binding mutant of X-Su(H), we show that X-Su(H) activity is required for induction of ESR-1. Finally, expression of the DNA binding mutant in embryos leads to a neurogenic phenotype as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos.
3190	Frequency and severity of central nervous system lesions in hemophagocytic lymphohistiocytosis.	9028310	We have retrospectively assessed the neurological manifestations in 34 patients with hemophagocytic lymphohistiocytosis (HLH) in a single center. Clinical, radiological, and cerebrospinal fluid (CSF) cytology data were analyzed according to treatment modalities. Twenty-five patients (73%) had evidence of central nervous system (CNS) disease at time of diagnosis, stressing the frequency of CNS involvement early in the time course of HLH. Four additional patients who did not have initial CNS disease, who did not die early from HLH complications, and who were not transplanted, also developed a specific CNS disease. Therefore, all surviving and nontransplanted patients had CNS involvement. Initially, CNS manifestations consisted of isolated lymphocytic meningitis in 20 patients and meningitis with clinical and radiological neurological symptoms in nine patients. For these nine patients, neurological symptoms consisted of seizures, coma, brain stem symptoms, or ataxia. The outcome of patients treated by systemic and intrathecal chemotherapy and/or immunosuppression exclusively (n = 16) was poor, as all died following occurrence of multiple relapses or CNS disease progression in most cases. Bone marrow transplantation (BMT) from either an HLA identical sibling (n = 6) or haplo identical parent (n = 3) was performed in nine patients, once first remission of CNS and systemic disease was achieved. Seven are long-term survivors including three who received an HLA partially identical marrow. All seven are off treatment with normal neurological function and cognitive development. In four other patients, BMT performed following CNS relapses was unsuccessful. Given the frequency and the poor outcome of CNS disease in HLH, BMT appears, therefore, to be the only available treatment procedure that is capable of preventing HLH CNS disease progression and that can result in cure when performed early enough after remission induction.
3191	Structural and functional analysis of hypoxia-inducible factor 1.	9027737	Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix protein that activates transcription of hypoxia-inducible genes, including those encoding: erythropoietin, vascular endothelial growth factor, heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1. Hypoxia response elements from these genes consist of a HIF-1 binding site (that contains the core sequence 5'-CGTG-3') as well as additional DNA sequences that are required for function, which in some elements include a second HIF-1 binding site. HIF-1 is a heterodimer. The HIF-1 alpha subunit is unique to HIF-1, whereas HIF-1 beta (ARNT) can dimerize with other bHLH-PAS proteins. Structural analysis of HIF-1 alpha revealed that dimerization with HIF-1 beta (ARNT) requires the HLH and PAS domains, DNA binding is mediated by the basic domain, and that HIF-1 alpha contains a carboxyl-terminal transactivation domain. Co-transfection of HIF-1 alpha and HIF-1 beta (ARNT) expression vectors and a reporter gene containing a wild-type hypoxia response element resulted in increased transcription in non-hypoxic cells and a superinduction of transcription in hypoxic cells, whereas HIF-1 expression vectors had no effect on the transcription of reporter genes containing a mutation in the HIF-1 binding site. HIF-1 alpha and HIF-1 beta (ARNT) protein levels were induced by hypoxia in all primary and transformed cell lines examined. In HeLa cells, the levels of HIF-1 alpha and HIF-1 beta protein and HIF-1 DNA-binding activity increased exponentially as cellular oxygen tension decreased, with maximum values at 0.5% oxygen and half-maximal values at 1.5 to 2% oxygen. HIF-1 alpha and HIF-1 beta (ARNT) mRNAs were detected in all human, mouse, and rat organs assayed and mRNA expression was modestly induced in rodents subjected to hypoxia. HIF-1 alpha protein levels were induced in vivo when animals were subjected to anemia or hypoxia. The HIF1A gene was mapped to human chromosome 14q21-q24 and mouse chromosome 12.
3192	Cdk2-dependent phosphorylation of Id2 modulates activity of E2A-related transcription factors.	9029153	The helix-loop-helix (HLH) protein Id2 is thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcription factors. Id2 acts by forming heterodimers that are unable to bind to specific (E-box) DNA sequences. Here we show that this activity can be overcome by phosphorylation of a serine residue within a consensus target site for cyclin-dependent kinases (Cdks). In vitro, Id2 can be phosphorylated by either cyclin E-Cdk2 or cyclin A-Cdk2 but not by cyclin D-dependent kinases. Analogous phosphorylation occurs in serum-stimulated human diploid fibroblasts at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. The phosphorylation of Id2 in these cells correlates with the restoration of a distinct E-box-dependent DNA-binding complex, suggesting that the levels of this complex are modulated by both the abundance and phosphorylation status of Id2. These data provide a link between cyclin-dependent kinases and bHLH transcription factors that may be critical for the regulation of cell proliferation and differentiation.
3193	Successful bone marrow transplantation from an HLA-identical unrelated donor in a patient with hemophagocytic lymphohistiocytosis.	9116618	We report a 3-year-old girl with hemophagocytic lymphohistiocytosis (HLH) who received BMT from an HLA-identical unrelated donor when the disease was active. She had entered remission in response to chemotherapy consisting of etoposide and methylprednisolone. After a relapse, her disease became refractory to chemotherapy, and splenectomy was performed with marginal improvement. She underwent BMT from an HLA-identical unrelated donor, conditioned with CY, TBI and anti-thymocyte globulin (ATG). She has been in complete remission for 18 months since the BMT. This result suggests that BMT from an HLA-identical unrelated donor should be considered for HLH even if the disease is active.
3194	Investigations on human lutropin from pituitary gland and urine.	9523009	Two newly isolated monoclonal anti-beta hLH antibodies did not recognize both human chorionic gonadotropin (hCG) and three synthetic peptide fragments corresponding to beta hLH sequence. These antibodies were applied for ELISA of human luteinizing hormone (hLH), however, sensitivities of these assays were much lower than those with two other monoclonal antibodies obtained earlier in our laboratory. No significant differences between assays of hLH from pituitary and urine with 6 different pairs of monoclonal antibodies (4 anti-beta and 1 anti-alpha-subunit) were noticed. The highest hLH concentration in urine samples collected during 53 menstrual cycles of 6 women was demonstrated at different times of the day. In 14 out of 52 "preovulatory urine" portions, preserved for several weeks, over 80% increase of assayed hLH was shown. The highest assays of hLH from pituitary and urine were noted at pH 7.5-9.0. Using affinity chromatography the whole pituitary hLH was bound to ConA-Sepharose column and was eluted with alpha-methyl-D-mannoside as a single peak. However, a small part of hLH from urine was eluted from the column as non retarded peak. When HPLC analysis with DEAE column was performed, pituitary hLH was separated in two fractions while lutropin from urine was eluted as a single peak.
3195	[Pre-ovulatory peaks of gonadotropins. Recent data].	9453972	From recent data we know that: The duration of the LH peak seems to be more important than its amplitude for the induction of ovulation. Ovulation induction by hCG is not physiological; the absence of an FSH surge, and the long duration of LH activity would contribute to some of the luteal phase abnormalities. Recombinant hLH, on the other hand could give better results provided its action last about 48 hours. The results, however, are not as expected, probably because of the type of isoforms injected. The circulating FSH and LH isoforms found during the preovulatory peak of gonadotropins have a much higher in vitro biological activity than those found at other periods of the cycle. The production of FSH and LH recombinant of different biological activity is in process. The mechanism of the preovulatory peak of gonadotropins is still not fully understood. The respective role of GnRH and of the ovarian steroids has yet to be precisely determined in the human being. Ovarian peptides very probably also play a role.
3196	Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vitro, radioreceptor activity and immunoactivity.	9418979	To establish whether sialic acid content is responsible for an observed 7-8-fold variability in bioactivity in vitro of highly purified human pituitary luteinizing hormone (hLH) isoforms, the bioactivity in vitro, radioreceptor activity and immunoactivity of hLH isoforms were determined before and after enzymatic desialylation. Three immunofluorometric assays with different hLH specificities allowed characterization of 13-24 pituitary hLH isoform preparations of pI 7.03-8.98 in terms of sialic acid content (1-5 sialic acid residues per LH molecule), bioactivity in vitro (4030-30,000 I.U. mg[-1]), radioreceptor activity (6420-25,400 I.U. mg[-1]) and hLH immunoactivity (2900-4400 to 18,300-27,300 I.U. mg[-1]). Significant positive correlations between sialic acid content and either immunoactivity or in vitro bioactivity were observed, whereas radioreceptor activity showed a curvilinear response. Following more than 90% removal of sialic acid, both in vitro bioactivity and radioreceptor activity were increased, although specific activity still differed between isoforms; immunoactivities were unaffected. It is concluded that the presence of the sialic acid residue(s) on hLH isoforms does partially contribute to the in vitro bioactivity and radioreceptor activity of the isoforms, but that hLH immunoactivity is independent of sialic acid content.
3197	No germline mutations in the dimerization domain of MXI1 in prostate cancer clusters. The CRC/BPG UK Familial Prostate Cancer Study Collaborators. Cancer Research Campaign/British Prostate Group.	9376279	There is evidence that predisposition to cancer has a genetic component. Genetic models have suggested that there is at least one highly penetrant gene predisposing to this disease. The oncogene MXI1 on chromosome band 10q24-25 is mutated in a proportion of prostate tumours and loss of heterozygosity occurs at this site, suggesting the location of a tumour suppressor in this region. To investigate the possibility that MXI1 may be involved in inherited susceptibility to prostate cancer, we have sequenced the HLH and ZIP regions of the gene in 38 families with either three cases of prostate cancer or two affected siblings both diagnosed below the age of 67 years. These are the areas within which mutations have been described in some sporadic prostate cancers. No mutations were found in these two important coding regions and we therefore conclude that MXI1 does not make a major contribution to prostate cancer susceptibility.
3198	SHARPs: mammalian enhancer-of-split- and hairy-related proteins coupled to neuronal stimulation.	93612829532582	In the mammalian central nervous system, a diverse group of basic helix-loop-helix (bHLH) proteins is involved in the determination of progenitor cells and, subsequently, in regulating neuronal differentiation. Here we report the identification of a novel subfamily of bHLH proteins, defined by two mammalian enhancer-of-split- and hairy-related proteins, termed SHARP-1 and SHARP-2. In contrast to known bHLH genes, detectable transcription of SHARP genes begins at the end of embryonic development marking differentiated neurons that have reached a final position, and increases as postnatal development proceeds. In the adult, SHARP genes are expressed in subregions of the CNS that have been associated with adult plasticity. In PC12 cells, a model system to study neurite outgrowth, SHARP genes can be induced by NGF with the kinetics of an immediate-early gene. Similarly, within 1 h after the administration of kainic acid in vivo, SHARP-2 is induced in neurons throughout the rat cerebral cortex. This suggests that neuronal bHLH proteins are also involved in the "adaptive" changes of mature CNS neurons which are coupled to glutamatergic stimulation.
3199	Developmental regulation of the Drosophila Tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins.	9254904	Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was know about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. We have recently shown that the 355-bp proximal enhancer contains a binding site for the Drosophila homologue of vertebrate MEF2 and that MEF2 acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, does not contain any consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting the MEF2 may interact with other factors to regulate muscle genes in Drosophila. In this study we have used mutation analysis and germ-line transformation to analyze cis-acting elements within the muscle activator region that regulate its expression in transgenic flies. We have identified a 71-bp region that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has allowed us to identify the sites in this region at which multiple proteins complex and interact. We propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression.
3200	Expression and function of Enhancer of split bHLH proteins during Drosophila neurogenesis.	9171445	The products of the Enhancer of split complex are required during neurogenesis for neural fate to be limited to a subset of cells within the ectoderm. Deletions which remove the complex lead to neural hypertrophy. The complex encodes seven related basic-helix-loop-helix transcription factors which are expressed in response to Notch activation. They accumulate in the cells surrounding the delaminating neuroblast where they prevent cells from adopting the neural fate, most likely by antagonising either directly or indirectly the actions of the proneural genes encoded by the achaete-scute complex. The individual roles of the seven different Enhancer of split proteins remains unclear, since their functions are at least partially redundant. However, the Enhancer of split complex is required in many other processes where Notch is active; the function of the individual proteins may relate to their roles in other developmental decisions or to their expression in distinct regions.
3201	Heteronuclear (1H, 13C, 15N) NMR assignments and secondary structure of the basic region-helix-loop-helix domain of E47.	9007990	E47 is an immunoglobulin enhancer DNA-binding protein that contains a basic region-helix-loop-helix (b/HLH) domain. This structural motif defines a class of transcription factors that are central to the developmental regulation of many tissues. Its function is to provide a dimerization interface through the formation of a parallel four-helix bundle, resulting in the juxtaposition of two basic DNA-recognition alpha-helices that control sequence-specific DNA-binding. In order to gain insight into the biophysical nature of b/HLH domains, we have initiated structural studies of the E47 homodimer by NMR. Sequence-specific resonance assignments have been obtained using a combination of heteronuclear double- and triple-resonance NMR experiments. The secondary structure was deducted from characteristic patterns of NOEs, 13C (alpha)/beta chemical shifts, and measurements of 3JHNH alpha scalar couplings. Except for the basic region recognition helix, the secondary structural elements of the E47 homodimer are preserved in the absence DNA when compared with the co-crystal structure of E47 bound to DNA (Ellenberger T, Fass D, Arnaud M, Harrison SC, 1994, Genes & Dev 8:970-980). As expected, the DNA-binding helix is largely unstructured, but does show evidence of nascent helix formation. The HLH region of E47 is structured, but highly dynamic as judged by the rapid exchange of backbone hydrogen atoms and the relatively weak intensities of many of the NOEs defining the dimerization helices. This dynamic nature may be relevant to the ability of E47 both to homodimerize and to heterodimerize with MyoD, Id, and Tal1.
3202	Mutations of the TWIST gene in the Saethre-Chotzen syndrome.	89881678988156	Saethre-Chotzen syndrome (acrocephalo-syndactyly type III, ACS III) is an autosomal dominant craniosynostosis with brachydactyly, soft tissue syndactyly and facial dysmorphism including ptosis, facial asymmetry and prominent ear crura. ACS III has been mapped to chromosome 7p21-22. Of interest, TWIST, the human counterpart of the murine Twist gene, has been localized on chromosome 7p21 as well. The Twist gene product is a transcription factor containing a basic helix-loop-helix (b-HLH) domain, required in head mesenchyme for cranial neural tube morphogenesis in mice. The co-localisation of ACS III and TWIST prompted us to screen ACS III patients for TWIST gene mutations especially as mice heterozygous for Twist null mutations displayed skull defects and duplication of hind leg digits. Here, we report 21-bp insertions and nonsense mutations of the TWIST gene (S127X, E130X) in seven ACS III probands and describe impairment of head mesenchyme induction by TWIST as a novel pathophysiological mechanism in human craniosynostoses.
3203	Structural and functional characterisation of hFSH and hLH isoforms.	9027351	Human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) are gonadotropins which are secreted as multiple forms by the pituitary. Evidence supporting the structural and functional heterogeneity of 15 purified hFSH isoforms and 20 purified hLH isoforms from pituitary extracts will be presented. Gonadotropin isoforms were purified by a combination of preparative isoelectric focusing and ion-exchange chromatography. The protein mass of each isoform was determined by amino acid analysis, which also correlated (data for hLH) (r = 0.999, P < 0.001, n = 15) with the UV area under the curve at 280 nm of the isoforms following gel-filtration HPLC. The alpha and beta subunits of FSH and LH were shown to be intact by SDS-PAGE under reducing condition, with no evidence of proteolytic nicking or presence of contaminating proteins. hFSH radioreceptor activity varied over a seven-fold range, and a positive correlation (r = 0.85, P < 0.001, n = 9) was observed between FSH receptor activity and the sialic acid (SA) content (1.5-13.7 mol SA/mol hFSH) of the isoforms, as determined by an HPLC-based microfluorometric assay. FSH in vitro activities varied over a similar range with a high correlation (r = 0.82, n = 15) with receptor activities, suggesting that the initial association of the hormone with the receptor is the key interaction with less differences attributed to subsequent effects in the signaling pathway. A similar result was seen with the hLH isoforms. To explore FSH/LH in vivo, the circulating half-life (LH/FSH) and the in vivo bioactivity (LH) using an acute in vivo assay was investigated. The clearance of hLH and hFSH showed a bi-exponential pattern for all isoform preparations with the proportion of the slower dissociating component (t 1/2 50-60 min) increasing three-fold with increasing sialic acid content of the isoform. The more rapidly cleared component (t 1/2 approx 10 min) is attributed to hepatically cleared gonadotropin, rather than gonadotropin equilibration between body compartments. The in vivo assay procedure for LH was based on the 24 h integrated plasma testosterone levels in rats following administration of graded doses of hLH isoform or standard. A 16-fold range in vivo activities between LH isoforms (n = 14) was observed. A comparison between hLH in vitro and in vivo activities showed a good correlation (r = 0.75) with the slope of the regression line (1.39) not significantly different from unity. These results suggest that in this acute in vivo assay method, the differences in circulating half-lives between hLH isoforms although large is not a key factor in their in vivo activity. However, in chronic in vivo assay systems the differences in clearance rates between isoforms may be important in their subsequent biological response. It is concluded that structural heterogeneity of FSH and LH contributes to functional differences, with a key interaction occurring at the receptor level. The contribution of sialic acid to these activities was also investigated.
3204	Metabolism of hCG and hLH to multiple urinary forms.	9027350	Human chorionic gonadotropin (hCG) is synthesized primarily in the placenta while human luteinizing hormone (hLH) is produced in the pituitary. Both hormones are highly homologous in structure and both appear to be altered to analogous molecular forms as the hormones are proteolytically processed, or metabolized, from tissue of origin, through the circulation, and finally to the urine. Placental hCG is excreted into urine as heterodimeric hormone, heterodimeric nicked hCG, free subunits (some nicked), and predominantly as the hCG beta core fragment. A pituitary form of heterodimeric hCG, which is partly sulfated as is pituitary hLH, was recently isolated and is likely the form of hCG observed in the urine of healthy postmenopausal women and nonpregnant premenopausal women as well. A pituitary form of the hLH beta core fragment, highly analogous in structure to that of urinary hCG beta core fragment, has been used to develop specific monoclonal antibody assays to measure urinary hLH beta core fragment which is excreted at significantly higher molar concentrations than is hLH in the urine of ovulating women 1 or 2 days after the LH surge. This fragment of LH appears in the urine of postmenopausal women as well. The development of the capability to distinguish the hCG beta core fragment from the hLH beta core fragment in urine may have useful applications in tumor marker assays, pregnancy tests, and menopause. While hCG urinary assays have been widely employed, urinary assays for hCG and hLH metabolites are much less used since the urinary molecular forms are only partly known. Our studies of hCG and hLH urinary metabolites are directed towards improvement of the utility of urinary measurements of molecules derived from these hormones. Since many of the molecular forms of these two hormones in urine differ from their forms in blood, it may be necessary to produce new immunoassays as well as novel urinary reference preparations to accurately measure these molecules within their urinary matrix.
3205	Immunochemical mapping of gonadotropins.	9027341	As a glycoprotein hormone, human chorionic gonadotropic (hCG) is not a single molecular entity but this term rather comprises an array of molecular variants such as hCG, hCG beta, hCGn, hCG beta n, hCG beta cf, -CTPhCG, hCG beta CTP, deglyhCG, asialohCG, hCGav and the closely related molecules hLH, hLH beta and hLH beta ef. The advent of monoclonal antibodies (MCA), the availability of ultrasensitive detection systems and the recent determination of the crystal structure of hCG, made it possible to design special purpose diagnostic and clinical research immunoassays for hCG-like molecules. For more than a decade we and others have tried to refine epitope maps for hCG and related molecules by means of a large panel of MCA, naturally occurring metabolic variants of hCG (hCGn, hCG beta, hCG alpha, hCG beta cf, hCG beta CTP), homologous hormones and subunits of various species (e.g. hLH, hLH beta, hFSH, hTSH, oLH, rLH beta), chemically modified molecules (deglyhCG, asialohCG, tryptic and chymotryptic hCG beta and hCG alpha fragments) and synthetic peptides (octapeptides and longer). It appeared that all epitopes on molecular hCG-variants recognized by our MCA are determined by the protein backbone. Except for the two major epitopes on hCG beta CTP and parts of two antigenic domains on hCG alpha, epitopes on hCG-derived molecules are determined by the tertiary and quarternary structure. Operationally useful descriptive epitope maps were designed including information on assay suitability of antigenic determinants. On this basis we established ultrasensitive time-resolved fluoroimmuno-assays for hCG, hCG and hCGn, hCG beta and hCG beta n and hCG beta cf, hCG alpha and additional assays recognizing different spectra of hCG-variants. Such assay have been applied by us and others to the detection of pregnancy, early pregnancy loss, choriocarcinoma, testicular cancer, other cancers and prenatal diagnosis. However, as the molecular structure of many epitopes utilized in immunoassays of different laboratories was not resolved, comparability of results was not satisfactory. Consequently, attempts were made to compare schematic epitope maps from different research institutions. The situation has been much improved by solving the three-dimensional (3D) structure of hCG. It has been shown that hCG is a member of the structural superfamily of cystine knot growth factors like NGF, PDGF-B and TGF-beta. Each of its subunits is stabilized in its topology by three disulfide bonds forming a cystine knot. Moreover, it turned out that the disulfide bridges in their majority have previously been wrongly assigned. Computer molecular modeling of crystallographic coordinates of hCG and subsequent selective combined--PCR-based and immunological--mutational analyses of hCG beta expressed via the transmembrane region of a MHC molecule made it possible to more precisely localize epitopes on hCG-derived molecules. Although the entire surface of hCG has to be regarded as potentially immunogenic there seems to be hot spots where epitopes are clustered in antigenic domains. These are located on the first and third loops protuding from the cystine knots of both subunits and are possibly centered around the knot itself. Ultimate answers on epitope localizations will be given by the crystal structure determination of hCG complexed with different Fabs.
3206	Structural features of mammalian gonadotropins.	9027339	There are two species for which both pituitary and placental gonadotropins are readily available, humans and horses. The human gonadotropins are better characterized than equine gonadotropins. Nevertheless, the latter are very interesting because they provide exceptions to some of the general structure-function principles derived from studies on human and other mammalian gonadotropins. For example, separate genes encode the hLH beta and hCG beta subunits while a single gene encodes eLH beta and eCG beta. Thus, eCG and eLH differ only in their oligosaccharide moieties and eLH is the only LH that possesses the O-glycosylated C-terminal extension previously believed to be restricted to chorionic gonadotropins. Truncation experiments involving eLH beta and hCG beta have suggested the C-terminal extension has no effect on receptor binding. However, the largest of three eCG forms which differ only in the extent of O-glycosylation possessed reduced affinity for LH and FSH receptors. This result suggested that effects of O-glycosylation need to be considered when examining the glycosylation differences between eLH and eCG responsible for the 10-fold lower eCG receptor binding affinity compared with that of eLH. Contribution of alpha Asn56 N-linked oligosaccharides to the different biological activities of eLH and eCG has been evaluated following selective removal using peptide-N-glycanase digestion of native equine alpha-subunit preparations. Hormones-specific patterns of glycosylation were observed on alpha Asn56 of eLH, eFSH, and eCG. Removal of alpha Asn56 oligosaccharides increased the rate of subunit association, the extent of association, and receptor binding activity. Some unassociated alpha-subunit oligosaccharides were identified which may interfere with subunit association because they were more abundant in unassociated subunit oligosaccharide maps than in a total oligosaccharide map. This was most striking in the case of eCG alpha in which two minor peaks became the major oligosaccharide peaks detectable in the unassociated eCG alpha fraction following association with eLH beta and eFSH beta. The biological activities exhibited by hybrid hormones, eLH alpha reassociated with oLH beta and pLH beta, found to be greater than those of oLH and pLH provided an interesting exception to the general rule that the beta-subunit determines the potency of the heterodimer. LH receptor binding activities of eLH beta-chimeric ovine/equine alpha-subunits suggested that the equine alpha-subunit N-terminal domain may be responsible for this effect. Equine FSH has higher FSH receptor binding activity than human, ovine, and porcine FSH preparations. This probably results from two factors. First, the presence of the equine alpha-subunit promotes receptor binding as noted above. Second, the overall -2 charge of the eFSH beta determinant loop, which is less negative that the -3 observed in other species, results from the presence of an Asn residue at position 88 instead of Asp. This apparently facilitates binding to the FSH receptor.
3207	Cloning of the human platelet endothelial cell adhesion molecule-1 promoter and its tissue-specific expression. Structural and functional characterization.	8955189	Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a cell adhesion molecule involved in transendothelial migration and expressed by hemopoietic and endothelial cells. To understand the mechanisms underlying its regulated expression, a genomic clone containing 1555 bp of the 5'-flanking region and the first exon of the human PECAM-1 gene has been isolated. The 5'-flanking region of the PECAM-1 gene lacks a consensus TATA box, but contains consensus motifs for Sp1, EGR1, ets, helix-loop-helix (HLH) box, GATA, AP-2, C/EBP, YY1, CCACC, LyF-1, imperfect octamer, heptamer, high mobility group proteins (HMG) box, and nuclear factor-kappaB, as well as shear stress-, retinoic acid-, glucocorticoid-, and acute phase-responsive elements, and an Alu sequence. Successive 5' to 3' or 3' to 5' deletions revealed tissue-specific promoter activity within the two contiguous 0.22-kb NheI/BglII and 0.44-kb BglII/PstI fragments. The transcriptional activity displayed by the 0.22-kb NheI/BglII fragment was specific for the myeloid lineage, whereas the promoter activity of the 0.44-kb BglII/PstI fragment was apparently restricted to endothelial cells. The transcriptional activity of the 0.22-kb NheI/BglII fragment was confirmed by 5' RACE (rapid amplification of 5' cDNA ends) and S1 nuclease protection experiments that revealed previously unidentified transcription start sites. The 0.22-kb NheI/BglII promoter exhibited PMA inducibility in myeloid cells and contained a PMA-responsive element recognized by Sp1 and EGR-1 transcription factors. Isolation and characterization of the human PECAM-1 promoter represent an initial step in elucidating the controlled expression of the PECAM-1 gene.
3208	The TEL/platelet-derived growth factor beta receptor (PDGF beta R) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGF beta R kinase-dependent signaling pathways.	8962143	The TEL/PDGF beta R fusion protein is the product of the t(5;12) translocation in patients with chronic myelomonocytic leukemia. The TEL/PDGF beta R is an unusual fusion of a putative transcription factor, TEL, to a receptor tyrosine kinase. The translocation fuses the amino terminus of TEL, containing the helix-loop-helix (HLH) domain, to the transmembrane and cytoplasmic domain of the PDGF beta R. We hypothesized that TEL/PDGF beta R self-association, mediated by the HLH domain of TEL, would lead to constitutive activation of the PDGF beta R tyrosine kinase domain and cellular transformation. Analysis of in vitro-translated TEL/ PDGF beta R confirmed that the protein self-associated and that self-association was abrogated by deletion of 51 aa within the TEL HLH domain. In vivo, TEL/PDGF beta R was detected as a 100-kDa protein that was constitutively phosphorylated on tyrosine and transformed the murine hematopoietic cell line Ba/F3 to interleukin 3 growth factor independence. Transformation of Ba/F3 cells required the HLH domain of TEL and the kinase activity of the PDGF beta R portion of the fusion protein. Immunoblotting demonstrated that TEL/PDGF beta R associated with multiple signaling molecules known to associate with the activated PDGF beta R, including phospholipase C gamma 1, SHP2, and phosphoinositol-3-kinase. TEL/PDGF beta R is a novel transforming protein that self-associates and activates PDGF beta R-dependent signaling pathways. Oligomerization of TEL/PDGF beta R that is dependent on the TEL HLH domain provides further evidence that the HLH domain, highly conserved among ETS family members, is a self-association motif.
3209	E-box sequence and context-dependent TAL1/SCL modulation of basic helix-loop-helix protein-mediated transcriptional activation.	8940159	TAL1/SCL is a basic helix-loop-helix (bHLH) oncoprotein that is expressed in several cell lines including many hematolymphoid cells, but not in T- and B-lineage cells. The TAL1 gene was originally discovered as being transcriptionally activated by chromosomal rearrangements in T-cell acute lymphoblastic leukemia (T-ALL). Here we have shown that TAL1 and the ubiquitously expressed murine bHLH transcription factor ALF1 formed heterodimers that, compared with ALF1 homodimers, had a more restricted E-box specificity and bound preferentially to the glucocorticoid-responsive E-box (Egre) motif (AACAGATGGT). Overexpression of the dominant inhibitory HLH protein Id1 in NIH3T3 cells reduced the transcriptional activity mediated by ALF1 homodimers, whereas the transcriptional activity mediated by TAL1/ALF1 heterodimers was resistant to Id overexpression. Our results show that ALF1 may serve as a dimerization partner for the bHLH oncoprotein TAL1 and form a complex with a distinctive DNA binding property. These findings support the hypothesis that the leukemic characteristics of the TAL1 oncoprotein could be mediated by activation of a set of target genes as heterodimeric complexes with ubiquitously expressed bHLH transcription factors such as ALF1 and that a principal role of TAL1 might be to neutralize an Id-mediated inactivation.
3210	The human H-twist gene is located at 7p21 and encodes a B-HLH protein that is 96% similar to its murine M-twist counterpart.	8995765	NaN
3211	Expression of the human chorionic gonadotropin-beta gene cluster in human pituitaries and alternate use of exon 1.	8954017	Previous studies have indicated that in addition to other glycoprotein hormones, the pituitary gland produces small amounts of hCG beta, the classical pregnancy and tumor marker. At the gene transcription level, definitive proof for hCG beta messenger ribonucleic acid transcription was still lacking, largely due to the 90% homology to hLH beta at the DNA sequence level, which renders specific hCG detection in the presence of a vast excess of LH difficult. We investigated both the presence of hCG beta messenger ribonucleic acid and the protein itself in normal human female postmenopausal (n = 4) and male pituitaries (n = 2). Reverse transcription-PCR and subsequent restriction enzyme analysis revealed that the hCG beta 3, 5, 7, and 8 genes coding for genuine hCG beta were transcribed in all pituitaries. Additionally, three alternatively spliced gene products derived from hCG beta genes 1 and 2 were detected and verified by single strand sequencing of the complementary DNAs. The most abundant fragment (244 bp) showed a point mutation (T-->A) in the splice donor site for the first intron, resulting in an alternate use of exon 1 and a frame shift in the open reading frame that might give rise to a hypothetical protein, 132 amino acids in length. With regard to protein synthesis, we confirmed the pituitary as the site of production for hCG beta by reverse phase high performance liquid chromatography and subsequent immunoradiometric assays, including a monoclonal antibody directed against the unique C-terminal extension of hCG beta.
3212	The semidominant Mi(b) mutation identifies a role for the HLH domain in DNA binding in addition to its role in protein dimerization.	8947051	The mouse microphthalmia (mi) locus encodes a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor called MITF (microphthalmia transcription factor). Mutations at mi affect the development of several different cell types, including melanocytes, mast cells, osteoclasts and pigmented epithelial cells of the eye. Here we describe the phenotypic and molecular characterization of the semidominant Microphthalmia(brwnish) (Mi(b)) mutation. We show that this mutation primarily affects melanocytes and produces retinal degeneration. The mutation is a G to A transition leading to a Gly244Glu substitution in helix 2 of the HLH dimerization domain. This location is surprising since other semidominant mi mutations characterized to date have been shown to affect DNA binding or transcriptional activation domains of MITF and act as dominant negatives, while mutations that affect MITF dimerization are inherited recessively. Gel retardation assays showed that while the mutant MITF(Mi-b) protein retains its dimerization potential, it is defective in its ability to bind DNA. Computer modeling suggested that the Gly244Glu mutation might disrupt DNA binding by interfering with productive docking of the protein dimer onto DNA. The Mi(b) mutation therefore appears to dissociate a DNA recognition function of the HLH domain from its role in protein dimerization.
3213	Neuronal type information encoded in the basic-helix-loop-helix domain of proneural genes.	8917575	The proneural genes encode basic-helix-loop-helix (bHLH) proteins and promote the formation of distinct types of sensory organs. In Drosophila, two sets of proneural genes, atonal (ato) and members of the achaete-scute complex (ASC), are required for the formation of chordotonal (ch) organs and external sensory (es) organs, respectively. We assayed the production of sensory organs in transgenic flies expressing chimeric genes of ato and scute (sc), a member of ASC, and found that the information that specifies ch organs resides in the bHLH domain of ato; chimeras containing the b domain of ato and the HLH domain of sc also induced ch organ formation, but to a lesser extent than those containing the bHLH domain of ato. The b domains of ato and sc differ in seven residues. Mutations of these seven residues in the b domain of ato suggest that most or perhaps all of these residues are required for induction of ch organs. None of these seven residues is predicted to contact DNA directly by computer simulation using the structure of the myogenic factor MyoD as a model, implying that interaction of ato with other cofactors is likely to be involved in neuronal type specification.
3214	Effect of mutagenesis of GPIIb amino acid 273 on the expression and conformation of the platelet integrin GPIIb-IIIa.	8916916	A G273D mutation immediately proximal to the first calcium binding domain of platelet GPIIb impairs the export of GPIIb-IIIa heterodimers to the platelet surface. To examine how this mutation might alter the structure of GPIIb, G273 was replaced by other amino acids and the resulting mutants were coexpressed with GPIIIa in COS-1 cells. Although replacement with Ala or Val had no effect on GPIIb-IIIa expression, replacement with Glu, Lys, Pro, or Asn caused intracellular retention of GPIIb-IIIa. Concurrently, the consequences of these replacements were examined by comparative modeling by introducing them into the analogous position of the first helix-loop-helix (HLH) motif of calmodulin, based on homology between the calcium binding domains of GPIIb and the calcium binding loops of HLH-containing proteins. The modeling revealed that as the side chain of the introduced amino acid increased in size, it progressively interfered with hydrophobic interactions between the incoming and outgoing helices of the motif. To test whether this observation also applies to GPIIb, V286, located immediately distal to the first GPIIb calcium binding domain, was replaced by Asp and Phe. Expression of these mutants in COS-1 cells also resulted in the intracellular retention of GPIIb-IIIa, suggesting that interactions between sequences that flank the first calcium binding domain of GPIIb affect its folding. Finally, the endoplasmic reticulum chaperone BiP was detected in immunoprecipitates of GPIIb-IIIa containing GPIIb with Ala, Val, Lys, or Pro, but not Gly, at position 273. This suggests that although BiP binding is a sensitive indication of the fidelity of GPIIb-IIIa folding, it is not sufficient to account for the intracellular retention of the heterodimer.
3215	Characterization of a monoclonal antibody reacting with the free human luteinizing hormone beta-subunit.	8958785	Measurement of serum luteinizing hormone (hLH) is important for the detection and follow-up of patients with pathological processes of the reproductive axis. Detection of the uncombined form of the beta-subunit of hLH, or free hLH beta, has proved to be of clinical interest in the recognition of gonadotroph adenomas. As no monoclonal antibody specific for the free hLH beta is at present available, we elicited monoclonal antibodies using free hLH beta as an immunogen. An antibody, named BLH01, was selected for its specific binding to the free beta-subunit, and its antibody-binding site was characterized at the molecular level, emphasizing the importance of amino acid residues located between the disulfide-bonded Cys93 and Cys100. This region has been demonstrated as being particularly critical for the specific binding of lutropic hormones to their receptors. Topographic assignment of the epitope recognized by BLH01 was then achieved by cross-matching studies based on a library of antibodies directed to hLH beta, and the location of some epitopes on the three-dimensional model of the beta-subunit is proposed. A two-site immunoassay based on BLH01 as capture antibody was then developed. This assay, using BLH01, may constitute a simple, sensitive and highly specific procedure for assessing the clinical usefulness of measuring free hLH beta, particularly for the diagnosis and follow-up of patients presenting with pituitary adenomas.
3216	Successful treatment of non-familial haemophagocytic lymphohistiocytosis with interferon and gammaglobulin.	8957958	Two cases of non-familial haemophagocytic lymphohistiocytosis (HLH) are presented in which treatment with interferon alfa and gammaglobulin was associated with complete clinical remission. In one case, serological evidence of recent Epstein-Barr virus infection was found. Natural killer cell activity was within normal limits in both children, compatible with a secondary form of HLH. The combination of interferon alfa and intravenous gammaglobulin requires further evaluation in the treatment of non-familial HLH.
3217	Net (ERP/SAP2) one of the Ras-inducible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif.	8918463	The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation.
3218	In vivo bioactivities and clearance patterns of highly purified human luteinizing hormone isoforms.	8895353	Previous studies have shown that highly purified isoforms of human pituitary LH exhibited a 20-fold range of in vitro bioactivities. The aim of this study was to determine the corresponding plasma half-lives, metabolic clearance rates (MCR), and in vivo bioactivities of these human (h) LH isoforms. Cannulated adult male rats were administered hLH isoforms as a bolus i.v. injection. For the half-life studies, blood was then serially collected over a 6-h period, and serum was assayed for hLH using a specific immunofluorometric assay. All hLH (n = 19) isoforms exhibited biexponential disappearance profiles with an initial fast half-life (t 1/2) for component A of 12.8 +/- 3.7 min, followed by a slow component B with t 1/2 of 58.9 +/- 4.4 min. The prevalence of component B in relation to component A increased significantly (r = 0.81, P < 0.001) over a 3-fold range when correlated with the sialic acid content of the isoform. Similarly, the MCR showed a significant correlation (r = 0.77, P < 0.001) with sialic acid content. The basis for the two t 1/2 components was then investigated. In the first experiment, rat plasma containing primarily component B was collected 90 min after hLH isoform administration and injected into a second animal. Only component B was observed with no evidence of component A, which indicates that the two t 1/2 components are not the product of the redistribution of the hLH isoform between body compartments. In the second experiment, component B was found to be dependent on sialic acid content, as desialylated hLH isoforms showed a rapid disappearance (t 1/2 = 8.6 +/- 3.1) with the component B proportion decreasing to < 10% of that of the nondesialylated control. This data indicates that sialic acid protects component B from rapid clearance. In addition, the proportion of the two components is dependent on sialic acid content, suggesting that the molecular location of the sialic acid on the carbohydrate moieties of hLH has a critical role in the clearance process. To determine the in vivo bioactivity of the hLH isoforms, an acute in vivo bioassay was developed in male rats. The assay was based on the hLH dose-dependent increase in total testosterone release in the same rat model as used in the plasma disappearance studies. Using the second International Standard (IS) hLH (0.3 IU-2.6 IU/kg) as standard, a linear dose-response of 24-h integrated serum testosterone levels was observed, with an index of precision of 0.11. Using this in vivo assay, a 16-fold range in in vivo bioactivities (3,200 to 51,100 IU/mg) was observed for 14 hLH isoforms. These in vivo bioactivities correlated with sialic acid content (r = 0.78, P < 0.001), MCR (r = 0.56, P < 0.05) and LH in vitro bioactivity (r = 0.75, P < 0.001) as determined using mouse Leydig cells in culture. Desialylation lead to over a 100-fold decrease in in vivo bioactivity of hLH. It is concluded that hLH isoforms are cleared in vivo by a two-component clearance mechanism, the proportion of which varies between isoforms and is dependent on sialic acid content of the isoform. These findings suggest that the molecular location of sialic acid on the hLH isoform is critical in defining the plasma disappearance of component B, whereas the mechanism of elimination of component A may well involve the hepatic GalNAc-sulphate receptor. Using an in vivo bioassay, the 16-fold difference in bioactivity between isoforms is attributed primarily to differences in their in vitro activity at the cellular level with a minor influence (< 2-fold) due to differences in in vivo clearance.
3219	Characteristic immune abnormalities in hemophagocytic lymphohistiocytosis.	8888739	Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by fever, hepatosplenomegaly, pancytopenia, and infiltration of vital organs by non-Langerhans histiocytes and is rapidly fatal without early diagnosis and institution of therapy. Immune dysregulation is thought to be responsible for the disease.Extensive immune evaluation was performed on 13 consecutive patients diagnosed with HLH over a 4-year period to characterize existing immunologic abnormalities in order to improve early diagnosis. Evaluation included quantitative immunoglobulins, immunophenotyping, mitogen-induced lymphoproliferation, natural killer (NK) cell function, and cytotoxic T cell lymphocytolysis (CTL).Immunoglobulin levels showed no consistent abnormality. Immunophenotyping showed an absolute decrease in number of B cells but normal numbers and proportional distribution of T cell subsets and NK cells. Most patients demonstrated decreased proliferative responses to mitogens (10/13) and severely decreased to absent T cell cytotoxicity (11/12) and NK cytotoxic function (13/13).Our results show that while humoral immunity is essentially intact, cellular immune function is significantly impaired in the vast majority of patients with HLH. The coincident finding of profoundly decreased T cell cytotoxicity along with absent NK cytotoxicity suggests that patients with active HLH may have global cytotoxic dysfunction. Since the majority of our patients were studied prior to starting therapy, we feel that these findings reflect the pathophysiologic process and are not therapy related. Unclear from the present work is whether these findings represent primary or secondary dysfunction. We conclude from these studies that profoundly decreased CTL function and absence of NK cell function are characteristic immunologic features of HLH and may serve as additional laboratory data, in conjunction with currently proposed diagnostic guidelines, to support a diagnosis of HLH.
3220	Molecular cloning of a myoD-like gene from the parasitic nematode, Trichinella spiralis.	8898330	The infective larval stage of the nematode Trichinella spiralis is an intracellular parasite of skeletal muscle cells. Infection with T. spiralis results in dedifferentiation of the host cell and the formation of a host/parasite complex. A gene encoding a T. spiralis Helix-Loop-helix (HLH) protein with homology to the myogenic transcription factor, MyoD, and to the Caenorhabditis elegans protein, CeMyoD, has been identified and partially characterized. The tsmyd-1 gene is expressed constitutively during the muscle-larval and adult stages. A purified recombinant Tsmyd-1 protein, expressed in Escherichia coli, binds to a high affinity mouse MyoD DNA binding site in vitro. The present study describes the first HLH gene to be identified in Trichinella.
3221	Histiocyte disorders.	9039734	Histiocyte disorders are characterised by tissue infiltration with cells of monocyte/macrophage lineage, with two disorders, Langerhans' cell histiocytosis (LCH) and haemophagocytic lymphohistiocytosis (HLH) accounting for the overwhelming majority of cases in childhood and, apart from monocyte variants of acute myeloid leukaemia, histiocytic malignancy is very rare. Although both LCH and HLH are considered reactive disorders, the prognosis of these conditions differs greatly, LCH is usually self limiting, with a mortality of 10%, but HLH is usually fatal, with a mortality of over 80% in the absence of bone marrow transplantation. Increased levels of cytokines have been demonstrated in these disorders, and may be responsible for many of the clinical features: it is unclear whether histiocytes themselves, or other immune cells, particularly T lymphocytes, are the abnormal cell population. Due to the rarity of histiocyte disorders, collaborative studies are essential to improve understanding and advance treatment.
3222	ETV6 gene rearrangements in hematopoietic malignant disorders.	9031109	Chromosomal abnormalities involving the short arm of chromosome 12 have been frequently observed in a broad spectrum of hematological malignancies. Recently, a gene located in this chromosomal region and implicated in leukemogenesis was identified. The gene, called ETV6 (previously known as TEL) is a new member of the ETS family, a group of genes thought to act as transcriptional activators. The gene spans 240 kb and consists of eight exons coding for a helix-loop-helix (HLH) and a DNA-binding domain. ETV6 was originally identified in a t(5;12)(q33;p13) occurring in a chronic myelomonocytic leukemia (CMML). Recent reports, however, show its involvement in a growing number of translocations associated with myeloid as well as lymphoid leukemias. At the molecular level fusions of ETV6 with PDGFRB (5q33), ABL (9q34), MNI(22q11) and AML1(21q22) have already been identified. Analysis of these chimeric proteins indicates that distinct domains of ETV6 can be involved in different fusion products, thus ETV6 can provide transcriptional and dimerization properties for partner genes, or the gene itself can act as an altered transcriptional factor. At least two clinico-pathological entities associated with ETV6 rearrangements have emerged as distinct disorders. The first one is a chronic myeloid malignancy characterized by t(5;12)(q33;p13), monocytosis and/or eosinophilia. The second entity is a type of childhood acute lymphoblastic leukemia (ALL) hallmarked by t(12;21)(p13;q22), and is shown to be the most frequent but cytogenetically largely undetectable chromosomal anomaly in childhood ALL.
3223	Evolution of proneural atonal expression during distinct regulatory phases in the developing Drosophila eye.	8939576	Receptors of the Notch family affect the determination of many cell types. In the Drosophila eye, Notch antagonises the basic helix-loop-helix (bHLH) protein atonal, which is required for R8 photoreceptor determination. Similar antagonism between Notch and proneural bHLH proteins regulates most neural cell determination, however, it is uncertain whether the mechanisms are similar in all cases. Here, we have analyzed the sensitivity of atonal expression to Notch signalling using a temperature-sensitive Notch allele, by the expression of activated Notch or of the ligand Serrate, and by monitoring expression of the atonal-dependant gene scabrous and of the Notch-dependent Enhancer of split genes.The atonal expression pattern evolves from general "prepattern' expression, through transient "intermediate groups' to R8 precursor-specific expression. Successive phases of atonal expression differ in sensitivity to Notch. Prepattern expression of atonal is not inhibited. Inhibition begins at the intermediate group stage, corresponding to the period when atonal gene function is required for its own expression. At the transition to R8 cell-specific expression, Notch is activated in all intermediate group cells except the R8 cell precursor. R8 cells remain sensitive to inhibition in columns 0 and 1, but become less sensitive thereafter; non-R8 cells do not require Notch activity to keep atonal expression inactive. Thus, Notch signaling is coupled to atonal repression for only part of the atonal expression pattern. Accordingly, the Enhancer-of-split m delta protein is expressed reciprocally to atonal at the intermediate group and early R8 stages, but is expressed in other patterns before and after.In eye development, inhibition by Notch activity is restricted to specific phases of proneural gene expression, beginning when prepattern decays and is replaced by autoregulation. We suggest that Notch signalling inhibits atonal autoregulation, but not expression by other mechanisms, and that a transition from prepattern to autoregulation is necessary for patterning neural cell determination. Distinct neural tissues might differ in their proneural prepatterns, but use Notch in a similar mechanism.
3224	A synthetic peptide representing residues 7 to 21 of human luteinizing hormone beta-subunit binds calcium, facilitates calcium uptake by liposomes and possesses sequence similarity to calcium-binding domains of calmodulin.	8884977	We have previously demonstrated that a synthetic peptide corresponding to residues 1 to 15 of hFSH-beta-subunit (hFSH-beta-(1-15)) shares sequence homology with the calcium-binding domains of calmodulin (CaM), binds calcium with an affinity similar to that of CaM-binding loop III (CaM III) and facilitates uptake of external calcium by liposomes and cultured rat Sertoli cells. We noted a sequence homologous to hFSH-beta-(1-15) between residues 7 to 21 of the beta-subunits of both hLH and hCG. A peptide amide representing this region of hLH-beta (hLH-beta-(7-21)) and an N-terminal truncated analog (hLH-beta-(10-21)) that more closely resembles the calcium-binding loops of calmodulin were synthesized and tested for their ability to bind 45Ca2+, and facilitate uptake of 45Ca2+ by liposomes. hLH-beta-(7-21) and hLH-beta-(10-21) bound significant amounts of 45Ca2+ with affinities of 1.2 +/- 0.4 and 0.7 +/- 0.3 mM, respectively, values virtually identical to that reported for a synthetic peptide corresponding to CaM III. The two peptides also facilitated specific, concentration-related and saturable uptake of 45Ca2+ by liposomes, which was sensitive to blockade by voltage-independent calcium-channel antagonists. Our data suggest that residues 7 to 21 of the beta-subunit of hLH may be associated with the previously demonstrated calcium requirement for specific binding of LH to its receptor, and that the calcium-binding property of this region is related to its ability to facilitate uptake of calcium into liposomes via the formation of calcium-conducting transmembrane channels.
3225	Point mutations of the Mxil gene are rare in prostate cancers.	8827088	Overexpression of the Myc genes promote cellular transformation. Max protein exerts a pivotal function with the Myc family, and Mxi1/Mad proteins play a positive and negative activity, respectively, on transcription. The function of Mxi1 suggests that it might be a tumor suppressor protein. The Mxi1 gene map to 10p24-25 and deletions of this locus are frequently observed in prostate cancers. The N-terminal helical motif, helix-loop-helix (HLH) and leucine zipper (ZIP) regions of the Mxi1 gene are functionally important. We analyzed most of the coding region of the Mxi1 gene, including these three important regions in 32 prostate cancers and three cell lines by PCR-single strand conformational polymorphism (SSCP). To enrich neoplastic cells, the microdissection was performed on clinical samples. We detected a silent mutation in the HLH region, but no point mutations reflecting the functional change of Mxi1 were found in human prostate cancers. Point mutations of the Mxi1 gene, if they occur, must be minor events in primary prostate cancers.
3226	High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA.	8811096	RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.
3227	Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during imaginal development.	8787746	The basic helix-loop-helix proteins of the Enhancer of split complex constitute a link between activation of the transmembrane receptor Notch and the resulting effects on transcription of downstream genes. The Suppressor of Hairless protein is the intermediary between Notch activation and expression of all Enhancer of split genes even though individual genes have distinct patterns of expression in imaginal discs. A comparison between the phenotypes produced by Notch, Suppressor of Hairless and Enhancer of split mutations in the wing and thorax indicate that Suppressor of Hairless and Notch requirements are indistinguishable, but that Enhancer of split activity is only essential for a subset of developmental processes involving Notch function. Likewise, the ectopic expression of Enhancer of split proteins does not reproduce all the consequences typical of ectopic Notch activation. We suggest that the Notch pathway bifurcates after the activation of Suppressor of Hairless and that Enhancer of split activity is not required when the consequence of Notch function is the transcriptional activation of downstream genes. Transcriptional activation mediated by Suppressor of Hairless and transcriptional repression mediated by Enhancer of split could provide greater diversity in the response of individual genes to Notch activity.
3228	Sin3 corepressor function in Myc-induced transcription and transformation.	8710905	Many basic-helix-loop-helix-leucine zipper (b-HLH-LZ) proteins, including the Myc family and non-Myc family, bind a common DNA sequence CACGTG, yet have quite different biological actions. Myc binds this sequence as a heterodimer with Max in the activation of both transcription and transformation. The Myc family members Mad and Mxi1 are known to suppress Myc-induced transcription and transformation and to dimerize with Max to form ternary complexes with the mammalian Sin3 transcriptional corepressor (mSin3). The b-HLH-LZ domain of TFEB, which cannot heterodimerize within the Myc family, does not suppress Myc-induced transcription or transformation. However, transfer of a 25- to 36-aa region from Mad or Mxi1, which interacts with mSin3, to the b-HLH-LZ of TFEB, mediated profound suppression of Myc-induced transcription and transformation. These results suggest that the DNA binding specificities of the Myc family and non-Myc family b-HLH-LZ proteins, in the context of the cellular genes involved in Myc-induced transformation, are shared. The results also demonstrate that targeting mSin3 to CACGTG sites via a non-Myc family DNA binding domain is sufficient to oppose Myc activity in growth regulation.
3229	Involvement of the E2A basic helix-loop-helix protein in immunoglobulin heavy chain class switching.	8960119	The basic helix-loop-helix (bHLH) transcriptional factor E2A has previously been shown to play a critical role in early B cell development, with E2A knockout mice and Id1 transgenic mice showing an arrest at the pro-B cell stage of development. More recent data suggest that E2A, through an interaction with the immunoglobulin heavy chain 3' enhancer, might also regulate later events in B cell development such as heavy chain class switching. The patterns of E2A protein expression in secondary lymphoid tissues support a role in later stages of B cell maturation. In particular, immunostaining reveals upregulation of E2A protein in cells of the dark zone of the germinal center, the site of immunoglobulin heavy chain class switching. To examine the role of E2A in class switching, the inhibitory HLH protein Id1 was expressed in B cell lines which normally undergo spontaneous and inducible switching from IgM to IgA. The forced expression of Id1 in these cell lines effectively blocked class switching. This Id1 blockade of class switching did not occur via downregulation of immunoglobulin heavy chain germline transcription or through inhibition of cell cycling. Furthermore, Id1 inhibited spontaneous and, to a lesser degree, cytokine inducible class switching. From these data, we conclude that E2A plays an important role in the class switching process.
3230	Muscle cell differentiation is inhibited by the helix-loop-helix protein Id3.	8853903	Id3 (originally named HLH462) belongs to the Id family of the helix-loop-helix transcription factors. Members of the Id family do not contain basic DNA binding regions adjacent to the helix-loop-helix dimerization domain and are, therefore, hypothesized to act as negative regulators of other helix-loop-helix proteins by preventing the formation of functional dimers. We have investigated the potential role of Id3 in the control of muscle cell differentiation. Id3 mRNA is expressed at a high level in proliferating myoblasts and is down-regulated following induction of differentiation. We show that stable overexpression of Id3 mRNA inhibits differentiation of the Sol 8 muscle cell line. Both the HLH and COOH-terminal domains of Id3 are necessary and sufficient for its dominant-negative activity in muscle cells. DNA-binding activity present in nuclear extracts prepared from Id3-overexpressing cells was significantly reduced when compared to the wild-type or vector-transfected cells. Finally, we show by in situ hybridization that the Id3 mRNA is co-expressed with the myogenic regulatory factor myogenin in somites and developing muscle during embryogenesis, although unlike the myogenic regulatory factors, Id3 is also expressed in many other locations in the embryo. These data support a model in which Id3 negatively regulates muscle differentiation by inhibiting the DNA-binding activities of the myogenic regulatory factors.
3231	The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands.	8756291	We examined the function of the intracellular domains of the two known Drosophila Notch ligands, Delta and Serrate, by expressing wild-type and mutant forms in the developing Drosophila eye under the sevenless promoter. The expression of intracellularly truncated forms of either Delta (sev-DlTM) or Serrate (sev-SerTM) leads to extra photoreceptor phenotypes, similar to the eye phenotypes associated with loss-of-function mutations of either Notch or Delta. Consistent with the notion that the truncated ligands reduce. Notch signalling activity, the eye phenotypes of sev-DlTM and sev-SerTM are enhanced by loss-of-function mutations in the Notch pathway elements, Notch, Delta, mastermind, deltex and groucho, but are suppressed by a duplication of Delta or mutations in Hairless, a negative regulator of the pathway. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split m delta, a target of Notch signalling, is down-regulated by the truncated ligands highly expressed in neighbouring cells. We conclude that the truncated ligands act as antagonists of Notch signalling.
3232	Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia.	8754809	TEL is a member of the Ets family of transcription factors which are frequently rearranged in human leukemia. The mechanism of TEL-mediated transformation, however, is unknown. We report the cloning and characterization of a chromosomal translocation associated with acute myeloid leukemia which fuses TEL to the ABL tyrosine kinase. The TEL-ABL fusion confers growth factor-independent growth to the marine hematopoietic cell line Ba/F3 and transforms Rat-1 fibroblasts and primary murine bone marrow cells. TEL-ABL is constitutively tyrosine phosphorylated and localizes to the cytoskeleton. A TEL-ABL mutant containing an ABL kinase-inactivating mutation is not constitutively phosphorylated and is nontransforming but retains cytoskeletal localization. However, constitutive phosphorylation, cytoskeletal localization, and transformation are all dependent upon a highly conserved region of TEL termed the helix-loop-helix (HLH) domain. TEL-ABL formed HLH-dependent homo-oligomers in vitro, a process critical for tyrosine kinase activation. These experiments suggest that oligomerization of TEL-ABL mediated by the TEL HLH domain is required for tyrosine kinase activation, cytoskeletal localization, and transformation. These data also suggest that oligomerization of Ets proteins through the highly conserved HLH domain may represent a previously unrecognized phenomenon.
3233	Regulation of tyrosinase gene expression by cAMP in B16 melanoma cells involves two CATGTG motifs surrounding the TATA box: implication of the microphthalmia gene product.	8707852	In melanocytes and in melanoma cells, upregulation of melanogenesis, by cAMP elevating agents, results from a stimulation of tyrosinase activity that has been ascribed to an increase in tyrosinase protein and messenger amount. However, the mechanism by which cAMP elevating agents increase tyrosinase mRNA remains to be elucidated. In this study, using a luciferase reporter plasmid containing the 2.2-kb fragment 5' of the transcriptional start site of the mouse tyrosinase gene, we showed that cAMP elevating agents lead to a strong stimulation (20-fold) of transcriptional activity of the tyrosinase promoter. Deletions and mutations in the mouse tyrosinase promoter showed that the M-box 70-bp upstream from the TATA-box and the E-box located downstream the TATA-box, near to the initiator site, are involved in the regulation of the tyrosinase promoter activity by cAMP. Additionally, we showed that microphthalmia, a b-HLH transcription factor associated with pigmentation disorders in mouse, binds to these regulatory elements and modulates the transcriptional activity of the tyrosinase promoter. Since cAMP stimulates the binding of microphthalmia to the M-box and to the E-box; it is tempting to propose that microphthalmia, through its interaction with cis-acting elements surrounding the TATA-box, plays a key role in the regulation of the mouse tyrosinase gene expression by cAMP.
3234	Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes of Drosophila.	8757393	The Enhancer of split and achaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis in Drosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.
3235	Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin in mucosal cells from human fallopian tubes.	8770916	The present study investigated the regulation of cyclooxygenase-2 (COX-2) gene by human CG (hCG) in mucosal cells from human fallopian tubes. The mucosal cells contained a major [4.3 kilobase (kb)] and several minor (3.6, 2.4, and 1. 8 kb) messenger RNA (mRNA) transcripts of LH/hCG receptors and also an 80-kDa receptor protein. The receptor protein can bind 125I-hCG. Culturing mucosal cells with increasing concentrations of highly purified hCG resulted in a dose- and time-dependent increase in steady-state levels of a 4.4-kb mRNA transcript and 72-kDa protein of COX-2. Whereas hLH and hCG could mimic each other in increasing COX-2 protein levels, FSH, TSH, PRL, and isolated alpha- and beta-subunits of hCG had no effect, suggesting that the hCG effect is hormone specific and requires the conformation of native hormone. Culturing mucosal cells with increasing concentrations of hCG also resulted in a dose-dependent increase in media PGE2, levels, suggesting that the COX-2 protein increased by hCG is catalytically active. To determine the molecular mechanism of hCG action responsible for increasing the steady-state COX-2 mRNA levels, we measured the transcription rate of the COX-2 gene by nuclear run-on assay and the stability of its transcripts by an actinomycin D blocking method. The results showed that although hCG treatment had no effect on the transcription rate of COX-2 gene, it significantly increased the stability of COX-2 transcripts from 3.7 h in the control to 7.3 h after treatment. In summary, we conclude that tubal mucosal cells contain LH/hCG receptor transcripts and the receptor protein that can bind hCG. Culturing these cells with exogenous hCG and LH can up-regulate the expression of COX-2 gene by increasing the stability of transcripts. Through this up-regulation, LH and hCG may influence tubal functions that are important for early pregnancy in women.
3236	Analyses of loss-of-function mutations of the MITF gene suggest that haploinsufficiency is a cause of Waardenburg syndrome type 2A.	8659547	Waardenburg syndrome type 2 (WS2) is a dominantly inherited disorder characterized by a pigmentation anomaly and hearing impairment due to lack of melanocyte. Previous work has linked a subset of families with WS2 (WS2A) to the MITF gene that encodes a transcription factor with a basic-helix-loop-helix-leucine zipper (bHLH-Zip) motif and that is involved in melanocyte differentiation. Several splice-site and missense mutations have been reported in individuals affected with WS2A. In this report, we have identified two novel point mutations in the MITF gene in affected individuals from two different families with WS2A. The two mutations (C760--> T and C895--> T) create stop codons in exons 7 and 8, respectively. Corresponding mutant alleles predict the truncated proteins lacking HLH-Zip or Zip structure. To understand how these mutations cause WS2 in heterozygotes, we generated mutant MITF cDNAs and used them for DNA-binding and luciferase reporter assays. The mutated MITF proteins lose the DNA-binding activity and fail to transactivate the promoter of tyrosinase, a melanocyte-specific enzyme. However, these mutated proteins do not appear to interfere with the activity of wild-type MITF protein in these assays, indicating that they do not show a dominant-negative effect. These findings suggest that the phenotypes of the two families with WS2A in the present study are caused by loss-of-function mutations in one of the two alleles of the MITF gene, resulting in haploinsufficiency of the MITF protein, the protein necessary for normal development of melanocytes.
3237	Elevated circulating levels of interleukin-1 receptor antagonist but not IL-1 agonists in hemophagocytic lymphohistiocytosis.	8614386	The familial form of hemophagocytic lymphohistiocytosis (HLH) is an inherited disease with disturbed immunomodulation and characterized by fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, and coagulopathy, i.e., findings which are similar to many of the reported biological effects of the inflammatory cytokines. Due to the previously shown hypercytokinemia in active HLH with elevated levels of interleukin (IL)-6, tumor necrosis factor-alpha, and interferon-gamma, it has been suggested that cytokine dysregulation may be of pathophysiological importance. Here we have assayed the serum levels of the members of the IL-1 ligand family, the two agonists IL-1 alpha and IL-1 beta and the antagonist IL-1 receptor antagonist (IL-1ra), in nine children with HLH and cerebrospinal fluid (CSF) specimens from four children. Serum IL-1ra was elevated in all patients with active disease to a degree which correlated well with disease activity. Furthermore, the levels decreased day by day during treatment of a patient who suffered a relapse. Moreover, high levels of IL-1ra were also detected in CSF during active disease. However, IL-1 beta levels were all within normal limits and circulating IL-1 alpha levels were normal in all but two patients.
3238	SV40 large T antigen interferes with adult myosin heavy chain expression, but not with differentiation of human satellite cells.	8660914	The growth of muscle fibers during late development as well as in regeneration following muscle injury is the result of the proliferation and differentiation of satellite cells. However, all human cells, including satellite cells, show a limit in their proliferation. In order to define a cellular system with enhanced proliferative capacity, human satellite cells were transfected with a construct containing large T antigen from SV40 under the control of the human vimentin promoter. Vimentin is normally expressed during proliferation, and its expression is down-regulated as differentiation proceeds. In transfected cells, the construct is regulated like the endogenous vimentin gene. The effect of exogenous T antigen expression on both the proliferation and differentiation of human satellite cells was investigated. T antigen expression reduced the doubling time of human satellite cells from 36 to 20 h and increased the final proliferative capacity from 46 to 69 mean population doublings. When differentiation was triggered, although T antigen did not prevent the formation of myotubes, fusion was delayed. A similar delay was observed in the appearance of myogenin protein, one of the HLH regulatory factors, but not in the corresponding mRNA. Finally, T antigen has an effect on adult myosin isoform expression, since both adult slow and fast isoforms were only detected in myotubes negative for T antigen. These results led us to propose a model of the possible interactions between T antigen and muscle-specific factors.
3239	Id2 expression increases with differentiation of human myeloid cells.	8652837	Id proteins are helix-loop-helix (HLH) transcriptional factors that lack the basic DNA binding domain. The Id proteins have been reported generally to function as inhibitors of cell differentiation, and their gene expression is often downregulated during cell differentiation. We examined the expression of human Id mRNAs by Northern hybridization in 11 human myeloid cell lines, several myeloid cell lines induced to differentiate, fresh myeloid leukemia samples, and normal human myeloid cells. Id2 mRNA was expressed in myelomonoblastic and monoblastic leukemic cells (PLB-985, THP-1, and U-937) but was weakly expressed in myeloblastic leukemic cells (KG-1 and HL-60). Id2 mRNA levels markedly increased with induction of differentiation of myeloid blasts (HL-60, PLB-985, THP-1, and U-937) toward either granulocytes or macrophages. Examination of fresh acute myeloid leukemic samples from 22 individuals also showed prominent Id2 mRNA expression in those samples having more differentiated blasts. Using the French-American-British classification, only 2 of 8 M0/M1 samples expressed Id2 mRNA; however, 10 of 13 M2/M3/M4 samples expressed it. In normal human myeloid cells, Id2 mRNA was expressed in cultured macrophages from bone marrow and in mature granulocytes and monocytes from peripheral blood. The half-life of Id2 mRNA was short (1 hour), and its expression was inducible by cessation of protein synthesis. Id3 mRNA was moderately expressed in monoblastic cell lines (THP-1 and U-937), and levels decreased with their differentiation. Almost no Id3 expression was detectable in either other myeloid leukemia lines, fresh leukemic samples, or normal human myeloid cells by Northern analyses. Id1 mRNA was not detected by polymerase chain reaction in either leukemic or normal myeloid cells except in K562 myeloid/erythroid cells. These results showed that Id2 mRNA was constitutively expressed in more mature myeloid blast cells and level markedly increased with terminal myeloid differentiation, suggesting that Id2 protein may inhibit an HLH transcriptional complex that normally represses myeloid differentiation.
3240	Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins.	8649774	TAL1 is a basic helix-loop-helix (bHLH) protein involved in hematopoietic development. In T cell acute lymphoblastic leukemic cells, TAL1 is aberrantly overexpressed and is thought to contribute to oncogenesis. To identify proteins that interact with TAL1 in mediating leukemogenesis, we used TAL1 as a bait in a two-hybrid interaction screen, and isolated a cDNA clone that encodes a unique GTP binding protein, DRG. The interaction between DRG and TAL1 was confirmed both in vitro and in vivo. DRG was also shown to bind in vitro to two TAL1-related proteins, TAL2 and Lyl1. Mutational analyses showed that the HLH domain of TAL1 was necessary and sufficient for its interaction with the C-terminus of DRG. Furthermore, while DRG and E47 compete to interact with TAL1, TAL1 binds to DRG and E47 in a mutually exclusive manner. In rat embryonic fibroblast transformation assays, DRG stimulated the cotransforming activity of c-myc and ras. Based on these results, DRG appears to be a potential target for TAL-like oncoproteins.
3241	[The expression of Pax7 in C2C12 cell line and its inducible inactivation].	9387795	Pax7 is a member of mouse Pax gene family. It was expressed from day 8 to 17 p. c. during murine embryogenesis, mainly restricted to the central nerve systems. Additional Pax7 expression could be followed during myogenesis from the dermamyotome of the somites to the skeletal muscle tissues. For the first time, we demonstrated that Pax7 is expressed in mouse myoblast cell line, C2 C12. The expression of Pax7 was blocked when C2 C12 cells were induced to differentiate into myotube. Moreover, cells of mesodermal origin, in particular of the mouse embryo fibroblast cell line C3 H10 T1/2 which were especially permissive to the action of myogenic HLH proteins, did not express Pax7. These results suggest that Pax7 may be a suppressive factor for the myoblast differentiation of C2 C12 cells in vitro.
3242	How is myogenesis initiated in the embryo?	8928226	Skeletal myoblasts are derived from paraxial mesoderm, but how myoblasts acquire their identity is still a matter of speculation. The characterization of molecular markers and, in some cases, the analysis of mutations in the corresponding genes, has now made it possible to ask specific questions about this process. Specification of somite cell fate depends on epigenetic factors. Adjacent tissues, such as the neural tube, notochord, dorsal ectoderm and lateral mesoderm, act either positively or negatively on the different myogenic precursor populations in the somite. Candidate molecules for this complex signalling activity include sonic hedgehog and the Wnt proteins as positive signals, and BMP4 as a possible inhibitor. Although it is generally assumed that induction is required, some observations suggest that embryonic cells might have a tendency to undergo myogenesis as a 'default' pathway. By analogy with Drosophila, where the neurogenic genes affect myogenesis, the vertebrate homologues of notch and its ligands could be candidate molecules for a repression or derepression mechanism. Similar studies with cultured muscle cells also implicate other HLH factors as potential inhibitors of the MyoD family and, hence, of inappropriate myogenesis.
3243	Mutational analyses of the extracellular domain of the full-length lutropin/choriogonadotropin receptor suggest leucine-rich repeats 1-6 are involved in hormone binding.	8776736	Previous studies have demonstrated that the amino-terminal extracellular domain of the lutropin/choriogonadotropin receptor (LHR) is sufficient for conferring high affinity binding and binding specificity. The present study was undertaken to further delineate those regions involved in hormone binding. Since the extracellular domains of the gonadotropin receptors are defined by multiple leucine-rich repeat motifs, LHR deletion mutants were constructed in which individual or multiple leucine-rich repeats were deleted from the full-length receptor. These mutants were transiently expressed in mammalian 293 cells, assayed for protein expression by Western blotting of cell extracts, and then tested for hormone binding in both intact cells and detergent-solubilized cell extracts. Western blot analyses confirmed the expression of all LHR deletion mutant proteins in the transfected cells. Although human (h) CG binding activity was not detected for any of the mutants when intact cells were assayed, mutants in which leucine-rich repeats 9-14 were collectively deleted, or repeats 7 or 8 were individually deleted, expressed binding activity when the cells were first solubilized in detergent. Of significance, rat (r) LHR(delta LRR 9-14) exhibited high affinity hCG and hLH binding, comparable to wild type rLHR. Detergent extracts from cells expressing rLHR(delta LRR 8) and rLHR(delta LRR 7) bound hCG and hLH, albeit with reduced affinities compared with the wild type receptor. All three of these deletion mutants, rLHR(delta LRR 9-14), rLHR(delta LRR 8), and rLHR(delta LRR 7), exhibited normal binding specificity as they did not bind hFSH even at very high concentrations. Thus, deletion of these regions in the carboxyl portion of the extracellular domain of the LHR did not remove any potentially inhibitory elements that would normally prevent hFSH binding. Deletion of either the 11 amino-terminal acids before LRR 1 or LRRs 1, 2, 3, 4, 5, or 6 individually from the full-length rLHR resulted in the total absence of detectable hCG binding activity in detergent-solubilized extracts, in spite of stable protein expression. These results suggest that the amino terminus and LRRs 1-6 are absolutely essential for gonadotropin binding to the rLHR.
3244	Haemophagocytic lymphohistiocytosis associated with constitutional inversion of chromosome 9.	8703807	Familial haemophagocytic lymphohistiocytosis (HLH) is considered an autosomal recessive disease, although the putative gene responsible for the disease has not yet been localized. Identification of the involved gene may elucidate the pathogenesis of the disease and is essential for prenatal testing in affected families. We present an infant with HLH and constitutional inversion 9 (p23q31) in cells from bone marrow, lymphocytes and fibroblasts. The parents had normal karyotypes. It may be speculated that one of the parents was a carrier of HLH and a de novo inversion occurred in chromosome 9 from the non-carrier parent. This would imply that the putative HLH-related gene is located at one of the two breakpoints on chromosome 9.
3245	Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphohistiocytosis.	8703806	Clinical features in patients with haemophagocytic lymphohistiocytosis (HLH) have been demonstrated to be characterized by hypercytokinaemia. Previously, we reported the impact of high serum levels of interferon (IFN)-gamma and soluble IL-2 receptor (sIL-2R) on patient outcome; however, it was not known if serum levels of interleukin (IL)-6 also could be a prognostic factor. In a study during the active phase of disease in 25 cases of HLH in children and young adults (median age 3 years, range 0.1-23 years), we noted 12 cases which showed serum IL-6 > 100 (normal < 4.0) pg/ml. Five of these cases showed hyper-IL-6-naemia alone without hyper-IFN-gamma-naemia (group A) whereas seven cases showed both hyper-IL-6- and IFN-gamma-naemia (group B). Patient outcome did not differ between the patients with IL-6 > 100 pg/ml and those with IL-6 < 100 pg/ml, suggesting that high serum concentrations of IL-6 alone do not necessarily indicate poor prognosis in patients with HLH. Among the cases with hyper-IL-6-naemia ( > 100 pg/ml), underlying disorders causing haemophagocytosis were found to be different between groups A and B.
3246	Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.	8649364	Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.
3247	Cooperative interaction between AhR.Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene.	8647831	Expression of CYP1A1 gene is regulated in a substrate-inducible manner through at least two kinds of regulatory DNA elements in addition to the TATA sequence, XRE (xenobiotic responsive element), and BTE (basic transcription element), a GC box sequence. The trans-acting factor on the XRE is a heterodimer consisting of arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt), while Sp1 acts as a regulatory factor on the BTE. We have investigated how these factors interact with one another to induce expression of the CYP1A1 gene. Both in vivo transfection assays using Drosophila Schneider line 2 (SL2) cells, which is devoid of endogenous Sp1, AhR, and Arnt, and in vitro transcription assays using baculovirus-expressed AhR, Arnt, and Sp1 proteins revealed that these factors enhanced synergistically expression of the reporter genes driven by a model CYP1A1 promoter, consisting of four repeated XRE sequences and a BTE sequence, in agreement with previous observation (Yanagida, A., Sogawa, K., Yasumoto, K., and Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-1475). We have proved by coimmunoprecipitation assays and DNase I footprinting that both AhR and Arnt interact with the zinc finger domain of Sp1 via their basic HLH/PAS domains. When either the AhR.Arnt heterodimer of Sp1 was bound to its cognate DNA element, DNA binding of the second factor was facilitated. Survey of DNA sequences in the promoter region shows that the XRE and GC box elements are commonly found in the genes whose expressions are induced by polycyclic aromatic hydrocarbons, suggesting that the two regulatory DNA elements and their cognate trans-acting factors constitute a common mechanism for induction of a group of drug-metabolizing enzymes.
3248	A homology model of the Id-3 helix-loop-helix domain as a basis for structure-function predictions.	8645731	The function of the dominant negative Id (inhibitor of differentiation) helix-loop-helix (HLH) proteins is to dimerize with, and prevent the DNA binding of basic HLH (bHLH) transcription factors. A three-dimensional homology model was constructed for the HLH domain of human Id3 based on the X-ray crystal structures of the E47, MyoD, and Max bHLH proteins. The model showed that, in contrast to bHLH proteins, Id proteins appear able to dimerize without DNA stabilization because of better packing of the hydrophobic core, and the absence of destabilizing polar loop residues and of repulsive positive charges in the monomer interface at the base of the four alpha-helix bundle. This prediction was tested by in vitro protein-binding experiments, which showed that Id3 did indeed self-associate. It also showed that the inability of Id proteins to bind DNA arises from the non-basic, poorly defined, random coil structure of the region corresponding to that responsible for bHLH DNA-binding. A model of the Id1 protein was constructed and revealed a potential site of charge-charge repulsion in the hypothetical homodimer interface that may explain its observed inability to form homodimers.
3249	Functional analysis of the human androgen receptor promoter.	8793857	Although androgen receptor levels vary widely during development, our previous studies have suggested that a single promoter is active in a number of human and rat tissues and cell lines. To examine the elements controlling androgen receptor expression, we performed deletion mapping and site-directed mutagenesis of the human androgen receptor promoter and assayed these promoter fusions in human cell lines that produce the androgen receptor as well as those deficient in the androgen receptor, both in the presence and absence of androgen. Our studies identify a region (-74 to +87) surrounding the site of transcription initiation that constitutes the core of the androgen receptor promoter. When assayed in T47D cells (an AR-expressing cell line), this segment was sufficient to direct the expression of reporter genes at levels similar to that observed for larger promoter fragments, and further deletions led to an appreciable loss of promoter activity. DNA sequences surrounding this region appear to modulate the activity of this minimal promoter in a cooperative fashion. Site-directed mutagenesis and mobility shift assays demonstrated the importance of regulatory regions upstream of the transcription initiation site including an SP1 binding site and two segments with similarities to consensus HLH protein binding sites (E-boxes). Androgen treatment did not cause a decrease in activity of any of the transiently transfected promoter fusions tested, suggesting that the promoter does not contain the information necessary for autoregulation or that a posttranscriptional or posttranslational mechanism is responsible for the regulation of AR mRNA by ligand. Furthermore, the androgen receptor promoter fusions displayed differing transcriptional activities when transfected into two cell lines deficient in androgen receptor expression suggesting heterogeneity in the mechanisms responsible for this deficiency.
3250	Replicative senescence, aging and growth-regulatory transcription factors.	8864060	Normal somatic cells invariably enter a state of permanent growth arrest and altered function after a finite number of divisions. This phenomenon is termed cellular or replicative senescence. Replicative senescence is thought to be a tumor-suppressive mechanism, and a contributing factor in aging. Three features distinguish senescent from presenescent cells: an irreversible block to cell proliferation, increased resistance to apoptotic death, and changes in differentiated functions. Senescence entails an altered pattern of gene expression, much of which is due to altered transcription. At least three growth regulatory transcriptional modulators are repressed in senescent cells: the c-fos component of the AP1 transcription factor, the Id1 and Id2 helix-loop-helix (HLH) proteins that negatively regulate basic HLH transcription factors, and the E2F-1 component of the E2F transcription factor. Failure to express any one of these modulators is very likely sufficient to arrest cell proliferation. Loss of these modulators may also explain many of the functional changes shown by senescent cells. In the case of c-fos repression, the resulting decline in AP-1 activity may be exacerbated by an altered ratio of AP-1 components to a protein known as QM or Jif. QM interacts with the c-jun component of AP-1 and suppresses AP-1 activity. We cloned QM from a senescent fibroblast cDNA library, and found that it was neither cell cycle- nor senescence-regulated. However, QM suppressed the growth of murine and human fibroblasts when overexpressed. Thus, an altered balance between positive factors (e.g., AP-1 components) and negative factors (e.g., QM) may lead to the growth arrest, as well as the changes in differentiated gene expression, that are a hallmark of senescent cells.
3251	European collaborative study of LH assay: 3. relationship of immunological reactivity, biological activity and charge of human luteinizing hormone.	8796333	This report describes the results of the third part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The aim of the present work was to establish the link between immunoreactivity and biological activity of human LH, thus allowing to determine the antigenic domains of the molecule involved in the induction of the biological effect. The relationship between immunoreactivity and electric charge of hLH was also studied. This work allowed to further apprehend hLH isomorphism and its role in discrepancies observed among hLH assays and clinical status. It also made the feasibility of measuring biologically active isoforms by an immunological method to be assessed. The effect of 36 mAb with known epitopic specificity, was evaluated on both hLH binding to rat membrane receptor and hLH induced production of testosterone by porcine Leydig cells. All the epitopes located on the beta subunit were found to be strongly involved in the biological activity whereas 4/9 and 10/18 epitopes present on the alpha subunit or specific for the holomolecule respectively appeared weakly involved. Assaying biological hLH using immunological method would require that mAb specific for all the epitopes involved in the receptor activation be tested, and thus appears presently unsuitable for routine clinical evaluation. In the previous work some LH immunoassays were found to underestimate LH concentrations (J. Endocrinol. Invest 1994, 17: 397-406 and 407-416). The mAb used in liquid phase in these kits were found in the present work to be directed against the domains of LH weakly involved in the activation of the receptor and would suggest that bioactive LH would be misevaluated by these kits. The immunoreactivity of hLH isoforms separated by isoelectric focusing (IEF) in liquid phase was also determined. IEF allowed to separate three groups of hLH isoforms but none of them exhibited a specific discriminating pattern of immunoreactivity when they were tested against a panel of mAb. It suggests that, in our experimental conditions, the electric charge and the immunoreactivity of hLH were not closely linked.
3252	Genomic organization of TEL: the human ETS-variant gene 6.	8743990	We have constructed a detailed map of the genomic region containing the ETS-variant gene 6 (ETV6), involved in translocations and deletions associated with hematologic malignancies. Thirty-eight cosmids were characterized belonging to two contigs spanning 340 kb, and an EcoRl restriction map was developed. The gap between the two contigs, 2 kb in size, was closed by PCR. The contigs contain the complete coding sequence and the 5' and 3' UTRs of ETV6. Eight exons accounting for the ETV6 cDNA sequence were identified. The helix-loop-helix (HLH) motif is coded by exons 3 and 4, whereas exons 6-8 code for the ETS DNA-binding domain. All introns show consensus 5' donor and 3' acceptor splice sites. Introns 1 and 2 span 100 and 82 kb, respectively, and introns 3-7 range from 15 to 1.3 kb. An alternative exon 1 (exon 1B) is localized in intron 2. The 5' end of the ETV6 gene is associated with a CpG island characterized by the presence of four Notl, four Sacll, and three BssHll recognition sites and several SP1- and AP2-binding motifs. Alternative polyadenylation at the 3' end of the ETV6 gene generates the three transcripts of 6200, 4300, and 2400 nucleotides, respectively. The ETV6 gene spans 240 kb and is flanked at its 5' and 3' end by D12S1697 and D12S98, respectively. The markers D12S1095 and D12S89 are located in the first intron. Two new DNA polymorphisms were identified in the ETV6 gene, which will be useful for the analysis of loss of heterozygosity reported for the ETV6 gene in leukemia.
3253	Influence of imposed feed intake patterns during lactation on reproductive performance and on circulating levels of glucose, insulin, and luteinizing hormone in primiparous sows.	8726735	Thirty-six primiparous sows were assigned to one of five treatments designed to mimic the lactation feed intake patterns observed in a previous study of commercial farms: high energy intake (HHH) or low energy intake during each week of a 3-wk lactation (LLL), or reduced intake during wk 1 (LHH), wk 2 (HLH) or wk 3 of lactation (HHL). The metabolizable energy intake of sows was either 16.5 (H) or 6.5 (L) Mcal/d. Diets were equal in lysine, providing 45 g/d. The HHH sows had a shorter (P < .05) weaning-to-estrus interval (9 +/- 3.2 d) than the sows in the LLL (23 +/- 3.5 d), HLH (22 +/- 3.5 d), and HHL (18 +/- 3.2 d) groups. The LH pulse frequencies of HHH (d 14: 1.5 pulses/8 h; d 21: 2.1 pulses/8 h) were greater (P < .05) than those of LLL (.2 pulses/8 h) and HLH (.5 pulses/8 h) on d 14 and those of LLL (0 pulses/8 h) and HHL (.9 pulses/8 h) on d 21 of lactation. No differences (P > .1) in mean serum concentrations of LH were observed among treatment groups either on d 21 of lactation or on d 1 postweaning. When fed a low-energy diet, sows had lower (P < .05) concentrations of serum insulin and plasma glucose than did sows fed a high-energy diet. Our results indicate that energy intake during lactation influences circulating insulin and glucose levels and LH pulse frequency and amplitude during midlactation and during the postweaning period.
3254	Helix-loop-helix proteins LYL1 and E2a form heterodimeric complexes with distinctive DNA-binding properties in hematolymphoid cells.	8628307	LYL1 is a basic helix-loop-helix (HLH) protein that was originally discovered because of its translocation into the beta T-cell receptor locus in an acute lymphoblastic leukemia. LYL1 is expressed in many hematolymphoid cells, with the notable exceptions of thymocytes and T cells. Using the yeast two-hybrid system to screen a cDNA library constructed from B cells, we identified the E-box-binding proteins E12 and E47 as potential lymphoid dimerization partners for LYL1. The interaction of LYL1 with E2a proteins was further characterized in vitro and shown to require the HLH motifs of both proteins. Immunoprecipitation analyses showed that in T-ALL and other cell lines, endogenous LYL1 exists in a complex with E2a proteins. A preferred DNA-binding sequence, 5'-AACAGATG(T/g)T-3', for the LYL1-E2a heterodimer was determined by PCR-assisted site selection. Endogenous protein complexes containing both LYL1 and E2a bound this sequence in various LYL1-expressing cell lines and could distinguish between the LYL1 consensus and muE2 sites. These data demonstrate that E2a proteins serve as dimerization partners for the basic HLH protein LYL1 to form complexes with distinctive DNA-binding properties and support the hypothesis that the leukemic properties of the LYL1 and TAL subfamily of HLH proteins could be mediated by recognition of a common set of target genes as heterodimeric complexes with class I HLH proteins.
3255	Contraceptive vaccines.	8736723	The basic principle of a contraceptive (or anti-fertility) vaccine is to use the body's own immune defence mechanisms to provide protection against an unplanned pregnancy. Factors such as: economic production, convenience of use, relatively long-lasting but reversible action, low failure rate, and the avoidance of mechanical devices or exogenous hormones make this approach a potentially attractive option for family planning programmes in both developing and developed countries. The major efforts in research and development have involved the prospect of active immunization against specific antigens of sperm, ovum, zygote and early embryo, and the pregnancy hormone hCG. Several anti-hCG vaccines have entered clinical trials and show promising results. These vaccines operate by preventing or interrupting pregnancy at the peri-implantation stage probably by neutralizing the luteotrophic action of hCG. The most refined vaccine is one directed against the unique C-terminal peptide on the beta-subunit of hCG. This vaccine provokes antibodies that are specific for hCG and do not cross react with hLH. Future research efforts aim to optimize the anti hCG approach, utilize new vaccine delivery systems, and broaden the spectrum of target antigens of potential utility for contraceptive vaccines.Immunological contraception has numerous potential advantages over currently available methods of family planning: choice of several sites of action in the reproductive process, including in the male; freedom from menstrual, systemic, metabolic, or endocrine sequelae; lack of interference with sexual response or activity; ease of administration, enabling distribution by paramedical personnel; sustained and defined duration of action; high intrinsic efficacy; reduced user failure; and economical production. On the other hand, diverse groups have expressed concerns about the potential for abuse by health authorities. At present, potentially feasible approaches to contraceptive vaccine development involve target antigens in the gametes, zygote, blastocyst, and early developing placenta. Recent advances in molecular genetics and hybridoma technology have provided impetus to anti-sperm and anti-ovum vaccine development. Moreover, several promising human chorionic gonadotropin (hCG) vaccines have entered clinical trials. These vaccines prevent or interrupt pregnancy at the pre-implantation stage, probably by neutralizing the luteotrophic action of hCG. Future research will attempt to optimize the anti-hCG approach, utilize new vaccine development systems, and broaden the spectrum of target antigens.
3256	Identification of a human LIM-Hox gene, hLH-2, aberrantly expressed in chronic myelogenous leukaemia and located on 9q33-34.1.	8649822	We describe the isolation of human LH-2, a putative transcription factor containing two cysteine-rich regions (LIM domains) and a homeobox (Hox) DNA-binding domain. High levels of hLH-2 expression were observed in all cases of chronic myelogenous leukaemia (CML) tested, regardless of disease status. hLH-2 was mapped to chromosome 9Q33-34.1, in the same region as the reciprocal translocation that creates the BCR-ABL chimera of the Philadelphia chromosome (Ph'), the hallmark of CML; hLH-2 was retained on the derivative 9 chromosome and is therefore centromeric of c-ABL. The proximity of hLH-2 to the breakpoint on chromosome 9 raises the possibility of cis-activation by the t(9;22)(q34;q11) translocation. In addition to finding hLH-2 expression in all cases of CML, expression was observed in lymphoid malignancies and myeloid cell lines, but not in primary cases of acute myelogenous leukaemia. The role of hLH-2 in the development or progression of leukaemia is not known. However, hLH-2 may prove useful as a marker of CML for monitoring residual disease.
3257	Developmental potential of embryos produced by in-vitro fertilization from gonadotrophin-releasing hormone antagonist-treated macaques stimulated with recombinant human follicle stimulating hormone alone or in combination with luteinizing hormone.	8671277	We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.
3258	A splice variant of the ITF-2 transcript encodes a transcription factor that inhibits MyoD activity.	8631961	Proteins of the basic helix-loop-helix (bHLH) family are transcription factors that bind DNA containing the E box motif (CANNTG) found in the promoters of many muscle-specific genes. ITF-2 is a bHLH protein with widespread expression that is thought to form active heterodimers with MyoD, a muscle-specific bHLH transcription factor. We have isolated cDNAs derived from two alternatively spliced forms of mouse ITF-2, termed MITF-2A and -2B. These proteins differ in their N termini. Neither MITF-2A nor -2B transactivated the cardiac alpha-actin promoter, which contains an E box, when transfected into nonmuscle cells. In fact, MITF-2B inhibited MyoD activation of the cardiac alpha-actin promoter. This inhibitory activity required the N-terminal 83 amino acids since MITF-2A showed no inhibitory activity, and a mutant MITF-2B with deletion of the N-terminal 83 amino acids failed to inhibit MyoD-mediated transcriptional activation. MyoD activity was also inhibited by Id, a HLH protein, and this inhibition was reversed by the addition of excess E12 or MITF-2A. However, the inhibition of MyoD activity by MITF-2B was not reversed with E12 or MITF-2A. While Id is thought to inhibit MyoD by binding and sequestering potential dimerization partners, MITF-2B appears to inhibit MyoD activity by forming an inactive heterodimer with MyoD. Thus, differentially spliced transcripts of mouse ITF-2 encode different proteins that appear to dimerize with MyoD and activate or repress transcription.
3259	Discrimination between different E-box-binding proteins at an endogenous target gene of c-myc.	8600028	c-myc plans a key role in regulating mammalian cell proliferation and apoptosis. The gene codes for a transcription factor, Myc, that belongs to the helix-loop-helix/leucine zipper (HLH/LZ) family of proteins. Myc heterodimerizes with a partner protein termed Max; the heterodimeric complex binds to CAC(G/A)TG (E-box) sequences and activates transcription from these sites. However, several other HLH/LZ proteins, including USF and TFE-3, bind to and trans-activate from the same element, yet have no documented effect on cell proliferation or apoptosis. Therefore, it is likely that mechanisms exist that discriminate between these proteins for activation of natural target genes of Myc. We now show that trans-activation from the E-box in the rat prothymosin-alpha intron enhancer is indeed specific for Myc, and identify both the distance from the start site of transcription and a second E-box element adjacent to that recognized by Myc as critical determinants of specificity. Surprisingly, transcription activation domains required for Myc to activate from this distal enhancer position differ from previously mapped domains and closely correlate with those domains essential for transformation. As observed in transformation assays, Myc and Max strongly synergize in activation from a distal enhancer position. Our data suggest that trans-activation from the prothymosin intron enhancer is a faithful reflection of the transforming properties of the Myc protein.
3260	Regulation of Id-HLH transcription factor function in third messenger signalling.	8674722	NaN
3261	Hemophagocytic lymphohistiocytosis. Report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society.	8637226	Hemophagocytic lymphohistiocytosis (HLH) is a rare, often fatal, disease of early infancy. The diagnosis of HLH is frequently delayed or made at autopsy because no genetic or biologic marker has been identified. To improve the classification and treatment of HLH, the Histiocyte Society has established an 'International Registry for HLH'. Data collected included family history, clinical and laboratory features at the onset of illness, and treatment outcome. Stringent diagnostic criteria (ie fever, splenomegaly, cytopenia, hypertriglyceridemia, and/or hypofibrinogenemia, and hemophagocytosis without evidence of malignancy) were used for patient selection. One hundred and twenty-two patients (61 males, 61 females) were enrolled from 17 centers in 11 countries. The rate of parental consanguinity was 24%. A positive family history was reported in 49% of cases including two pairs of affected male twins. The median age at disease onset was 2.9 months, with no difference between familial and sporadic cases. Age at onset was similar in affected sibs from 10 of 14 families, but in four up to 3-year differences were observed. Hemophagocytosis was present at diagnosis in 75%. An associated infection (usually by common viral pathogens) was reported in 50 of the 122 (41%) cases, of which 25 had familial disease. Natural killer activity was impaired in 36 of 37 patients studied. Chromosome analysis was normal in all tested patients. A decreased frequency of HLA-B7 and B8 alleles and increased frequency of HLA-B21 and DQ3 were observed. The estimated 5-year survival (SE) was 21% (18.7) for all patients. It was 66% (37.8) for patients who received allogeneic bone marrow transplant and 10.1% (9.6) for patients treated with chemotherapy alone (P=0.0001). None of the previously proposed prognostic indicators (age, associated infection, cerebrospinal fluid pleocytosis, family history) correlated with treatment outcome.
3262	The functions of Myc in cell cycle progression and apoptosis.	9552384	c-myc has emerged as one of the central regulators of mammalian cell proliferation. The gene encodes a transcription factor of the HLH/leucine zipper family of proteins that activates transcription as part of a heteromeric complex with a protein termed Max. In mammalian fibroblasts, Myc acts as an upstream regulator of cyclin-dependent kinases and functionally antagonises the action of at least one cdk inhibitor, p27. Myc also induces cells to undergo apoptosis, and the relationship between Myc-induced cell cycle entry and apoptosis is discussed.
3263	[The testis as eutopic production site of human growth hormone, placental lactogen and prolactin: possible autocrine/paracrine effects on testicular function].	89927878992785	The human (h) protein hormones, growth hormone (hGH-N) and prolactin (hPRL), are mainly produced in the pituitary, whereas the human placenta expresses the other four members of the protein hormone gene family, designated placental lactogens (PL-A, PL-B, PL-L) and growth hormone variant (GH-V), GH-N stimulates somatic growth, supports nitrogen-, phosphate- and potassium retention and promotes lipolytic and anabolic metabolism, whereas PRL acts on the mammary gland and induces mammogenesis, lactogenesis and galactopoesis. Both hyperprolactinemia and growth hormone deficiency affect the onset of puberty and reproduction in man and mice. In addition to the glycoprotein hormones, these hormones play a role in the maintenance of testicular function. Our group previously demonstrated eutopic production of glycoprotein hormones hLH (human luteinizing hormone) and hCG (human chorionic gonadotropin) in the testis. We have now extended our investigations to the local testicular expression of protein hormones. By means of the molecular biology techniques of the reverse transcription-polymerase chain reaction (RT-PCR), Southern blot and by restriction endonuclease analyses of the generated PCR products we demonstrated the eutopic expression of genes coding for the protein hormones. GH-N gene transcripts were detected only in the pituitary and abundant PL-A/B and a few GH-V gene transcripts were demonstrable in the placenta. In contrast, in the testis GH/PL and PRL genes are transcribed. Since testicular protein hormone gene expression is rather low, these hormones may act locally and not as systemic factors; they presumably modulate the LH/CG-mediated testosterone biosynthesis and/or may act on the spermatogenesis.
3264	The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts.	8934878	Normal somatic cells of higher organisms do not divide indefinitely. After a finite number of divisions, normal cells irreversibly cease proliferation by a process termed replicative or cellular senescence. Replicative senescence is controlled by multiple, dominant-acting genes about which very little is known. The only genes known to reactivate DNA synthesis in senescent cells are viral oncogenes encoding proteins that bind and inactivate the p53 and retinoblastoma (pRb) tumor suppressor proteins. SV40 T antigen is the best studied of these viral oncoproteins. T[K1] is a T antigen point mutant that selectively is defective in binding pRb and the pRb-related proteins p107 and p130. We show that T[K1] stimulated quiescent human fibroblasts to synthesize DNA nearly as well as wild-type T but was incapable of stimulating senescent cells. We tested several growth regulatory genes that are repressed in senescent cells for ability to restore activity to T[K1]. These included c-fos, c-jun, Id-1, Id-2, E2F-1, and cdc2. Only the helix-loop-helix (HLH) protein, Id-1, restored the ability of T[K1] to reactivate DNA synthesis in senescent cells. This activity of Id-1 was not shared by Id-2, a related protein, and depended on an intact HLH domain. It did not appear that Id-1 interacted directly with pRb or p107. Constitutive Id-1 expression failed to rescue proliferating cells from growth inhibition by pRb, p107, or p130, and failed to interact with pRb in the yeast two hybrid system. Because Id proteins negatively regulate basic-HLH (bHLH) transcription factors, we suggest that senescent cells express one or more bHLH factor that cooperates with pRb, or pRb-related proteins, to suppress proliferation.
3265	Monoclonal antimyogenin antibodies define epitopes outside the bHLH domain where binding interferes with protein-protein and protein-DNA interactions.	8900045	We have developed a panel of monoclonal antibodies against rat myogenin, a skeletal muscle regulatory protein of the bHLH family. Some of these monoclonals have been widely used by others, and details of their production are presented. Mapping the epitopes by immunoprecipitation of myogenin deletion mutants demonstrates that this panel recognizes epitopes spanning the entire molecule outside the HLH region. Four antibodies against epitopes outside the bHLH region interfere with the binding of myogenin/E-protein heterodimers to DNA sequences containing the myogenin heterodimer consensus recognition site. Three of these epitopes are partially masked in the heterodimers; antibody binding to these epitopes reduces the interactions between myogenin and E12. This suggests that surfaces outside the HLH dimerization domain may contribute to the stability of myogenin/E12 complexes. The binding of one antibody to its epitope did not appear to affect the myogenin/E12 interaction but nonetheless interfered with the binding of the complex to DNA, suggesting that this epitope lies near to a surface occupied by DNA.
3266	A one-step, separation-free amperometric enzyme immunosensor.	8746187	A new one-step, separation-free, amperometric enzyme immunosensor is described. The sensor consists of an antibody electrode that is low cost, disposable, and operates without washing or separation steps. The immunosensor combines the following signal-amplification systems: enzyme-channeling immunoassay; accumulation of the redox mediators (I2/I-); cyclic regeneration of an enzyme (peroxidase) substrate at the (polyethylenimine) polymer/electrode interface; and control of the hydrodynamic conditions at the interface of the antibody electrode. The immunological reactions were monitored electrochemically in situ, and the binding curves were directly visualized on a computer screen. The complete immunoassay can be performed in 5-20 min depending on the complexity of the immunological reactions. Model systems using rabbit IgG and human luteinizing hormone (hLH) in a 'sandwich' immunoassay revealed that the immunosensor can detect concentrations of hLH in human serum as low as 1 ng ml-1.
3267	Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signalling in Drosophila.	8565827	Like the neuroblasts of the central nervous system, sensory organ precursors of the peripheral nervous system of the Drosophila thorax arise as single spaced cells. However, groups of cells initially have neural potential as visualized by the expression of the proneural genes achaete and scute. A class of genes, known as the 'neurogenic genes', function to restrict the proportion of cells that differentiate as sensory organ precursors. They mediate cell communication between the competent cells by means of an inhibitory signal, Delta, that is transduced through the Notch receptor and results in a cessation of achaete-scute activity. Here we show that mutation of either the bHLH-encoding genes of the Enhancer of split complex (E(spl)-C) or groucho, like Notch or Delta mutants, cause an overproduction of sensory organ precursors at the expense of epidermis. The mutant cells behave antonomously suggesting that the corresponding gene products are required for reception of the inhibitory signal. Epistasis experiments place both E(spl)-C bHLH-encoding genes and groucho downstream of Notch and upstream of achaete and scute, consistent with the idea that they are part of the Notch signalling cascade. Since all competent cells produce both the receptor and its ligand, it was postulated that Notch and Delta are linked within each cell by a feedback loop. We show, that, like mutant Notch cells, cells mutant for E(spl)-C bHLH-encoding genes or groucho inhibit neighbouring wild-type cells causing them to adopt the epidermal fate. This inhibition requires the genes of the achaete-scute complex (AS-C) which must therefore regulate the signal Delta. Thus there is a regulatory loop between Notch and Delta that is under the transcriptional control of the E(spl)-C and AS-C genes.
3268	Splicing removes the Caenorhabditis elegans transposon Tc1 from most mutant pre-mRNAs.	8524324	The transposable element Tc1 is responsible for most spontaneous mutations that occur in many Caenorhabditis elegans strains. We analyzed the abundance and sequence of mRNAs expressed from five different Tc1 insertions within either hlh-1 (a MyoD homolog) or unc-54 (a myosin heavy chain gene). Each of the mutants expresses substantial quantities of mature mRNA in which most or all of Tc1 has been removed by splicing. Such mRNAs contain small insertions of Tc1 sequences and/or deletions of target gene sequences at the resulting spliced junctions. Most of these mutant mRNAs do not contain premature stop codons, and many are translated in frame to produce proteins that are functional in vivo. The number and variety of splice sites used to remove Tc1 from these mutant pre-mRNAs are remarkable. Two-thirds of the Tc1-containing introns removed from hlh-1 and unc-54 lack either the 5'-GU or AG-3' dinucleotides typically found at the termini of eukaryotic introns. We conclude that splicing to remove Tc1 from mutant pre-mRNAs allows many Tc1 insertions to be phenotypically silent. Such mRNA processing may help Tc1 escape negative selection.
3269	The AD1 transactivation domain of E2A contains a highly conserved helix which is required for its activity in both Saccharomyces cerevisiae and mammalian cells.	8524288	A conserved region, designated the AD1 domain, is present in a class of helix-loop-helix (HLH) proteins, E proteins, that includes E12, E47, HEB, E2-2, and a Xenopus laevis HLH protein closely related to E12. We demonstrate that the AD1 domain in E2A and the conserved region of E2-2 activate transcription in both yeast and mammalian cells. The AD1 domain contains a highly conserved putative helix that is crucial for its transactivation properties. Circular dichroism spectroscopy data show that AD1 is structured and contains distinctive helical properties. In addition, we show that a synthetic peptide corresponding to the conserved region is unstructured in aqueous solution at neutral pH but can adopt an alpha-helical conformation in the presence of the hydrophobic solvent trifluoroethanol. Amino acid substitutions that destabilize the helix abolish the transactivation ability of the AD1 domain. Both structural and functional analyses of AD1 reveal striking similarities to the acidic class of activators. Remarkably, when wild-type and mutant proteins are expressed in mammalian cells and Saccharomyces cerevisiae, identical patterns of transactivation are observed, suggesting that the target molecule is conserved between S. cerevisiae and mammals. These data show that transactivation by E proteins is mediated, in part, by a strikingly conserved peptide that has the ability to form a helix in a hydrophobic solvent. We propose that the unstructured domain may become helical upon interaction with its cellular target molecule.
3270	Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects.	8543157	Mammalian hairy and Enhancer of split homolog-1 (HES-1) encodes a helix-loop-helix (HLH) factor that is thought to act as a negative regulator of neurogenesis. To directly investigate the functions of HES-1 in mammalian embryogenesis, we performed a targeted disruption of the HES-1 locus. Mice homozygous for the mutation exhibited severe neurulation defects and died during gestation or just after birth. In the developing brain of HES-1-null embryos, expression of the neural differentiation factor Mash-1 and other neural HLH factors was up-regulated and postmitotic neurons appeared prematurely. These results suggest that HES-1 normally controls the proper timing of neurogenesis and regulates neural tube morphogenesis.
3271	MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.	8524857	MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.
3272	HLH beta core fragment immunoreactivity in the urine of ovulating women: a sensitive and specific immunometric assay for its detection.	21153216	Recently, we isolated an hLH beta core fragment (hLHβcf) from human pituitaries. This molecule is homologous to the hCG beta core fragment (hCGβcf), which may be a marker of normal pregnancy, Down syndrome, and certain cancers. We now report antibodies to the hLHβcf, four of which have been applied in sensitive immunoradiometric assays for urinary measurements. One of the antibodies recognizes an epitope on the hLHβcf, which is not present on the hCGβcf, hLH, or hLHβ. This specific hLHβcf antibody acts cooperatively with other newly-developed antibodies reported here to produce an assay with a sensitivity of 1 fmol/ml of hLHβcf. The specificity of these new IRMA systems will make it possible to measure the hLHβcf in urine in the presence of hLH, hLH beta, or the hCGβcf. Although the hLHβcf used to develop specific antibodies was purified from pituitaries, the assays developed recognize this metabolite in urine. Measurements of heterodimeric hLH as compared to hLHβcf in the urine of cycling women indicated that the concentration of hLHβcf rose as high as 6-7 times the concentration of hLH starting a day after the midcycle surge. The new measuring systems allow the precise quantitation of this hLH metabolite in urine.
3273	Expression of Id2 and Id3 mRNA in human lymphocytes.	8632670	Helix-loop-helix (HLH) transcription factors are involved in cellular growth and differentiation. The Id (inhibitor of DNA binding and differentiation) HLH proteins, in a dominantly negative fashion, regulate transcriptional activities of basic HLH proteins. We examined by northern hybridization the expression of Id2 and Id3 mRNA in human leukemia/lymphoma lines and patient samples, as well as resting and activated normal human lymphocytes from peripheral blood (PBL). The Id2 mRNA was abundantly expressed in 5/12 T-cell and 3/4 B-cell lines, and Id3 mRNA was detected in 4/12 T-cell and 3/4 B-cell lines. Interestingly, Id2, but not Id3, mRNA was strongly expressed in 4/5 T-cell lines infected with human T-cell leukemia virus type I (HTLV-I) (ATL-1k, MT-2, S-LB1) and type II (Mo). Another unexpected finding was that T-cell leukemias and T-cell lines often expressed either Id2 or Id3 mRNA. In addition, resting PBL constitutively expressed prominent levels of Id2 mRNA, but not Id3 mRNA. Upon PHA-stimulation, Id2 expression decreased and Id3 levels increased with biphasic kinetics. Taken together, our studies revealed three unexpected findings which require further analysis: (1) expression of Id2 mRNA is often associated with lymphocytic transformation by HTLV-I or -II; (2) T-cells usually express either Id2 or Id3 mRNA, but B-cells often express both simultaneously; (3) non-dividing, normal PBL express high levels of Id2 and no Id3 mRNA; and with the onset of cellular proliferation, levels of Id2 mRNA decrease while levels of Id3 mRNA increase, suggesting that regulation of expression of these closely related genes is disparate.
3274	Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression.	8524259	The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix (bHLH) proteins function as transcriptional repressors. We have examined the mechanisms of repression used by the Hairy and E(SPL) proteins by assaying the antagonism between wild-type or altered Hairy/E(SPL) and Scute bHLH proteins during sex determination in Drosophila melanogaster. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. We show that the Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. In addition, we show that a Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity in our assays. We propose that transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.
3275	Induction of apoptosis by the c-Myc helix-loop-helix/leucine zipper domain in mouse 3T3-L1 fibroblasts.	7499403	The cellular proto-oncogene c-myc is involved in cell proliferation and transformation but is also implicated in the induction of programmed cell death (apoptosis). The c-Myc protein is a transcriptional activator with a carboxyl-terminal basic region/helix-loop-helix (HLH)/leucine zipper (LZ) domain. It forms heterodimers with the HLH/LZ protein Max and transactivates gene expression after binding DNA E-box elements. We have studied the phenotype of dominant-negative mutants of c-Myc and Max in microinjection experiments. Max mutants with a deleted or mutated basic region inhibited DNA synthesis in serum-stimulated 3T3-L1 mouse fibroblasts. In contrast, mutants of c-Myc expressing only the basic region/HLH/LZ or HLH/LZ domains rapidly induced apoptosis at low and high serum levels. Co-expression of the HLH/LZ domains of c-Myc and Max failed to do so. We suggest that the c-Myc HLH/LZ domain induces apoptosis by specific interaction with cellular factors different to Max.
3276	Preferential heterodimeric parallel coiled-coil formation by synthetic Max and c-Myc leucine zippers: a description of putative electrostatic interactions responsible for the specificity of heterodimerization.	7490766	The oncoprotein c-Myc must heterodimerize with Max to bind DNA and perform its oncogenic activity. The c-Myc-Max heterodimer binds DNA through a basic helix-loop-helix leucine zipper (b-HLH-zip) motif and it is proposed that leucine zipper domains could, in concert with the HLH regions, provide the specificity and stability of the b-HLH-zip motif. In this context, we have synthesized the peptides corresponding to the leucine zipper domains of Max and c-Myc with a N-terminal Cys-Gly-Gly linker and studied their dimerization behavior using reversed-phase HPLC and CD spectroscopy. The preferential formation of a fully helical parallel c-Myc-Max heterodimeric coiled-coil was observed under air-oxidation and redox conditions at neutral pH. We show that the stability and the helicity of the disulfide-linked c-Myc-Max heterostranded coiled-coil is modulated by pH, with a maximum around pH 4.5, supporting the existence of stabilizing and specific interhelical electrostatic interactions. We present a molecular model of the c-Myc-Max heterostranded coiled-coil describing potential electrostatic interactions responsible for the specificity of the interaction, the main feature being putative buried electrostatic interactions between a histidine side-chain (in the Max leucine zipper) and two glutamic acid side-chains (in the c-Myc leucine zipper) at the heterodimer interface. This model is supported by the fact that the apparent pKa (as determined by [1H]-NMR spectroscopy) of this histidine side-chain at 25 degrees C is 0.42 (+/- 0.05) pKa units higher in the folded form than in the unfolded form. This indicates that the charged histidine side-chain contributes approximately 0.57 (+/- 0.07) kcal/mol (2.38 (+/- 0.30) kJ/mol) of stabilization free energy to the c-Myc-Max heterostranded coiled-coil through favorable electrostatic interaction.
3277	Identification of non-tissue-specific helix-loop-helix genes in Xenopus laevis.	8522197	We have isolated four non-tissue-specific helix-loop-helix (HLH) transcription factors from Xenopus laevis (Xl). While some are clearly orthologous to known mammalian HLH proteins, others have dramatic amino-acid differences in otherwise highly conserved protein domains. We propose that these changes arose following the tetraploidization of Xl approx. 30 Myr ago.
3278	Protein characterization and targeted disruption of Grg, a mouse gene related to the groucho transcript of the Drosophila Enhancer of split complex.	8573724	The Grg gene encodes a 197 amino acid protein homologous to the amino-terminal domain of the product of the groucho gene of the Drosophila Enhancer of split complex. Analysis with a polyclonal antisera specific for the Grg protein revealed that Grg is a 25 kd nuclear protein that can participate in specific protein-protein interactions. A null mutation of the Grg gene was constructed by gene targeting. Mice homozygous for this mutation completed embryogenesis and were born, but exhibited varying degrees of post-natal growth deficiency. No dosage-sensitive genetic interaction was detected between the Notch1 and Grg genes in mice heterozygous for a Notch1 mutant allele and homozygous for the Grg null mutation.
3279	Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity.	7590239	We have investigated the functional relationships among three loci that are required for multiple alternative cell fate decisions during adult peripheral neurogenesis in Drosophila: Notch (N), which encodes a transmembrane receptor protein, Suppressor of Hairless [Su(H)], which encodes a DNA-binding transcription factor, and the Enhancer of split gene complex [E(spl)-C], which includes seven transcription units that encode basic helix-loop-helix (bHLH) repressor proteins. We describe several lines of evidence establishing that Su(H) directly activates transcription of E(spl)-C genes in response to N receptor activity. Expression of an activated form of the N receptor leads to elevated and ectopic E(spl)-C transcript accumulation and promoter activity in imaginal discs. We show that the proximal upstream regions of three E(spl)-C genes contain multiple specific binding sites for Su(H). The integrity of these sites, as well as Su(H) gene activity, are required not only for normal levels of expression of E(spl)-C genes in imaginal disc proneural clusters, but also for their transcriptional response to hyperactivity of the N receptor. Our results establish Su(H) as a direct regulatory link between N receptor activity and the expression of E(spl)-C genes, extending the known linear structure of the N cell-cell signaling pathway.
3280	Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.	7565756	Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression.
3281	Helix-loop-helix transcription factors regulate Id2 gene promoter activity.	7589553	Id-like helix-loop-helix (HLH) transcription factors are involved in the regulation of proliferation and differentiation of several cell types. We isolated 5' regulatory region of mouse Id2 gene and demonstrated that it contains several E-box clusters. These E-boxes mediate stimulatory effects of basic-HLH (bHLH) transcription factors ME1, ME2, and NSCL1 on Id2 promoter activity. Co-expression of Id2 blocks the stimulatory effect of bHLH transcription factors which suggests the presence of feedback loops in Id2 transcriptional regulation. Overexpression of NSCL1 in F9 cells blocks the downregulation of Id2 gene expression during retinoic acid induced differentiation. Our data demonstrate that bHLH transcription factors regulate Id2 gene expression.
3282	MIDA1, a protein associated with Id, regulates cell growth.	7559602	We have isolated cDNA clone encoding a protein that can associate with Id, a helix-loop-helix (HLH) protein. This protein is named MIDA1 (Mouse Id Associate 1), and its predicted amino acid sequence consists of Zuotin (a putative Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats similar to c-Myb oncoprotein. MIDA1 associates with the HLH region of Id with the conserved region adjacent to eukaryotic DnaJ conserved motif within the Zuotin homology region, although it does not have any canonical HLH motif. The addition of antisense oligonucleotide of MIDA1 inhibited growth of murine erythroleukemia cells without interfering with erythroid differentiation, indicating that it regulates cell growth.
3283	Engineering magnesium selectivity in the helix-loop-helix calcium-binding motif.	7487056	Engineering magnesium selectivity into the helix-loop-helix (hlh) cation binding site is relatively unstudied in the calmodulin superfamily of calcium-regulated proteins, which include parvalbumin, oncomodulin, troponin C, calbindin, and calmodulin. Studies using a 33-residue synthetic peptide model of the hlh cation binding motif have indicated that magnesium will induce structural change in those peptide motifs containing three or four acid residues in chelating positions with a single-acid-pair on the Z-axis. Decreasing the cation binding cavity size in Z-axis acid-paired motifs through replacement of chelating residues in the +Z or -X metal ion coordinating positions in the loop region by glutamic acid has been successful in decreasing the calcium ion affinity. The same changes did not create or enhance magnesium binding in the 33-residue model hlh cation binding motif.
3284	The leucine zippers of the HLH-LZ proteins Max and c-Myc preferentially form heterodimers.	7577944	c-Myc and Max are members of a subfamily of the helix-loop-helix transcription-regulating proteins. Their function is mediated by switches in the dimerization partners; c-Myc does not homodimerize in vivo but competes with Mad, another member of the subfamily, to form heterodimers with Max, leading to either activation or repression of transcription. Max is also able to form homodimers. In an attempt to identify which regions of the proteins carry the information to determine specific recognition of the dimerization partner, we have investigated the dimerization properties of synthetic peptides corresponding to the leucine zipper sequence of Max and c-Myc using circular dichroism and nuclear magnetic resonance techniques. We show that the heterodimer is obtained readily by simply mixing the peptides and that at neutral pH it is more stable than the homodimer of the Max leucine zipper. We have shown in a previous paper [Muhle-Goll, C. et al. (1994) Biochemistry 33, 11296-11306] that the leucine zipper of c-Myc does not form stable homodimers under these conditions. Thus, the leucine zipper regions of these two proteins by themselves display the same behavior as the entire proteins. However, even the heterodimer is less stable than dimers of leucine zippers of the basic leucine zipper family such as GCN4 and Fos-Jun. The specificity of the interaction between different monomers can be explained by polar interactions. We investigate the structural role of the polar and charged residues in the hydrophobic interface by molecular-modeling studies.
3285	Human chorionic gonadotropin-beta subunit gene expression in cultured human fetal and cancer cells of different types and origins.	862042586204018646720	The authors' previous investigations using living cultured human cancer cells and cells isolated from cancer tissues, analytical flow cytometry, and monoclonal antibodies directed to epitopes located in five different sites of the human chorionic gonadotropin (hCG) molecule, identified the presence of membrane-associated hCG, its subunits and fragments, by cells from all cancers, irrespective of type and origin, indicating that the expression of these sialoglycoproteins is a common phenotypic characteristic of cancer. Although benign neoplasms do not express these compounds, cultured human embryonic and fetal cells also express the same materials. To corroborate these findings, five fetal cell lines and 28 cancer cell lines were randomly selected from those previously studied, to determine the presence of translatable levels of hCG-beta (hCG beta) mRNA.All cell lines were grown under identical conditions. Determination of hCG beta mRNA was made by extracting the total RNA from the cells, followed by synthesis of cDNA with RNase H- reverse transcriptase and polymerase chain reaction amplification using specific hCG beta-luteinizing hormone-beta (hLH beta) primers. The presence of amplified hCG beta cDNA was corroborated by hybridization of the product with an hCG beta-specific oligonucleotide and Southern blot analyses of the hybridization products. Gestational choriocarcinoma cells and HeLa adenocarcinoma of cervical cells, known producers of biologically active hCG, were positive control subjects, and human pituitary cells were used as negative control subjects.The results showed single and multiple hCG beta gene activation by the fetal cells and the different types of cancer, indicating that at any given time, there is the possibility of activation of as many as four genes of the six genes of the hCG beta-hLH beta gene cluster, even though alternative gene splicing cannot be ruled out.In addition to the authors' previous findings, the results of these studies support the concept that cancer is a problem of development and differentiation, and, to the authors' knowledge, prove definitively for the first time that synthesis and expression of hCG, its subunits, and its fragments, is a common biochemical denominator of cancer, providing the scientific basis for studies of its prevention and/or control by active and/or passive immunization against these sialoglycoproteins.
3286	Human genome--chromosome no. 19.	7585875	Chromosome 19 is short but has higher relative density of genes than other chromosomes. Increasing number of the genes coding for proteins implicated in the pathogenesis of various human diseases have been mapped on chromosome 19. Mutations of low density lipoprotein receptor (LDL-R) result in one of the most frequent mendelian inherited disorder-familial hypercholesterolemia. Mutations of insulin receptor (INSR) are causative for rare syndromes of insulin resistance and some of non insulin dependent diabetes mellitus (NIDDM). Erythropoietin receptor (EPOR) mutations are causative for rare primary familial and congenital polycythemias (PFCP). Defects of one of the largest gene in the human genome RYR 1 (ryanodine receptor gene) (> 240 kb in size) accounts for majority of malignant hyperthermia (MH) and central core disease (CCD). All these disorders represent group of receptor diseases. The amplification of GCT trinucleotide repeats in myotonic dystrophy protein kinase (DMPK) gene is causative for myotonic dystrophy (DM) and represents a new class of human gene mutations: trinucleotide repeat mutations. Apolipoprotein E (APOE) locus plays a role in pathogenesis of the late onset familial Alzheimer's disease. Translocation of EA2 gene which encodes two helix-loop-helix (HLH) transcription proteins and its fusion with PBXI or hepatic leukemia factor (HLF) leads to the leukemogenesis in subgroup of ALL. Interestingly adeno-associated virus (AAV), currently widely used as vector for gene therapy has unique capability of specific integration into human chromosome 19q.
3287	Thyrotropic action of human chorionic gonadotropin.	8563483	Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors. In these cases, greatly increased human chorionic gonadotropin (hCG) levels and suppressed TSH levels suggest that hCG has thyrotropic activity. Recent investigations have clarified the structural homology not only in the hCG and TSH molecules but also in their receptors, and this homology suggests the basis for the reactivity of hCG with the TSH receptor. The clinical significance of the thyrotropic action of hCG is now also recognized in normal pregnancy and hyperemesis gravidarum. Highly purified hLH binds to recombinant hTSH receptor and is about 10 times as potent as purified hCG in increasing cAMP. The beta-subunits of hCG and hLH share 85% sequence identity in their first 114 amino acids but differ in the carboxy-terminal peptide because hCG beta contains a 31-amino acid extension (beta-CTP). A recombinant mutant hCG that lacks beta-CTP showed almost identical potency to LH on stimulation of recombinant hTSH receptor. If intact hCG were as potent as hLH in regard to its thyrotropic activity, most pregnant women would become thyrotoxic. One of the roles of the beta-CTP may be to prevent overt hyperthyroidism in the first trimester of pregnancy when a large amount of hCG is produced by the placenta. Nicked hCG preparations, obtained from patients with trophoblastic disease or by enzymatic digestion of intact hCG, showed approximately 1.5- to 2-fold stimulation of recombinant hTSH receptor compared with intact hCG. This suggests that the thyrotropic activity of hCG may be influenced by the metabolism of the hCG molecule itself. Deglycosylation and/or desialylation of hCG enhances its thyrotropic potency. Basic hCG isoforms with lower sialic acid content extracted from hydatidiform moles were more potent in activating adenylate cyclase, and showed high bioactivity/immunoactivity (B/I) ratio in CHO cells expressing human TSH receptors. This is consistent with the finding that the beta-CTP truncated hCG with higher thyrotropic potency is substantially deglycosylated and desialylated in the beta-subunit relative to intact hCG because all four O-linked glycosylation sites occur within the missing C-terminal extension. The desialylated hCG variant also interacts directly with recombinant hTSH receptors transfected into human thyroid cancer cells. There is thyroid-stimulating activity in sera of normal pregnant women, and this correlates with serum hCG levels. The thyroid gland of normal pregnant women may be stimulated by hCG to secrete slightly excessive quantities of T4 and induce a slight suppression of TSH, perhaps being about 1 mU/L less than nongravid levels, but not high enough to induce overt hyperthyroidism. Maternal thyroid glands may secrete more thyroid hormone during early pregnancy in response to the thyrotropic activity of hCG that overrides the normal operation of the hypothalamic-pituitary-thyroid feedback system. Biochemical hyperthyroidism associated with hyperemesis gravidarum has been attributed to hCG. In patients with hyperemesis gravidarum, thyrotropic in serum correlated with hCG immunoreactivity, and the severity of vomiting as indicated by clinical and biochemical parameters correlated with the degree of thyroid stimulation. To understand the thyrotropic action of hCG, it is necessary to know whether hCG activates the same domain of the TSH receptor as does TSH. The identification of the molecular structure of the hCG isoform with the highest thyrotropic potency will resolve the enigma of gestational thyrotoxicosis and the hyperthyroidism associated with trophoblastic disease and hCG-producing tumors.
3288	Behavior of extramacrochaetae mutant cells in the morphogenesis of the Drosophila wing.	8562423	The gene extramacrochaetae (emc) encodes a non-basic Helix-loop-helix (HLH) protein that interacts and antagonises other basic-HLH proteins. The expression pattern of emc, and the phenotype of lethal emc alleles indicate that this gene is operative in several developmental processes. Here we study the requirements for emc during cell proliferation and vein differentiation in the wing. Mosaic analysis of hypomorphic conditions of emc reveals the tendency of mutant cells to proliferate along the veins as long stripes. Large clones abuting two adjacent veins obliterate the corresponding inter-vein, affecting the size and shape of the whole wing. Thus, the emc gene participates in the control of cell proliferation within inter-vein regions in the wing. Similar effects were found in the haltere and in the leg. The behavior of emc cells in genetic mosaics indicate that (1) proliferation is locally controlled within inter-vein sectors, (2) cells proliferate according to their genetic activity along preferential positions in the wing morphogenetic landscape and (3) cell proliferation in the wing is integrated by 'accommodation' between mutant and wild type cells.
3289	Basal testosterone secretion and response to human luteinizing, follicle-stimulating, and growth hormones in culture of cells isolated from testes of infants and children.	8559615	Little is known on the hormonal regulation of the early postnatal phase of Leydig cell activation in the human. Testosterone secretion by prepubertal testicular cells in culture was studied in two different age groups, 0-7-mo-old (group 1) and 16-36-mo-old (group 2) boys. A mixed cell preparation was isolated from testes collected at necropsy and maintained in culture for 6 d. Cells were cultured in serum-free medium in basal conditions and under the stimulation of human (h)LH, hFSH, or recombinant hGH, and the secretion of testosterone was determined on d 6 by RIA. In basal conditions, cells of group 1 secreted more testosterone (median 5.83 pmol/10(6) cells.d, n = 7) than cells of group 2 (median 1.73, n = 5), p < 0.05, reflecting the steroidogenic potential of the testes in vivo. Under hLH stimulation, cells of group 1 responded by increasing testosterone secretion significantly. Surprisingly, hFSH stimulation elicited a similar response in cells of group 1. Because FSH receptors are presumably located in Sertoli cells, it is concluded that these cells secreted a paracrine factor that stimulated testosterone secretion by Leydig cells. On the other hand, recombinant hGH also stimulated the secretion of testosterone by cells of group 1. Recombinant hGH could have interacted with either GH or prolactin receptors of testicular cells. Cells of group 2 did not respond to any stimulus. Because serum levels of LH, FSH, GH, and prolactin are higher during the first months of life than later in childhood, it is possible that the early postnatal activation of the testis is under multiple pituitary hormone influence.
3290	Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins.	7567454	Somatic sex determination in Drosophila depends on the expression of Sex-lethal (Sxl), whose level is determined by the relative number of X chromosomes and sets of autosomes (X:A ratio). The first step in regulation of Sxl expression is transcriptional control from its early promoter and several genes encoding transcription factors of the helix-loop-helix (HLH) family such as daughterless (da), sisterless-b (sis-b), deadpan (dpn) and extramacrochaetae (emc) have been implicated. By the use of transfection assays and in vitro binding experiments, here we show that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Interestingly, our data indicate that repression by the dpn product of da/sis-b dependent activation results from specific binding of dpn protein to a unique site within the promoter. This contrasts with the mode of emc repression, which inhibits the formation of the da/sis-b heterodimers. These results reveal the molecular mechanisms by which Sxl gene transcription is positively or negatively regulated to control somatic sex determination.
3291	Expression pattern of chick achaete-scute homologue (CASH) associated with mid-embryogenesis (stages 23 to 28).	28306060	Using the conserved basic helix-loop-helix (bHLH) domain of AS-C and MASH, we amplified by polymerase chain reaction, the HLH region of a chick AS-C/MASH homologue from cDNA prepared from poly (A+) RNA isolated from a region rich in cardiac neural crest cells. A full-length cDNA clone was obtained from a stage 23 chick head cDNA library using this conserved sequence as a probe. Comparison of this clone with MASH1 revealed strong homologies in the bHLH domain and carboxyl terminal region of the conceptually translated protein. The clone was named chick achaete-scute homologue (CASH). This transcript has a single open reading frame that codes for a protein of 218 residues. The deduced amino acid sequence of CASH is 100% identical to MASH1 in the bHLH region and 83.9% identical over the entire protein. The bHLH domain of CASH is highly conserved within the AS-C family. Northern analysis indicates that CASH is expressed transiently in a defined spatial pattern. The highest expression is in the head between stages 23 and 26. Whole mount in situ hybridization suggests that CASH is expressed at stage 23 in the dorsal ventricular zone and central sensory pathways and later in the ventral ventricular zone and ventral horn. By stage 28, it is expressed ubiquitously in the peripheral nervous system including all of the neurons derived from the the neural crest and epipharyngeal placodes, as well as neural crest-derived chromaffin cells in the adrenal medulla. Amplification by polymerase chain reaction indicates that CASH is expressed in the cardiac neural fold and continues to be expressed in cultured cardiac neural crest cells.
3292	Anti-cooperative biphasic equilibrium binding of transcription factor upstream stimulatory factor to its cognate DNA monitored by protein fluorescence changes.	7642609	Upstream stimulatory factor USF is a human transcriptional activation factor, which uses a basic/helix-loop-helix/ leucin zipper (b/HLH/Z) motif to homodimerize and recognize specific sequences in the promoter region of both nuclear and viral genes transcribed by RNA polymerase II. Steady state fluorescence spectroscopy demonstrated that the basic/helix-loop-helix/leucin zipper domain of USF binds its DNA targets with high affinity and specificity, whereas removal of the leucine zipper yielding the basic/helix-loop-helix minimal DNA binding region reduces both affinity and specificity. Stopped flow method provided kinetic evidence for a two-step binding process involving rapid formation of a protein-DNA intermediate followed by a slow isomerization step, which is consistent with the basic region undergoing a random coil to alpha-helix folding transition on specific DNA recognition. The leucine zipper is also necessary for USF to function as a bivalent homotetramer, capable of binding two distinct recognition sites simultaneously and mediating DNA looping under physiologic conditions. Titration studies revealed that the first binding event has a equilibrium constant Keq = (2.2 +/- 2.0) x 10(9) M-1 for major late promoter DNA, whereas the second binding event occurs with a remarkable reduced affinity, Keq = (1.2 +/- 0.8) x 10(8) M-1. This anticooperative feature of DNA binding by the homotetramer suggests that USF stimulates transcription by mediating DNA looping between nearby recognition sites located in class II nuclear and viral gene promoters.
3293	Determination of sequences responsible for the differential regulation of Myc function by delta Max and Max.	7630640	The DNA-binding, transcriptional activation and transforming activities of the Myc protein require dimerization with Max. Max can form also homodimers which are able to bind the same DNA sequence as Myc/Max heterodimers and suppress Myc-induced transcription and transformation. We have recently identified a naturally occurring truncated form of Max, delta Max, which in a rat embryo fibroblast enhances transformation by Myc and Ras. Like Max, this delta Max protein contains a b-HLH-Zip domain, except that the end of the leucine zipper is replaced by five delta Max-specific amino acid residues. Delta Max also lacks the C-terminal sequences of Max including a nuclear localisation signal. Here we have dissected the regions responsible for the specific effects of Max and delta Max in Ras-Myc cotransformation of rat embryo fibroblasts. Our results indicate that the suppressive activity of Max requires C-terminal acidic and basic regions and an intact leucine zipper. Replacement of the end of the leucine zipper with the delta Max-specific sequence is responsible for the enhancement of transformation by delta Max. Surprisingly, delta Max does not require the DNA-binding basic region for enhancement of transformation and has no effect on Myc-induced transcription activation from Myc/Max-binding site-containing promoter construct.
3294	Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development.	7671815	Trophoblast cells are the first lineage to form in the mammalian conceptus and mediate the process of implantation. We report the cloning of a basic helix-loop-helix (bHLH) transcription factor gene, Hxt, that is expressed in early trophoblast and in differentiated giant cells. A separate gene, Hed, encodes a related protein that is expressed in maternal deciduum surrounding the implantation site. Overexpression of Hxt in mouse blastomeres directed their development into trophoblast cells in blastocysts. In addition, overexpression of Hxt induced the differentiation of rat trophoblast (Rcho-1) stem cells as assayed by changes in cell adhesion and by activation of the placental lactogen-I gene promoter, a trophoblast giant cell-specific gene. In contrast, the negative HLH regulator, Id-1, inhibited Rcho-1 differentiation and placental lactogen-I transcription. These data demonstrate a role for HLH factors in regulating trophoblast development and indicate a positive role for Hxt in promoting the formation of trophoblast giant cells.
3295	Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells.	7623812	Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.
3296	Luteinizing hormone/chorionic gonadotropin bioactivity in the common marmoset (Callithrix jacchus) is due to a chorionic gonadotropin molecule with a structure intermediate between human chorionic gonadotropin and human luteinizing hormone.	7492691	Chorionic gonadotropin (CG), a pregnancy-specific heterodimeric hormone found in primates, is responsible for CL rescue with pregnancy maintenance. Of the primates, the human and baboon gene sequences are the only structures so far determined. In order to study the structure and function of CG in other primates, we have isolated and sequenced the coding regions for the two subunits of marmoset CG (mCG) by the reverse transcription/polymerase chain reaction method. Study of multiple clones confirmed a high degree of homology with the human sequences (88% and 80% for the alpha and beta nucleotide sequences, respectively). Marmoset CG alpha has an extra four amino acids compared to hCG alpha, whereas the mCG beta sequence has a 3-bp deletion that maintains the reading frame and C-terminal amino acid sequence. Most of the differences between hCG beta and mCG beta peptides occur in the C-terminal region, which includes the loss of two of the O-linked glycosylation consensus sequences and the presence of an N-linked glycosylation consensus sequence. When mCG alpha and beta were co-expressed in CHO cells, assembly of biologically active hormone was confirmed by induced steroid secretion by MA10 cells. Partially purified mCG beta was used to raise anti-mCG antibodies. To date, an antibody has been obtained that is capable of detecting recombinant mCG beta, recombinant mCG dimer, and mCG dimer secreted by cultured marmoset trophoblast. Marmoset CG alpha and beta were also detectable at the transcriptional level in cultured trophoblast by in situ hybridization. This suggests that the LH/CG bioactivity reported from marmoset placentae and embryos is due to a molecule with structural features common to hLH (glycosylation pattern) and hCG (CG beta C-terminal structure).
3297	Chromatin organization, meiotic status and meiotic competence acquisition in mouse oocytes from cultured ovarian follicles.	7473419	Changes in chromatin organization, meiotic status and the development of meiotic competence in oocytes retained within mouse ovarian follicles from day 0 to day 6 in culture were examined. The effects of exposure for 24 h to human luteinizing hormone (hLH) during the last day in culture was also determined. Preantral follicles from 22- to 24-day-old (prepubertal) mice develop antra and undergo significant growth from day 0 to day 4 in culture, after which the growth rates slow. The statistical significance of meiotic progression was examined using exact logistical regression analysis, which is particularly useful when the data are sparse and unbalanced. The transition from rimmed to unrimmed germinal vesicle stages was found to occur between day 2 and day 4 of follicle culture and was not influenced by exposure to hLH. Treatment with hLH caused a significant increase in the proportion of intrafollicular oocytes resuming meiosis. Assays of meiotic competence performed in vitro in oocytes retrieved from cultured follicles demonstrated that the transition from an unrimmed to a rimmed state is closely coincident with the acquisition and expression of meiotic competence. Forty-six per cent of competent oocytes from follicle cultures at day 3 progressed to metaphase II. These results indicate that the follicle culture system used in these studies supports the transformation of enclosed oocytes from a precompetent to a competent state and can maintain meiotic arrest for up to 6 days in culture. However, an increasing proportion of oocytes exhibit abnormal meiotic progression with continued follicle culture beyond 4 days.
3298	A structurally abnormal breakpoint cluster region gene in a transcription factor, hLH-2-negative human leukemia cell line.	7600533	hLH-2, a transcription factor that contains double cysteine rich regions (LIM motifs) and a homeobox (Hox) DNA-binding domain shows aberrant high expression in all cases of chronic myelogenous leukemia (CML). This gene has been mapped to the chromosome 9q33-34.1, the same region as the reciprocal translocation that creates the breakpoint cluster region (BCR)-ABL chimera of the Philadelphia chromosome (Ph'). To investigate the possible involvement between the BCR-ABL fusion gene and hLH-2 in the pathogenesis of CML, an hLH-2-negative CML cell line, JK-1 that carries double Ph' chromosomes, was examined. Like other CML cells, high BCR-ABL fusion mRNA levels are expressed, but no transcript of hLH-2 was detected in JK-1 cells as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Compared with the CML cell line, K-562, an additional rearrangement of the BCR gene was observed in JK-1 as determined by Southern blot hybridization; however, the hLH-2 gene was normal. These findings raise interesting questions about the possible roles of either the abnormal BCR gene or other genetic events such as the complex chromosomal abnormalities that result in hLH-2 being turned off in JK-1 cells.
3299	Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1.	7760836	Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses beta-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). We show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they arrested growth and formed three-dimensional structures similar to those of control cells. Id-1-expressing cells did not, however, express beta-casein. Moreover, 8 to 10 days after receiving differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. We suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium.
3300	Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations.	7632831	This study was conducted to investigate the relationships between oocyte meiotic competence, follicle size, and occyte-somatic cell associations in the marmoset monkey (Callithrix jacchus). Follicles were excised from ovaries of nonstimulated adult cyclic females (n = 6) collected on Day 7 of the follicular phase. Follicles were separated into size groups: large preantral (260-400 microns), periantral (420-640 microns), small antral (660-1000 microns), large antral (1020-2000 microns), and preovulatory (> 2000 microns). Partially naked and cumulus/granulosa-enclosed oocytes (n = 473) were released from follicles and cultured in Waymouth's medium with 10% fetal calf serum, 1 microgram/ml human (h) FSH, and 10 micrograms/ml hLH. Somatic cells remaining after 46 h were removed, and oocytes were fixed after 48 h and mounted for viewing. Chromatin staining and microtubulin fluorescence labeling were used to assess progression of meiotic maturation and spindle normality. The follicle size distribution and oocytesomatic cell associations are reported. Competencies of oocytes to achieve germinal vesicle breakdown (GVBD) and metaphase II (MII) increased significantly (p < 0.001) with follicular size but not with the association of somatic cells. Marmoset oocytes from antral follicles resumed (GVBD) and completed (MII) meiotic maturation with high frequencies (98% and 72%, respectively), with no significant differences among size groups of antral follicles. GVBD competence was virtually absent in oocytes from preantral follicles (2%) and was acquired coincidentally with antrum formation (60%), although MII competence was attained after the completion of antrum formation. Partially naked oocytes from small antral follicles matured with a high incidence of spindle and meiotic abnormalities (44%). Marmoset oocyte meiotic competencies are notably higher than in any other nonhuman primate species studied, and a possible explanation for this phenomenon in relation to the stage of antrum formation is offered.
3301	Human prepubertal testicular cells in culture: steroidogenic capacity, paracrine and hormone control.	7626444	The neonatal human Leydig cell undergoes a transient period of activation during the first months of life. The biological significance of this activation is unknown. Furthermore, little is known about the hormonal regulation of this biological process, even though it coincides with an elevation of LH levels in serum. In order to study the function of human prepubertal testicular culture cells, obtained during the neonatal period, a method for maintaining primary culture cells (isolated from testes collected at necropsy) in culture was developed. Within 24 h after death, testes were collected from 1-36-month-old subjects. Subjects were divided into two age groups, based on the presence or absence of fetal Leydig cells: 1-7-month-old infants (group 1) and 12-36-month-old children (group 2). Testes were digested with collagenase, and cells were seeded in multi-well dishes. Cells were grown in serum-free conditioned media supplemented with 5 mg/l vitamin C, 0.2 IU/l vitamin E and 10% fetal bovine serum for 2 days. Cells were then grown for an additional 4 days in serum-free media in the presence or absence of hLH (40 IU/l), hCG (135 IU/l), rh FSH (1.5 IU/l), rhGH (0.12 IU/l) or insulin (0.9 mumol/l). Concentrations of steroids in media were determined by RIA on day 6 of culture. In basal conditions cells of group 1 (n = 11) secreted more testosterone, androstendione, 17-hydroxyprogesterone, progesterone and dehydroepiandrosterone (mean +/- SE: 6.76 +/- 1.86, 7.37 +/- 1.82, 61.9 +/- 1.86, 5.75 +/- 1.74 and 8.51 +/- 3.23 pmol/10(6) cells/24 h, respectively) than cells of group 2 (n = 5) (2.95 +/- 1.15, 1.50 +/- 2.75, 1.44 +/- 2.75, 0.78 +/- 1.74 and 3.23 +/- 1.32, respectively). Under hLH stimulation, cells of group 1 increased testosterone, androstendione and 17-hydroxyprogesterone secretions (to 38.2 +/- 0.89, 13.5 +/- 1.17 and 51.7 +/- 3.23), while progesterone secretion remained unchanged (2.82 +/- 1.20). Cell response to rhFSH and rhGH was similar to that of hLH. On the other hand, medium collected from cultures of cells isolated from a Sertoli cell tumor was able to stimulate testosterone secretion in subcultures of control testicular cells in a way similar to that of hCG. In conclusion, (1) these prepubertal human testicular cells can be maintained in primary culture for several days keeping their in vivo steroidogenic potential; (2) cells isolated from young infants can respond to hLH in culture; (3) response to rhFSH is probably mediated by a paracrine factor; (4) response to rhGH is observed in the absence of gonadotropins.(ABSTRACT TRUNCATED AT 400 WORDS)
3302	Inhibition of cell fate in Drosophila by Enhancer of split genes.	7547476	The neurogenic genes of Drosophila act during many different times and places during development. It is thought their role is to repress cell fate within a group of equivalent cells and thus allow the singling out of discrete numbers of precursors. Amongst the genes at the neurogenic locus, Enhancer of split is a family of seven related genes that encode proteins containing the basic helix-loop-helix motif characteristic of transcriptional regulators. Previous functional analyses of these genes have relied on deletions which eliminate many other genes. We have ectopically expressed two of the Enhancer of split basic helix-loop-helix genes, m5 and m8, to test their effect on the determination of the precursor cells of adult sensory organs. Ectopic expression of m5 or m8 before bristle precursor division results in loss of sensory bristles from all parts of the adult fly. Ectopic expression after bristle precursor division produces bristles with aberrant cuticular structures. We have also tested the effect of reducing Enhancer of split gene function using mitotic recombination and show that this de-represses the neural fate and produces supernumerary sensory bristle neurons. We conclude that the Enhancer of split basic helix-loop-helix genes inhibit neural fate during the selection of neural precursors, and that they also play a role in restricting the neuronal fate to one of the four progeny cells of the bristle precursor.
3303	A novel initiator regulates expression of the nontissue-specific helix-loop-helix gene ME1.	7784173	The mouse ME1 gene (HEB, REB and GE1, homologues in human, rat and chick, respectively) is a member of the nontissue-specific helix-loop-helix (HLH) gene family that includes E2A, E2-2 and Drosophila daughterless. We have examined the factors that control ME1 gene expression. ME1 is a single copy gene that spans > or = 150 kb of DNA and contains > 10 exons. Transcription was directed by an unusual initiator element that contained a 13 bp poly d(A) tract flanked by palindromic and inverted repeat sequences. Both RNase protection and primer extension analyses mapped the ME1 transcriptional start site to the center of the 13 bp poly d(A) tract. The ME1 initiator and its proximal sequences were required for promoter activity, supported basal levels of transcription, and contributed to cell type-specific gene expression. Other cis-elements utilized by the TATA-less ME1 promoter included a cluster of Sp1 response elements, E-boxes and a strong repressor. Collectively, our results suggest that the ME1 initiator and other cis-elements in the proximal promoter play an important role in regulating ME1 gene expression.
3304	Analysis of the Myc and Max interaction specificity with lambda repressor-HLH domain fusions.	7752223	The basic helix-loop-helix domain (bHLH) is present in a large class of transcriptional regulators involved in developmental processes and oncogenesis. It determines DNA binding and specific homo- and heterodimeric protein associations, crucial for protein function. Myc and Max belong to a subset of HLH proteins, containing a leucine zipper (LZ) adjacent to the bHLH domain. They differ in dimerization and functional properties such as DNA binding and transcriptional activation, and their association is required for malignant transformation by Myc. To analyze the interaction specificity of Myc and Max bHLH-LZ domains, we developed a simple Escherichia coli genetic system, which uses the amino-terminal lambda phage cI repressor as a reporter for dimerization and allows an easy detection of dimeric interactions. By reciprocal exchanges of different Myc and Max subdomains (helix 1, helix 2 and leucine zipper), we showed that the recognition specificity of Max homodimers as well as of Myc/Max heterodimers is entirely determined by the helix 2-leucine zipper region, the major role being played by the leucine zipper. The Myc LZ was found to prevent homodimeric interactions, thus explaining Myc inability to homodimerize efficiently. Moreover, we showed that the system is valid as well for reproducing the interaction of HLH proteins not containing a leucine zipper and that the chimerical proteins maintain sequence-specific DNA binding.
3305	Clonal and non-clonal karyotypically abnormal cells in haemophagocytic lymphohistiocytosis.	7786795	We studied chromosomes in bone marrow (BM) or peripheral blood cells of nine patients with haemophagocytic lymphohistiocytosis (HLH); three of them had a family history of HLH and four others underwent concurrent Epstein-Barr virus (EBV) infection. In addition to a large population of normal mitotic cells, karyotypically abnormal clonal cells were found in two patients, abnormal clonal cells and a nonclonal (single) abnormal cell in one, and nonclonal abnormal cells in three. All the six patients with chromosome abnormalities died of progressive disease; one of them also had EBV infection and EBV-associated clonal proliferation. Two of three patients with EBV infection and only normal mitotic cells in BM completely recovered from the disease. Although HLH did not show histological and/or haematological evidence of a neoplastic disease, clonal chromosome abnormalities and the fatal clinical outcome found in some of the patients suggest that the disease may be heterogenous and include malignancy. HLH patients with karyotypically abnormal clonal cells in BM should warrant more intensive chemotherapy than that presently being applied to them and should be considered as candidates for BM transplantation.
3306	Early detection of secretogranin-II (SgII) in the human fetal pituitary: immunocytochemical study using an antiserum raised against a human recombinant SgII.	7720657	Secretogranin-II (SgII) is a protein contained within secretory granules of mainly gonadotrophs. The purpose of this study was to determine whether SgII immunoreactivity (SgII-IR) in the human fetal pituitary was temporally related to gonadotropin immunoreactivity. A specific antihuman SgII antiserum was thus required. A complementary DNA clone with an open reading frame for human (h) SgII was synthesized by reverse transcription-polymerase chain reaction from pituitary total RNA. This clone was used to obtain the SgII polypeptide (-9 to 152) as a fusion protein, in a heterologous expression prokaryotic system. Antisera against the fusion protein were raised in rabbits and checked for specificity and sensitivity through Western blotting. Human fetal pituitaries from week 6 of gestation onward were used for immunocytochemical studies. Consecutive semithin sections were treated with the specific antisera against hSgII, beta-endorphin, and hPRL and with monoclonal antibodies to hCG alpha, hLH, and hFSH. SgII immunoreactivity appeared at week 8 and was restricted to pituitary cells expressing beta-endorphin (100% colocalization). At week 9, FSH-positive cells did not contain SgII. From week 10, gonadotrophs progressively exhibited SgII-IR, up to 50% of that in FSH-containing cells at week 26. The granin was never found in PRL cells whatever the stage of development. The present data demonstrate that SgII-IR is detected very early in fetal life; however, the positive cells are not gonadotrophs, but corticotrophs. Within gonadotrophs, SgII appears subsequent to hormones. At birth, more than 90% of SgII-IR cells are represented by corticotrophs and gonadotrophs.
3307	Molecular cloning of ID4, a novel dominant negative helix-loop-helix human gene on chromosome 6p21.3-p22.	7665172	Transcription factors containing a basic helix-loop-helix (bHLH) motif regulate the expression of tissue-specific genes in a number of mammalian and insect systems. DNA-binding activity of the bHLH proteins is dependent upon formation of homo- and/or heterodimers. Dominant negative HLH proteins (Id-related genes) also contain the HLH-dimerization domain but lack the DNA-binding basic domain. Consequently, Id proteins inhibit binding to DNA and transcriptional transactivation by heterodimerization with bHLH proteins. We report here the cDNA sequence of a novel human HLH gene (HGMW-approved symbol ID4) that lacks the basic domain. ID4 is differentially expressed in adult organs in four mRNA molecules, which are presumably a result of differential splicing and/or alternative usage of the polyadenylation sites. Transfection experiments indicated that enforced expression of Id-4H protein inhibits the trans-activation of the muscle creatine kinase E-box enhancer by MyoD. Finally, we localized the ID4 gene to the chromosome 6p21-p22 region.
3308	Identification of a regulatory function for an orphan receptor in muscle: COUP-TF II affects the expression of the myoD gene family during myogenesis.	7753622	COUP-TF II is an 'orphan steroid receptor' that binds a wide variety of AGGTCA repeats and represses thyroid hormone (T3) and retinoid dependent trans-activation; however, very little is known of its functional and/or developmental role during mammalian cell differentiation. T3 and retinoids have been demonstrated to promote terminal muscle differentiation via activation of the muscle specific myoD gene family (myoD, myogenin, myf-5 and MRF-4). The myoD gene family can direct the fate of mesodermal cell lineages, repress proliferation, activate differentiation and the contractile phenotype. Hence, we investigated the expression and functional role of COUP-TF II during muscle differentiation. Proliferating C2C12 myoblasts expressed COUP-TF II mRNA which was repressed when cells were induced to differentiate into post-mitotic multinucleated myotubes by serum withdrawal. Concomitant with the decrease of COUP-TF II mRNA was the appearance of muscle specific mRNAs (e.g. myogenin, alpha-actin). We show that Escherichia coli expressed full length and truncated COUP-TF II bound in a sequence specific manner to the T3 response elements (TREs) in the myoD and myogenin regulatory HLH genes [Olson (1992) Dev. Biol. 154, 261-272]; and the TRE in the skeletal alpha-actin contractile protein gene. COUP-TF II diminished the homodimeric binding of the thyroid hormone receptor and the heterodimeric binding of thyroid hormone and retinoid X receptor complexes to these TREs. Constitutive over-expression of COUP-TF II cDNA in mouse C2C12 myogenic cells suppressed the levels of myoD mRNA and blocked the induction of myogenin mRNA, whereas constitutive expression of anti-sense COUP-TF II cDNA significantly increased the steady state levels of myoD mRNA and hyper-induced myogenin mRNA. These studies demonstrate for the first time (i) that COUP-TF II, functions as a physiologically relevant antagonistic regulator of myogenesis via direct effects on the myoD gene family and (ii) direct evidence for the developmental role of COUP-TF II during mammalian cell differentiation.
3309	The E12 inhibitory domain prevents homodimer formation and facilitates selective heterodimerization with the MyoD family of gene regulatory factors.	7737127	Although the ubiquitous helix-loop-helix (HLH) protein E12 does not homodimerize efficiently, the myogenic factor MyoD forms an avid DNA-binding heterodimer with E12 through the conserved HLH dimerization domain. However, the mechanism which ensures this selective dimerization is not understood at present. In our functional studies of various amino acid changes in the E12 HLH domain, we found that a single substitution in E12 helix 1 can abolish the effect of the E12 inhibitory domain and results in the efficient DNA binding of the E12 homodimer. Competition experiments revealed that the inhibitory domain, in fact, blocks the dimerization of E12 rather than DNA binding. MyoD contains two glutamic residues in helix 2 that are required for efficient dimerization with E12. More importantly, these residues were not essential for dimerization with E12 mutants in which the dimerization inhibitory domain had been relaxed, or for dimerization with E47 which does not contain the inhibitory domain owing to the use of an alternative exon. The positions of these glutamic residues are conserved among the four myogenic factors. Thus, members of the MyoD family of gene regulatory proteins can overcome the E12 dimerization inhibitory domain through a mechanism involving, in part, the negatively charged amino acid residues in helix 2. This result describes a novel mechanism facilitating the selective formation of the MyoD(MRF)-E12 heterodimer that enhances dimerization specificity and may apply to other members of the E-protein family.
3310	Complete sequence of the gene encoding a chorionic gonadotropin-like protein from Xanthomonas maltophilia.	7537705	Our laboratory has previously reported that: (i) Xanthomonas maltophilia (Xm) produces a protein which has immunological resemblance to the beta-subunit of human chorionic gonadotropin (hCG) and (ii) possesses a high-affinity receptor which binds holo hCG, and the endogenous ligand, Xm chorionic gonadotropin (xCG), but does not bind human luteinizing hormone (hLH). We have also previously published a 492-bp partial nucleotide sequence of the gene (xcg) coding for xCG. We report herein the entire xcg sequence of 1362 bp, which codes for a 48-kDa protein. This sequence confirmed the 492-bp sequence, as well as two partial amino acid (aa) sequences which we have previously reported. The sequence has a region which is homologous to aa 56-139 of the beta-subunit of hCG, and a second region homologous to the C-terminal tail of hCG. This is the first report of a prokaryotic gene homologous to the hCG beta-subunit-encoding gene.
3311	Transcriptional activation by Myc is under negative control by the transcription factor AP-2.	7729426	The Myc protein binds to and transactivates the expression of genes via E-box elements containing a central CAC(G/A)TG sequence. The transcriptional activation function of Myc is required for its ability to induce cell cycle progression, cellular transformation and apoptosis. Here we show that transactivation by Myc is under negative control by the transcription factor AP-2. AP-2 inhibits transactivation by Myc via two distinct mechanisms. First, high affinity binding sites for AP-2 overlap Myc-response elements in two bona fide target genes of Myc, prothymosin-alpha and ornithine decarboxylase. On these sites, AP-2 competes for binding of either Myc/Max heterodimers or Max/Max homodimers. The second mechanism involves a specific interaction between C-terminal domains of AP-2 and the BR/HLH/LZ domain of Myc, but not Max or Mad. Binding of AP-2 to Myc does not preclude association of Myc with Max, but impairs DNA binding of the Myc/Max complex and inhibits transactivation by Myc even in the absence of an overlapping AP-2 binding site. Taken together, our data suggest that AP-2 acts as a negative regulator of transactivation by Myc.
3312	MATH-2, a mammalian helix-loop-helix factor structurally related to the product of Drosophila proneural gene atonal, is specifically expressed in the nervous system.	7744035	In Drosophila, multiple helix-loop-helix (HLH) factors play an essential role in neural development. Mammalian homologues of such Drosophila HLH factors have been recently characterized and provide useful information for the analysis of the mechanisms of mammalian neurogenesis. We report here the molecular characterization of a novel mouse HLH factor, designated MATH-2, that has a structural homology to the product of Drosophila proneural gene atonal. MATH-2 consists of 337 amino acid residues and contains an atonal-related basic HLH domain. However, outside of this domain, there is no significant sequence similarity between MATH-2 and Atonal. MATH-2 expression occurs by embryonic day 11.5 (E11.5), and is first detected in the wall of brain vesicles as well as in the spinal cord. It is expressed in the cortical plate and the mantle layer throughout the developing central nervous system but not in the ventricular zone. By E13.5, the expression becomes more prominent in the cortical plate of the cerebrum but decreases in the other regions. In the adult, the cerebrum produces a high level of MATH-2 RNA but other neural tissues produce only low levels. MATH-2 RNA is not detected in non-neural tissues, indicating that MATH-2 expression is specific to the nervous system. The gel mobility-shift analysis shows that MATH-2 can interact with several E-box sequences in collaboration with E47, a ubiquitously expressed HLH factor. These results raise the possibility that MATH-2 may be a trans-acting factor involved in the development and maintenance of the mammalian nervous system.
3313	One monoclonal antibody to human thyrotropin (TSH) receptor agonist of TSH hormone stimulates the proliferation of human thyroid cells.	7697737	The question of thyroid cell growth induction by monoclonal antibodies (mAbs) to human thyrotropin receptor (hTSH-R), which stimulate increases in cAMP thyroid cells, is under debate and their implication in Graves' disease (GD) is controversial. In order to address this issue, we used characterized reagents, i.e., mAbs to hTSH-R, and cloned human thyroid hybridoma cells (GEJ). Cell counting on Days 1, 2, and 3 after addition of mAb and Northern blot analysis of mRNA specific for the c-fos oncogene were used to assess the proliferation of the thyroid cells. MAbs to hTSH-R, obtained by immunization of DBA/1 mice with affinity-purified hTSH-R, were added to GEJ cells in concentrations varying from 0.66 to 660 nM. Their effects on GEJ cell growth were compared to those of human TSH, of hLH, and of control mAb. Cell counting and evaluation of GEJ c-fos transcripts showed that mAbs to hTSH-R induce significant GEJ cell growth, whereas they were ineffective on the control cell line. Among them, one mAb, 34A, exhibited an impressive activity on thyroid cell growth and induced cAMP production comparable to that induced by bovine or human TSH. Our data demonstrate the existence of immunoglobulin which stimulates thyroid growth and elevates cAMP. The higher the cAMP production, the greater the thyroid cell proliferation. Furthermore, the data suggest a possible role for agonistic anti-hTSH-R autoantibodies in the immunopathogenesis of GD.
3314	Id gene activity during Xenopus embryogenesis.	7619724	The activity of bHLH transcription factors that are involved in cell determination and differentiation is inhibited by Ids, HLH proteins lacking the basic amino acid sequence element. In order to determine the role of Id during development, we have isolated and characterized the Id genes expressed in Xenopus embryos. Three cDNAs were characterized: XIdIa and XIdIb, which are transcribed from one gene but differentially spliced in the 3' untranslated part, and XIdII which is transcribed from a second copy of the gene. One of the two forms of the differentially spliced mRNAs exhibits, 30 nucleotides upstream from the AATAAA site, a sequence box homologous to the cytoplasmic polyadenylation element (CPE) which is present also in Id2 and Id3 mRNAs from higher vertebrates. This raises the question of whether this CPE-like element may link Id mRNA polyadenylation and translation to the cell cycle metabolism. The Xenopus Id gene is transcribed at low level in oocytes and at high level in embryos, after midblastula transition, in a large number of tissues, including the notochord, neural tube, eye, ear, neural crest cells, presomitic mesoderm, myotomes, tailbud and dorsal fin. In myotomes, expression is high in the areas of proliferating myoblasts and decreases as terminal differentiation proceeds, consistent with a function in cell determination and differentiation and possibly also in cell cycle regulation.
3315	Hairy and emc negatively regulate morphogenetic furrow progression in the Drosophila eye.	7697718	The initial steps of pattern formation in the developing Drosophila eye involve the coordination of cell cycles, changes in cell shape, and the specification of the R8 photoreceptor cell. These events begin several cell rows ahead of the morphogenetic furrow and are positively regulated by secreted signaling proteins and the proneural HLH transcription factor atonal (ato). Two HLH regulatory proteins that function to suppress neuronal development in other tissues, extra macrochaetae (emc) and hairy (h), are expressed ahead of the morphogenetic furrow. While neither h nor emc is required for photoreceptor cell determination, in emc-h-clones the morphogenetic furrow and differentiated eye field advance up to eight ommatidial rows ahead of adjacent wild-type tissue. This indicates that morphogenetic furrow progression and neuronal differentiation are negatively regulated by a combination of anteriorly expressed HLH regulatory proteins.
3316	Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation.	7892203	Arnt (Ah receptor nuclear translocator) is a member of a transcription factor family having characteristic motifs designated bHLH (basic helix-loop-helix) and PAS and was originally found as a factor forming a complex with Ah receptor (AhR) to bind the specific xenobiotic responsive element (XRE) sequence for induction of drug-metabolizing P4501A1. We have examined interaction of Arnt with other PAS proteins--Drosophila Per, Sim, and AhR--by the coimmunoprecipitation method. Arnt formed a homodimer with itself as well as heterodimers with the others by means of the PAS and HLH domains in a cooperative way. The Arnt homodimer binds the sequence of adenovirus major late promoter (MLP) with the E box core sequence CACGTG, suggesting that the CAC half of the XRE, CACGCN(A/T), recognized by the AhR-Arnt heterodimer is a target for Arnt. Cotransfection experiments using CV-1 cells with an Arnt expression plasmid and a MLP chloramphenicol acetyltransferase (CAT) reporter plasmid revealed that Arnt markedly activated CAT expression, indicative of a newly discovered regulatory role of Arnt.
3317	Possible existence of luteinizing hormone/chorionic gonadotropin receptors in human corpora lutea during early pregnancy.	9363234	In order to evaluate the possible existence of receptors for luteinizing hormone (LH)/chorionic gonadotropin (CG) in human corpora lutea of pregnancy, the specific binding of [125I]hLH to luteal pellets of 6-7 weeks' gestation and the effect of neuraminidase pretreatment on [125I]hLH binding were investigated. A relatively low capacity of hLH binding to luteal tissue of pregnancy was observed. In addition, the specific binding of hLH to luteal pellets significantly increased following neuraminidase pretreatment. The results obtained in this study suggest the possible existence of LH/CG receptors in human corpora lutea of pregnancy.
3318	The use of human recombinant gonadotropin receptors to search for immunoglobulin G-mediated premature ovarian failure.	7883837	Anti-FSH receptor antibodies, detected using animal systems, have been reported in a few patients with premature ovarian failure (POF). However, assays based on animal receptors may be inappropriate for detecting inhibiting antibodies in humans. Accordingly, we tested for interfering antibodies in patients with POF using a recombinant system expressing human (h) FSH and LH receptors. A mouse adrenal cell line transfected with the hFSH receptor (Y1-hFSHR) exhibits a dose-dependent increase in progesterone when exposed to hFSH. An embryonal kidney cell line transfected with the hLH receptor gene (hLHR-293) exhibits a dose-dependent increase in cAMP when exposed to hLH. We isolated immunoglobulins G (IgG) from 38 patients with POF and 14 normal women. We stimulated Y1-hFSHR and hLHR-293 cells with hFSH or hLH in the presence of these IgG and determined the resulting progesterone or cAMP response. The progesterone and cAMP responses obtained in the presence of IgG from patients with POF did not differ significantly from the responses in the presence of IgG from normal women. In contrast, antigonadotropin polyclonal antibodies isolated in the same manner as the above IgGs caused a greater than 90% reduction in the response of the Y1-hFSHR and hLHR-293 cells. We did not detect inhibitory antibodies in any of our 38 patients with POF. Therefore, if blocking antibodies interfering with gonadotropin-receptor interaction are a mechanism for POF, they account for a small minority of cases (< 8%).
3319	Immunological detection of membrane-associated human luteinizing hormone correlates with gene expression in cultured human cancer and fetal cells.	7867557	We have demonstrated the expression of membrane-associated hCG and its subunits and fragments by cells from 78 human cancer cell lines of different types and origins, indicating that such expression is a common phenotypic characteristic of cultured human malignant cells. Because human (h) LH beta has 80% homology with hCG beta and is coded by one of the seven genes in the gene cluster located in chromosome 19, it was important to determine whether hLH and its beta-subunit are also expressed as membrane-associated proteins by cells from human cancer cell lines. Thus, 11 cancer cell lines of different types and origins were adapted to grow in serumless medium, with Nutridoma-HU or SP as serum substitute, and analyzed by flow cytometry using two monoclonal antibodies directed to different conformational epitopes of intact hLH and a monoclonal antibody reacting with an epitope of hLH beta-free. The cells were also analyzed simultaneously for the expression of hCG and its subunits and fragments. Determination of translatable levels of hLH beta and hCG beta messenger RNAs (mRNAs) was performed in cells from some of the cancer cell lines, including the JEG-3 choriocarcinoma cell line, and in cells from a human fetal lung cell line. The analytical flow cytometry studies showed that in addition to the expression of membrane-associated hCG in all of its forms, expression of membrane-associated intact (holo) hLH and its free beta-subunit occurred in every case. These findings were corroborated by the presence of translatable levels of hLH beta and hCG beta mRNAs in all of the cancer cell lines analyzed, indicating that the expression of these membrane-associated glycoproteins is a phenotypic characteristic of human cancer cells and that the activation of the hCG beta-hLH beta gene cluster is nonselective. The presence of translatable levels of hCG beta-hLH beta mRNAs in the cultured human fetal lung cells punctuates once more the in vivo and in vitro biochemical similarities between fetal and cancer cells.
3320	A novel enhancer, the pro-B enhancer, regulates Id1 gene expression in progenitor B cells.	7862144	The helix-loop-helix (HLH) Id proteins have been reported to function as inhibitors of various differentiation programs. The HLH motif mediates dimer formation between Id and the basic HLH transcription factors. Since Id proteins lack the basic region responsible for DNA binding, the heterodimers cannot bind to DNA. Id proteins have also been found to be involved in early B-cell differentiation. They are expressed at high levels in progenitor B cells (pro-B cells), and the expression is diminished in pre-B cells and mature B cells. This expression pattern correlates inversely with basic HLH protein activity and immunoglobulin enhancer function in B-cell development. Regulation of Id expression may play an important role in transcriptional control of immunoglobulin genes and therefore in B-cell differentiation. We have characterized the regulatory elements of the Id1 gene. Using stable transfectants, transient transfection, and mobility shift assays, we have identified an 8-bp element designated PBE (pro-B enhancer) downstream of the Id1 gene that is responsible for a pro-B-cell-specific enhancer activity. A pro-B-cell-specific protein complex was found to bind to the 8-bp PBE element. Substitution mutagenesis at this binding site showed that it is indeed of functional importance in regulating the pro-B-cell-specific expression of the Id1 gene.
3321	MyoD and myogenesis in C. elegans.	7748176	One of the goals in developmental biology is the identification of key regulatory genes that govern the transition of embryonic cells from a pluripotent potential to a specific, committed cell fate. During vertebrate skeletal myogenesis, this transition is regulated by the MyoD family of genes. C. elegans has muscle analogous to vertebrate skeletal muscle and has a gene (hlh-1) related to the MyoD family. The molecular and genetic characterization of hlh-1 shows that it is very similar to the vertebrate MyoD family in many respects, including its expression pattern and DNA binding activity. The hlh-1 product is required for proper myogenesis, but it is not required for myogenic commitment during embryogenesis in the nematode. The role of this MyoD-related gene in nematode myogenesis is discussed and compared to those of the vertebrate MyoD family.
3322	Allogeneic bone marrow transplantation for hemophagocytic lymphohistiocytosis in Sweden.	7599555	Six children (aged 9 months to 10 and 5/12 years) with hemophagocytic lymphohistiocytosis (HLH) have undergone allogeneic BMT in Sweden. In two of the children unrelated donors were used. Parents were available as donors in two of the cases and siblings in the other two. Conditioning before BMT consisted of etoposide, busulfan and cyclophosphamide with the addition of ATG in two cases and OKT 3 in one case. For post-transplant immunosuppression, i.v. methotrexate and cyclosporin A (CsA) were used in five cases, and in one child CsA was combined with methylprednisolone. Of the six children, four are alive and well 2 and 3/12 to 3 and 1/12 years after BMT. One child, who had an unrelated donor with one DR-antigen mismatch, died 30 days after BMT of fulminant grade IV GVHD. Another patients, seropositive for CMV, received marrow from an unrelated HLA-A, -B, -DR and -DP identical donor. After an initially uneventful course, CMV was isolated from her leukocytes. Seven months after BMT she developed a progressive obstructive chronic bronchiolitis and succumbed to respiratory insufficiency 14 months after the transplant. This study supports the view that BMT is the treatment of choice in HLH, particularly if an HLA-identical related donor is available.
3323	Upstream stimulatory factor, a basic-helix-loop-helix-zipper protein, regulates the activity of the alpha-glycoprotein hormone subunit gene in pituitary cells.	7539886	In an effort to determine whether basic-helix-loop-helix (bHLH) proteins are important in pituitary-specific expression of the alpha-glycoprotein hormone subunit gene, we examined the effect of the dominant negative HLH protein, Id, on the activity of the alpha-subunit gene promoter in pituitary cells. Id over-expression reduces the expression of alpha-subunit reporter genes in either alpha T3-1 gonadotrope-derived or alpha TSH thyrotrope-derived cells. A deletion fragment containing nucleotides from -131 to +44 of the human alpha-subunit promoter is inhibited to a similar degree as a -244 to +44 fragment in alpha T3-1 cells. Nuclear proteins in alpha T3-1 cells bind two potential bHLH protein binding sites (E-boxes, alpha EB1 and alpha EB2) present in this fragment but not to mutations that specifically alter only these sequences. An antibody-specific for upstream stimulatory factor, a widely expressed bHLH-leucine zipper protein, is able to inhibit factor binding to the alpha EB2 sequences but not the alpha EB1 site. Mutating the alpha EB1 element of the alpha-subunit promoter decreases basal activity of this promoter to about 42% of control levels in alpha T3-1 cells. A mutation that abolishes upstream stimulatory factor binding, either alone or in combination with the alpha EB1 mutation, reduces basal activity of the promoter to approximately 21% of control levels in alpha T3-1 cells and abolishes the decrease in promoter activity seen when Id is overexpressed. These results demonstrate that the bHLH family of proteins are important regulators of alpha-subunit gene expression in pituitary cells.
3324	Pharmacokinetics of recombinant human luteinizing hormone after intravenous, intramuscular, and subcutaneous administration in monkeys and comparison with intravenous administration of pituitary human luteinizing hormone.	7852534	To assess the pharmacokinetics of recombinant human LH (rhLH) in monkeys, we measured serum LH levels after single iv injection and after single and repeated doses by the im or sc route. A single iv bolus of 400 IU/kg rhLH or pituitary hLH (phLH) in six cynomolgus monkeys resulted in parallel concentration-time curves. The initial and terminal half-lives of rhLH (0.8 and 11 h) were comparable to those of phLH (0.6 and 10 h). The serum levels of phLH were consistently higher due to the fact that the immunological dose of phLH was higher. Administration of increasing iv doses of rhLH (10, 63, and 400 IU/kg) to six monkeys showed that the pharmacokinetics are linear over this dose range. The total clearance for the two higher doses was 0.03 L/h.kg. Systemic bioavailability was 50% after a single sc injection of 400 IU/kg and 61% after a single im injection of the same dose. The peak concentration (180 IU/L) after im injection was reached after 2.7 h. This was higher and sooner than after sc injection (110 IU/L after 5.3 h). The terminal half-life by both routes was similar to that seen after iv injection (11 h). Daily sc or im administration of 63 IU/kg for 7 days confirmed these findings. There was no accumulation of rhLH. Some monkeys developed antibodies, especially after repeated administration. They were excluded from the analysis. No significant local or systemic adverse events occurred.
3325	A prospective study of CD45 isoform expression in haemophagocytic lymphohistiocytosis; an abnormal inherited immunophenotype in one family.	7851014	In a prospective study, CD45 isoform expression on T lymphocytes was analysed in seven patients with haemophagocytic lymphohistiocytosis (HLH) and their family members. Six patients, their parents and seven healthy age-matched controls showed the normal pattern of three subpopulations of CD45R expression. In one patient, his mother and one of his healthy brothers only two subpopulations could be identified, which might indicate that the maturation from a CD45RA/RO double-positive to a CD45RO single-positive phenotype does not occur in these individuals. It is not clear if this finding had any impact on the development of HLH in this patient and if the pattern of CD45RA and RO expression observed in this family might represent a predisposition to HLH. In another patient a marked increase of the CD45RO single-positive T cell population was observed during a period of increased disease activity. The clinical relevance of this observation and whether CD45RO expression could serve as a marker for disease activity, or in selected individuals as a susceptibility marker for HLH, remain unknown.
3326	Application of a sensitive HPLC-based fluorometric assay to determine the sialic acid content of human gonadotropin isoforms.	7608469	The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid (N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released by partial acid hydrolysis (0.1 M TFA, 80 degrees C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylenedioxybenzene) in conjunction with an internal standard (N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mol Neu5AC/mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoforms was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.
3327	Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro.	8848571	Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.
3328	Regulation of mammalian neural development by helix-loop-helix transcription factors.	8581982	Understanding of the molecular mechanisms of mammalian neural development has been greatly advanced by identification and characterization of the molecules homologous to the factors regulating Drosophila neurogenesis, which provides a powerful model system. Studies of Drosophila show that transcription factors with a helix-loop-helix (HLH) domain play an essential role in neurogenesis. Several lines of evidence demonstrate that mammalian homologues of the Drosophila HLH factors do also play an essential role in neural development. Mash-1, a mammalian HLH factor homologous to the products of Drosophila proneural genes achaete-scute complex, is a positive regulator of neurogenesis and required for differentiation of olfactory and autonomic neurons. In addition, HES-1, another mammalian HLH factor homologous to the products of Drosophila hairy and Enhancer of split, antagonizes the activity of Mash-1 and negatively regulates neurogenesis. Thus, positive and negative HLH factors interact with each other, and the balance between them is important for the developmental processes. Recent studies show that many other HLH factors exist expressed in the developing mammalian nervous system. In this article, the authors review mammalian HLH factors expressed in the nervous system and discuss the molecular aspect of mammalian neurogenesis.
3329	Immunoreactive human chorionic gonadotropin beta core fragment in human pituitary.	8536062	The human pituitary has been shown to produce small amounts of immunoreactive human chorionic gonadotropin (hCG) and its alpha- and beta- subunit (hCG alpha, hCG beta). The aim of the present studies was to further examine the various hCG-related materials in the human pituitary, particularly to search for the existence of pituitary hCG beta core fragment (hCG beta cf)--like material. HCG beta cf has been found in the urine of pregnant women, patients with trophoblastic tumors and also in postmenopausal women. Gel chromatography of pituitary extract on Superdex 200 showed three distinct peaks of hCG-related immunoreactivities, i.e. hCG, hCG beta and hCG beta cf, which were distinguishable from hLH and hLH beta peaks. HCG beta cf was recognized by a specific immunometric assay (crossreactivities with hCG beta 0.016%, hLH beta 0.04%), but unreactive in an hLH + hLH beta assay. It was purified and displayed physical properties similar to those of hCG beta cf derived from pregnancy urine. Apart from immunological differences between the small molecular weight forms or fragments of hCG and LH origin, reversed phase HPLC was able to physically discriminate between hCG beta cf and hLH beta fragment. The latter was much more abundant than the former in the pituitary extract. HCG beta cf showed microheterogeneity related to its sialic acid content. In conclusion, the present data indicate that immunoreactive hCG beta cf is present in human pituitary extracts. The physical and immunological properties of pituitary hCG beta cf are distinguishable from those of the more abundant hLH beta and its fragment, and compare favorably with those of urinary hCG beta cf of trophoblastic origin.
3330	The Pan basic helix-loop-helix proteins are required for insulin gene expression.	7760851	The rat I insulin enhancer contains two principal regulatory elements, the Nir and Far motifs of an identical 9-base pair sequence, which function both as positive and negative cis-acting elements. The Nir and Far elements are targets for DNA-binding proteins, which play a predominant role in the selective transcription of the insulin gene in endocrine beta-cells. In vitro DNA-binding studies have demonstrated the ability of several helix-loop-helix (HLH) proteins, including the Pan/E2A proteins, upstream stimulating factor, human beta-HLH factor (rat beta-HLH factor), and E2-2/ITF-2, to bind the Nir and Far enhancer motifs. The presence of the aforementioned different HLH proteins in endocrine beta-cells, all of which display similar binding affinities for the Nir and Far elements in vitro, raises the question of which HLH proteins actively participate in the transcriptional regulation of the rat insulin I gene in pancreatic endocrine beta-cells. To investigate the specific role that Pan proteins play in regulating insulin gene expression, we have created endocrine beta-cell stable integrants that constitutively express Pan antisense transcripts that selectively inhibit endogenous Pan protein synthesis in differentiated beta-cells. We demonstrate that diminished Pan protein levels in beta-cells, caused by Pan antisense transcripts, accompanies a dramatic attenuation of rat insulin gene transcription. We also show that the decrease in Pan protein expression correlates with a specific reduction of the beta-endocrine-specific Nir and Far element-binding activity, insulin enhancer factor 1.(ABSTRACT TRUNCATED AT 250 WORDS)
3331	Evolutionarily conserved positive and negative cis-acting elements control the blastoderm-specific expression of the Drosophila serendipity alpha cellularisation gene.	7748791	The serendipity alpha (sry alpha) cellularisation gene is only transcribed at the blastoderm stage, when this morphogenetic event takes place. We show that a 95 bp sry alpha upstream region is sufficient for blastoderm-specific expression of a lacZ reporter gene. This region encompasses four nucleotide motifs (I-IV, 5' to 3') conserved at similar relative positions in several Drosophila species. Removal of motif I leads to ectopic expression of lacZ in precursor cells of the PNS. Deletion of motif IV decreases the level of lacZ transcripts and modifies their banded pattern of accumulation late in cycle 14, whereas deletion of motifs II and III abolishes the sry alpha promoter activity. Motif III includes a consensus recognition site for b-HLH proteins. A point mutation in this E-box both severely reduces lacZ expression at blastoderm and prevents its ectopic expression in the PNS upon deleting motif I. These two effects depend upon da+ activity, suggesting that daughterless may positively control sry alpha transcription.
3332	Recombinant-human luteinizing hormone (r-hLH) as ovulatory stimulus in superovulated does.	7580012	To study the effects of r-hLH as ovulatory stimulus in does.New Zealand does, 18 wk old, in estrus, received 25 IU of pregnant mare serum gonadotropin (PMSG) followed at 48 h either by 50 IU of r-hLH (n = 20) or hCG (n = 20) to induce follicular growth and ovulation. All does were previously artificially inseminated to avoid endogenous LH surge. Half of the animals receiving r-hLH (n = 10) or hCG (n = 10) were killed at 72 h after the hormone administration, and the remaining half were killed at 14 days. At 72 h the number of corpora lutea and preovulatory follicles was determined, and fertilization rate, embryo quality, degree of embryonic development, and oviductal transit were all assessed. On day 14 the number corpora lutea and implanted embryos were counted, and implantation rate was determined. Median and interquartile ranges were calculated for each parameter.At 72 h the median for corpora lutea was 8 (7-10) in the r-hLH group vs 13 (10-14) in the hCG group (P = 0.009); preopvulatory follicles were 7 (6-10) vs 0 (0-0) (P = 0.0007); the percentage of good-quality embryos was 71.4% (54.5-75) vs 33.3% (25-37.5) (P = 0.001), for intermediate-quality embryos it was 25% (14.3-36.4) vs 33.3% (25-38.5), and the percentage of degenerated embryos was 0% (0-12.5) vs 33.3% (25-37.5) (P = 0.015), respectively. Fertilization rates were similar in both groups. Embryonic development was more homogeneous in the animals receiving r-hLH (8 to > or = 16 cells) compared to those receiving hCG (2 to > or = 16 cells). The median of embryos still in oviducts at 72 h was significantly higher in the hCG group [6 (4-13)] than in the r-hLH group [0 (0-4)] (P = 0.41). At 14 days the median of corpora lutea was higher in the hCG [12 (11-16)] than in the r-hLH group [10 (7-13)] (P = 0.008), but no differences were noted in the number of implanted embryos. Implantation rate was higher in the r-hLH group [100 (92.3-100)] than in the hCG group [87.5 (83.3-94.1)] (P = 0.056).At the studied dose an ovulatory stimulus with r-hLH induced fewer follicles to ovulate than hCG. Recombinant-hLH produced superior embryo morphological quality, a more homogeneous degree of embryo development, and more synchronous embryo transit than hCG. In spite of the larger number of ovulations following hCG, subsequent events essential for pregnancy were higher with r-hLH, offsetting differences in terms of implanted embryos at 14 days of pregnancy.
3333	The mycN/max protein complex in neuroblastoma. Short review.	7576956	The oncogenic activation by amplification of the MYCN gene is frequently observed in human neuroblastomas and occasionally in other tumours with neuronal qualities. As a consequence of amplification, elevated levels of the mycN protein are expressed. mycN contains a C-terminal basic region (BR) that can bind to DNA, and a helix-loop-helix (HLH)-leucine zipper (Zip) domain, which is responsible for the physical interaction with another HLH-Zip protein, max. This principle structure is conserved among all members of the MYC gene family. The resulting dimers can bind to the DNA sequence CACGTG. The mycN protein, but not max, contains, near the N-terminus, a region conferring the ability to activate the transcription of genes. mycN/max heterodimers probably activate and max/max homodimers repress transcription of, as yet, unidentified target genes. In neuroblastoma cells, where mycN is deregulated, the balanced interaction of BR-HLH-Zip proteins is probably perturbed, and, therefore, genes controlled by mycN might be abnormally expressed and thereby alter normal cell growth with the consequence of tumorigenesis.
3334	Influence of human luteinizing hormone on cell growth and CA 125 secretion of primary epithelial ovarian carcinomas in vitro.	7569684	In the present study, the influence of human luteinizing hormone(hLH) on the growth and the CA 125 secretion of primary ovarian carcinoma cell cultures derived from 11 previously untreated patients was investigated. Two different patterns of in vitro growth of unstimulated ovarian carcinoma cells could be detected: 5 cell cultures in which no changes of cell number were observed during an incubation period of 6 days (group A) and 6 cell cultures which grew without any hormonal support (group B). All tumors of group A showed enhanced cell proliferation when hLH was added to the culture medium, reaching a maximum at dosages of 100 mIU/ml on day 2 after incubation, while such an effect was not observed in group B. In most cases, no positive correlation was found between hLH-induced cell growth and CA 125 release. Our findings demonstrated a dose-dependent hLH-induced stimulation of epithelial ovarian tumors in vitro.
3335	Anti-human gonadotropin antibodies generated during in vitro fertilization (IVF)-related cycles: effect on fertility of rhesus macaques.	7563012	Administration of human gonadotropins such as hFSH, hLH, and hCG to rhesus macaques can result in formation of anti-human gonadotropin antibodies. To determine whether the presence of these antibodies interferes with subsequent fertility, sixteen female rhesus macaques (Macaca mulatta) with known antibody levels were bred with male rhesus macaques. The presence of antibodies did not interfere with conception or maintenance of pregnancy. Furthermore, antibody titers did not increase during gestation or following the resolution of pregnancy.
3336	Structural constraints for DNA recognition by Myc and other b-HLH-ZIP proteins: design of oncoprotein analogues.	7549463	DNA recognition is a critical property of many transcription factors, some of which play important roles in human disease. Disruption of this recognition may profoundly influence the biology of these factors. One such factor, the Myc oncoprotein, utilizes a basic/helix-loop-helix/leucine zipper motif to recognize the DNA target CACGTG. As discussed here, this recognition appears to occur through recognition by one face of a basic region alpha helix utilizing amino acid side chains highly conserved among CACGTG binding proteins. This basic domain alpha helix, however, requires DNA binding for stabilization. To circumvent this energetic requirement, analogues were produced that introduce multiple alanines, displaying substantially increased spontaneous alpha helicity and significantly enhanced DNA affinity. These studies simplify our understanding of the structural constraints for DNA recognition by this family and may serve as a template for the design of small molecule transcription-targeted therapeutics.
3337	Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta-subunit to the common alpha-subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG.	7539107	The hCG beta-subunit contains a carboxy-terminal extension bearing four serine-linked oligosaccharides [carboxy-terminal peptide (CTP)], which is important for maintaining its longer half-life compared with the other glycoprotein hormones. Previously, we enhanced the in vivo half-life of FSH by fusing the CTP to the carboxy end of FSH beta coding sequence. The alpha-subunit is common to the glycoprotein family. We constructed alpha-subunit CTP chimeras, since such analogs with the appropriate O-linked glycosylation and conformation would increase the in vivo stability of the entire glycoprotein hormone family. Two chimeras were constructed using overlapping polymerase chain reaction mutagenesis: a variant with CTP at the carboxy end and another analog with the CTP at the N-terminal region of the subunit, between amino acids 3 and 4. The latter design was based on models showing that the amino-terminal region of alpha is not involved in assembly with the beta-subunit, nor is it essential for receptor binding and signal transduction. These chimeras were cotransfected with the hCG beta gene into Chinese hamster ovary cells. The chimeras were secreted and combined efficiently with the CG beta-subunit, comparable to the wild type alpha-subunit. CG dimers containing the alpha-subunit chimera with CTP at the carboxy end of the subunit had a much lower binding affinity for the hLH-hCG receptor in vitro, whereas the binding of the dimer containing the CTP at the amino-terminal end of the subunit was similar to wild type hCG. Furthermore, the in vivo activity of this analog was enhanced significantly. Moreover, regardless of the two insertion points in the alpha-subunit, the CTP sequence was O-glycosylated. These data suggest that the entire signal for O-glycosylation is primarily contained within the CTP sequence and is not dependent on the flanking regions of the recipient protein. The transfer of CTP to the alpha-subunit of hCG results in an agonist with prolonged biological action in vivo. These data further support the rationale for using the CTP as a general target to increase the potency of bioactive glycoproteins.
3338	Structural organisation and chromosomal mapping of the human Id-3 gene.	7828896	The helix-loop-helix (HLH) family of transcription factors plays a central role in the regulation of cell growth, differentiation and tumourigenesis. Members of the Id (inhibitor of DNA binding) class of these nuclear proteins are able to heterodimerise with and thereby antagonise the functions of other transcription factors of this family. We report here on the genomic organisation of the human Id3 (HLH 1R21/heir1) gene. Comparison with the two other mammalian Id genes, Id1 and Id2, reveals a highly conserved protein coding gene organisation consistent with evolution from a common, ancestral Id-like gene. In addition, by using a yeast artificial chromosome (YAC) clone of Id3, we have fine-scale mapped the gene to chromosome band 1p36.1 by fluorescence in situ hybridisation (FISH) and, using the same FISH technique, we have detected heterogeneity in tumour-associated 1p36 chromosome translocations.
3339	Role of sialic acid residues in the in vitro superactivity of human choriogonadotropin (hCG) in rat Leydig cells.	7803516	The binding activity (B) of porcine Luteinizing Hormone (pLH) to rat LH receptor as well as its stimulating activity (S) of testosterone secretion by rat Leydig cells in vitro are similar to those of the homologous hormone rat LH (S/B = 1). By contrast, the human Chorionic Gonadotropin (CG) and hLH exhibit stimulating activities relative to rat LH that are considerably higher than their relative binding activities (S/B > 100) indicating that they have an abnormally high transducing efficiency (superactivity) after receptor binding. The heterologous hybrid alpha pLH x beta hCG is as superactive as native hCG and recombined alpha hCG x beta hCG whereas alpha hCG x beta pLH exhibits no superactivity, like native pLH and alpha pLH x beta pLH demonstrating that hCG superactivity is due to its beta-subunit. The removal of sialic acid residues with neuraminidase dramatically diminished hCG stimulating activity without impairing its receptor binding activity but the S/B ratio for asialo-hCG never reached values lower than 1. Similar treatments had no effect on the S/B ratios of non-superactive gonadotropins, pLH and equine CG. Sialic acid residues in the Asn beta 30 carbohydrate chains of hLH and hCG appear to be responsible for their superactivity in the in vitro stimulation of testosterone secretion by rat Leydig cells.
3340	An intermolecular disulfide bond stabilizes E2A homodimers and is required for DNA binding at physiological temperatures.	8001133	It is demonstrated in this report that purified E2A helix-loop-helix (HLH) proteins spontaneously form homodimers that are linked by an intermolecular disulfide bond. These homodimers bind DNA at physiological temperatures but fail to associate with either Id or MyoD. When the disulfide bond is reduced by an activity present in muscle cell lysates or disrupted by site-directed mutagenesis, the monomeric form of the protein is strongly favored at 37 degrees C. These E2A monomers cannot bind DNA but heterodimerize efficiently with Id and MyoD. It is also shown that an intermolecular disulfide bond cross-links E2A homodimers in B cells but not in muscle cells in which only heterodimers have been detected. These results suggest a novel mechanism for regulating the dimerization status and DNA binding properties of E2A HLH transcription factors.
3341	Role of the leucine zipper in the kinetics of DNA binding by transcription factor USF.	7982989	USF is a transcription factor characterized by a helix-loop-helix (HLH) DNA-binding domain that has been highly conserved through evolution. Vertebrate USFs contain an additional C-terminal leucine zipper (LZ) immediately adjacent to the HLH domain. This LZ is essential for efficient DNA binding by human USF. However, sea urchin has a USF family member that lacks a LZ and yet binds DNA efficiently. To clarify the role of the LZ in DNA binding by USF, we compared the properties of human and sea urchin USFs and found that the two proteins interacted with their specific sites on the DNA with identical affinities but very different kinetics. Association and dissociation rate constants of sea urchin USF were about 10-fold those of human USF. Domain-swapping experiments revealed that the LZ was responsible for the slower kinetics of human USF. USF heterodimers containing a single LZ displayed rates intermediate between those of dimers containing either two or no LZ, indicating that zipper-zipper interactions within USF dimers were not important for DNA binding. Temperature effects on DNA-binding parameters revealed a very high energy barrier for binding of human USF to DNA. Presence of a LZ increased the activation energy of the reaction.
3342	c-Myc inhibits myogenic differentiation and myoD expression by a mechanism which can be dissociated from cell transformation.	7970710	The c-Myc oncoprotein is a basic-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factor involved in regulating cell proliferation and differentiation. We have used retrovirus-mediated gene transfer to investigate the effect of ectopic c-Myc expression on the spontaneous differentiation of primary quail myoblasts in vitro. Unlike normal myoblasts, c-Myc-expressing myoblasts are unable to form myotubes or express muscle-specific genes, such as myosin, and show severely reduced expression of the myogenic regulatory factors myoD, myogenin, and myf5. The c-Myc leucine zipper (LZ) motif is essential for the differentiation block since myoblasts expressing a mutant with a partial deletion of this region, c-Myc delta 7, differentiate and express myoD family regulators and muscle-specific genes normally. Remarkably, c-Myc delta 7, like wild-type c-Myc, retains the capacity to transform the growth phenotype of myoblasts, and associates with the b-HLH-LZ Myc partner protein Max in transformed cells. We conclude that the block to myogenic differentiation induced by c-Myc can be dissociated from cell transformation per se, and that this attribute correlates more closely with down-regulation of myoD family gene expression. These findings are discussed in the light of current models of Myc function.
3343	The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo.	7821220	The Enhancer of split locus is required during many cell-fate decisions in Drosophila, including the segregation of neural precursors in the embryo. We have generated monoclonal antibodies that recognise some of the basic helix-loop-helix proteins encoded by the Enhancer of split locus and have used them to examine expression of Enhancer of split proteins during neurogenesis. The proteins are expressed in a dynamic pattern in the ventral neurogenic region and are confined to those ectodermal cells that surround a neuroblast in the process of delaminating. There is no staining in the neuroblasts themselves. We have also examined the relationship between Enhancer of split protein accumulation and the Notch signalling pathway. Protein expression is abolished in a number of neurogenic mutant backgrounds, including Notch, but is increased as a result of expressing a constitutively active Notch product. We conclude that Notch signalling activity is directly responsible for the accumulation of basic helix-loop-helix proteins encoded by the Enhancer of split locus.
3344	Haemophagocytic lymphohistiocytosis: experience at two U.K. centres.	7819097	Haemophagocytic lymphohistiocytosis (HLH) is a rare disorder of inappropriate macrophage activation. Both familial and sporadic forms, which may be infection-associated, are recognized. Between 1985 and 1991 we treated 23 cases of HLH (12 male, 11 female). There were eight familial cases, defined by a previously affected sibling and/or history of consanguinity, age 3 d to 15 months at presentation. The age of the remaining 15 cases varied from 1 month to 9.5 years. A potential viral trigger was identified in four cases (EBV, two; parvovirus B19, one; echovirus II, one) including one familial case. Six of eight (75%) patients who received supportive care alone, including all four familial cases, died within 6 months of presentation. Both long-term survivors in this group presented at an older age (7.5 and 8 years) and had proven or suspected virus-associated HLH. 15 patients were treated with etoposide (150-250 mg/m2 days 1-3 every 21 d) and methylprednisolone; 10 patients received intrathecal methotrexate in addition. In nine (60%) of these cases a complete (six) or partial (three) response was achieved, though one child suffered a fatal 'tumour lysis' syndrome. Overall mortality in the treated group was 66.6%, being highest (75%) in patients under 2 years at presentation compared to 33% in those over 2 years. Two of three familial and one of five sporadic cases relapsed and died 3 d to 20 months from diagnosis. Only one familial case survives at follow-up of 11 months. Of the five remaining survivors, two received allogeneic bone marrow transplantation (one matched related, one haploidentical) and are alive at 11 and 29 months. Three cases aged 2.5, 7.5 and 9.5 years remain in remission at 11, 20 and 25 months respectively. The high mortality of HLH supports a role for allogeneic BMT in selected cases, particularly those with a familial basis or under 2 years at presentation.
3345	Control of skeletal muscle-specific transcription: involvement of paired homeodomain and MADS domain transcription factors.	7779681	In the last few years, many aspects of skeletal muscle-specific gene regulation have been explained by the activity of the helix-loop helix (HLH) myogenic regulatory factors of the MyoD family, which are sequentially expressed during skeletal muscle formation. However, evidence is accumulating that muscle specific transcription requires functional interactions of these muscle-specific HLH factors with other regulatory proteins whose expression is not only restricted to skeletal muscle. These regulators include the SRF and MEF2 MADS domain and the MHox paired homeodomain transcription factors. Together with the aforementioned HLH factors, they build an increasingly complex network of regulatory factors. Two members of the Pax multigenic family of developmental control transcription factors, Pax-3 and 7, have been shown to be expressed not only in nervous tissue but also in skeletal muscle precursor cells. Their possible involvement in the control of muscle-specific transcription is discussed in light of known molecular properties of Pax gene products described in other biological systems.
3346	microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family.	7958932	The microphthalmia (mi) gene appears essential for pigment cell development and/or survival, based on its mutation in mi mice. It has also been linked to the human disorder Waardenburg Syndrome. The mi gene was recently cloned and predicts a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) factor with tissue-restricted expression. Here, we show that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family. Mi can also activate transcription through recognition of the M box, a highly conserved pigmentation gene promoter element, and may thereby determine tissue-specific expression of pigmentation enzymes. Six mi mutations shown recently to cluster in the b-HLH-ZIP region produce surprising and instructive effects on DNA recognition and oligomerization. An alternatively spliced exon located outside of the b-HLH-ZIP region is shown to significantly modulate DNA recognition by the basic domain. These findings suggest that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiT family interactions and transcriptional activities.
3347	Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete.	7958929	hairy (h) acts as a negative regulator in both embryonic segmentation and adult peripheral nervous system (PNS) development in Drosophila. Here, we demonstrate that h, a basic-helix-loop-helix (bHLH) protein, is a sequence-specific DNA-binding protein and transcriptional repressor. We identify the proneural gene achaete (ac) as a direct downstream target of h regulation in vivo. Mutation of a single, evolutionarily conserved, high-affinity h binding site in the upstream region of ac results in the appearance of ectopic sensory organs in adult flies, in a pattern that strongly resembles the phenotype of h mutants. This indicates that direct repression of ac by h plays an essential role in pattern formation in the PNS. Our results demonstrate that HLH proteins negatively regulate ac transcription by at least two distinct mechanisms.
3348	Elements regulating cell- and stage-specific expression of the C. elegans MyoD family homolog hlh-1.	7958441	We investigated the cis-acting sequences regulating expression of the Caenorhabditis elegans gene hlh-1, a homolog of the MyoD family of myogenic regulatory factors. The hlh-1 gene is expressed in mature body wall muscle, in clonal muscle precursors, in a set of early embryonic blastomeres (the MS-granddaughters), and in six glial-like cells called GLRs. The entire natural hlh-1 expression pattern is recapitulated in transgenic animals containing an hlh-1::lacZ fusion with 5.1 kb of hlh-1 sequences beginning upstream of the coding region and extending into the second exon. Deletions and rearrangements in this 5.1-kb sequence were assayed for their effects on reporter gene expression in transgenic animals. Deletions removing a segment 434-550 bp upstream of the coding region resulted in a loss of all aspects of the expression pattern, suggesting that at least one common regulatory factor is required for all expression. Deletions of other regulatory elements affected distinct aspects of the expression pattern; hence, each expression subpattern exhibits a different set of sequence requirements. Interspecies sequence comparison and lacZ expression constructs with the related nematode Caenorhabditis briggsae indicated that the C. briggsae and C. elegans genes contain equivalent sets of control signals. The results from this work implicate the hlh-1 promoter as an integration point for diverse temporal and spatial control signals.
3349	Hormone-binding properties and glycosylation pattern of a recombinant form of the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor expressed in mammalian cells.	7956911	The present studies were undertaken to characterize the properties of a recombinant truncated rat LH/CG (rLH/CG) receptor representing the extracellular domain (rLHR-t338) expressed in mammalian cells. Using detergent-soluble extracts of stably transfected cells, we show that the binding of ovine (o) LH to the extracellular domain is different from the binding of oLH to the full-length receptor. Thus, whereas the full-length receptor from either rat corpora lutea or stably transfected 293 cells binds oLH with two apparent affinities, rLHR-t338 binds oLH with only one affinity, which is intermediate between the high and low affinities of the full-length receptors. However, rLHR-t338 as well as the full-length receptor binds hCG and hLH with similar high affinities and by a model assuming the presence of only one binding site. These data are consistent with the possibility that the carboxyl-terminal half of the LH/CG receptor may contribute (either directly or indirectly) to a second low affinity hormone-binding site. The truncated receptor retains normal binding specificity, as it does not bind hFSH, demonstrating that the extracellular domain alone contains all of the necessary elements for conferring specificity among the gonadotropin hormones. Our studies also show that rLHR-t338 is glycosylated and migrates as two proteins of 48 and 44 kilodaltons (kDa) on sodium dodecyl sulfate gels (where the 44-kDa species is probably a partially degraded form of the 48-kDa protein). However, the N-linked oligosaccharides of the truncated receptor do not appear to be fully processed, as they remain in a high mannose form. rLHR-t338 is not secreted from cells, but remains trapped intracellularly. It can be detected, though, in detergent-soluble extracts of cells or in homogenates. Upon centrifugation of homogenates, it was found that about 90% of the recoverable truncated receptor can be obtained in the supernatant fraction. Thus, mammalian cells could provide a source of a water-soluble extracellular domain of the LH/CG receptor.
3350	Evolutionary relationships and functional conservation among vertebrate Max-associated proteins: the zebra fish homolog of Mxi1.	7936639	In mammals, current evidence supports the view that Myc-responsive activities are regulated in part through an intracellular balance between levels of transcriptionally-active Myc/Max heterodimers and those of transcriptionally-inert Max/Max, Mad/Max and Mxi1/Max complexes. To gain insight into the roles of Mad and Mxi1 in cellular growth and differentiation and to fortify key structure-function relationships from an evolutionary standpoint, low stringency hybridization screens were used to identify potential homologs of these Max-associated proteins in the zebra fish genome. A single class of cDNA clones that cross-hybridized both to human mad and mxi1 probes was shown to encode a putative protein with significantly greater homology to mammalian Mxi1 than to Mad, particularly in the basic and helix-loop-helix (bHLH) regions. The high degree of structural relatedness between vertebrate Mxi1 proteins apparent in molecular modelling studies was consistent with the findings that the HLH/leucine zipper (LZ) region of zMxi1 exhibited the same profile of dimerization specificities as its mammalian counterpart in the two-hybrid system and that zmxi1 could, like human mxi1 (Lahoz et al., 1994), suppress the oncogenic potential of mouse c-myc in a mammalian cell. Finally, a comparison of steady-state zc-myc and zmxi1 mRNA levels during zebra fish embryogenesis demonstrated (i) high levels of zc-myc relative to zmxi1 mRNA during initiation of organogenesis, a period characterized by intense growth and active differentiation and (ii) rising levels of zmxi1 mRNA during progression towards the terminally differentiated state. These contrasting patterns of developmental expression together with the capacity of zmxi1 to repress myc-induced transformation support a model for the regulation, by Max-associated proteins, of Myc functions in the control of normal cell development and neoplastic growth.
3351	Cloning and analysis of a new developmentally regulated member of the basic helix-loop-helix family.	7873406	We have isolated a basic helix-loop-helix (bHLH) family member from an embryonic chick-brain cDNA library. This 3.86-Kb cDNA, GbHLH1.4, exhibits extensive sequence similarity in the bHLH domain with Drosophila daughterless and the vertebrate cDNAs E12 and HTF4. Outside of the bHLH region the similarity is significantly reduced. GbHLH1.4 recognizes a 4.0-Kb mRNA and in situ hybridization analysis shows that GbHLH1.4 mRNA is widely expressed at early stages of development but becomes progressively restricted as embryogenesis proceeds. At later stages of embryonic development, mRNA transcripts are localized to several structures including the ventricular layers of the spinal cord and brain, the facial primordia, dorsal root ganglia and heart muscle and cardiac valves. Strikingly, GbHLH1.4 expression in chick embryos exhibits significant overlap with that reported for the murine negative HLH regulator, Id.
3352	Rhesus monkey oocyte maturation and fertilization in vitro: roles of the menstrual cycle phase and of exogenous gonadotropins.	7849190	Our objectives were to improve the efficiency of rhesus monkey oocyte maturation and fertilization in vitro and to determine the influence of menstrual cycle phase and exogenous gonadotropins on these processes. Immature oocytes were recovered from antral follicles of ovaries excised from unstimulated animals (n = 14). The highest yield and quality was associated with oocytes obtained from early follicular phase ovaries, and more of these oocytes underwent germinal vesicle breakdown (GVBD; 55%) and matured to metaphase II (MII; 40%) than did oocytes from late follicular (GVBD, 28%; MII, 9%) or luteal (GVBD, 13%; MII, 10%) phase ovaries. Exogenous human (h) FSH and hLH improved GVBD and MII levels for oocytes recovered from late follicular phase ovaries and increased GVBD, but not MII, for oocytes from luteal phase ovaries. MII levels for oocytes from early follicular phase ovaries were adversely affected. Early follicular phase oocytes underwent GVBD faster and attained MII sooner than did those from either late follicular or luteal phase ovaries. Whereas exogenous gonadotropins did not significantly affect oocyte fertilization, follicular phase oocytes tended to be fertilized at a higher rate (p = 0.07). These results demonstrate that higher maturation levels are obtained with oocytes from early follicular phase ovaries and that gonadotropins influence oocyte performance in a cycle phase-dependent manner.
3353	Haemophagocytic lymphohistiocytosis, interferon-gamma-naemia and Epstein-Barr virus involvement.	7819087	To clarify the correlation between Epstein-Barr virus (EBV) involvement and hypercytokinaemia in haemophagocytic lymphohistiocytosis (HLH), we analysed serum interferon-gamma levels and EBV-DNA in biological specimens obtained from 25 HLH cases (23 children and two adults). We found that HLH patients showed a wide range of serum IFN-gamma levels from 0.2 to 1300 U/ml, with a median 126 U/ml for EBV-DNA-positive (n = 9) and 4.5 U/ml for EBV-DNA-negative (n = 16) groups. The latter group could be classified further into a group with hyper-IFN-gamma-naemia (> 4.5 U/ml) (n = 8) and a group without hyper-IFN-gamma-naemia (n = 8). The survival of the hyper-IFN-gamma-naemic cases was significantly poorer than non-hyper-IFN-gamma-naemic cases. We conclude that EBV is probably involved in one third of the HLH cases, all of whom show hyper-IFN-gamma-naemia, and in the half of the HLH cases with hyper-IFN-gamma-naemia who have a rapidly fatal outcome.
3354	Immunohistochemical double-staining for Ah receptor and ARNT in human embryonic palatal shelves.	7716743	The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic-helix-loop-helix-PAS (HLH) proteins involved in transcriptional regulation. Polycyclic aromatic halogenated chemicals, of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent, bind to the AhR. In the cellular cytoplasm, the AhR exists as a complex with the heat shock protein HSP90 and other small peptides. This complex dissociates following ligand binding and then the ligand-bound AhR binds ARNT. The ligand-AhR-ARNT complex interacts with a specific, nuclear DNA sequence, the dioxin response element (DRE), altering transcription of a regulated gene. Studies in hepatoma cell lines indicate that both proteins are required for regulation of transcription. In this study, AhR and ARNT were localized immunohistochemically in human embryonic palatal cells and specific patterns of expression were seen for each protein. A double-staining protocol revealed that epithelial cells expressed both AhR and ARNT, but in mesenchyme and nasal spine cartilage individual cells were identified which expressed either AhR or ARNT. This heterogeneous pattern may be a means of suppressing transcriptional regulation and also suggests the existence of other, unidentified basic-helix-loop-helix partner(s). The heterogeneous expression pattern may also reflect a complex role for these HLH proteins as transcriptional regulators of embryonic development.
3355	The helix-loop-helix containing transcription factor USF activates the promoter of the CD2 gene.	7929376	T cell development within the thymus involves the ordered expression of a number of tissue-specific components such as the CD2 gene. Control of expression of this gene is regulated by a well characterized 3' enhancer together with a promoter and upstream elements. The CD2 promoter is typical of a group of T cell-specific promoters that lack a TATA box and use multiple sites for initiation of transcription. An "E box" motif CACGTG, located just upstream from the most 5' initiation start site, was found to contribute a major effect to the level of basal transcription of a reporter gene. Analysis of the proteins in T cell extracts that bound to this site revealed that the bHLH-LZ protein USF was the major component. A functional role for USF was established in transient transfection experiments. Thus, this protein restored full promoter activity following repression caused by cotransfection with the E box binding bHLH-LZ protein Max. Taken together, these results indicate that an E box motif is critical to expression of the CD2 gene during T cell development and that the HLH protein USF acts as a transcriptional activator of the CD2 promoter.
3356	Receptor binding and functional properties of chimeric human follitropin prepared by an exchange between a small hydrophilic intercysteine loop of human follitropin and human lutropin.	7929221	The family of pituitary/placental glycoprotein hormones includes follicle-stimulating hormone (follitropin, FSH), luteinizing hormone (lutropin, LH), chorionic gonadotropin (choriogonadotropin, CG), and thyroid-stimulating hormone (thyrotropin, TSH). These glycoproteins are heterodimeric, with an alpha-subunit of identical primary structure and a hormone-specific beta-subunit which is believed to confer receptor specificity. Previous studies demonstrated that substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta, residues 94-145, conferred to human (h) CG an FSH receptor binding specificity. To more finely map the LH/FSH receptor binding specificity determinant, hFSH beta residues 88DSDS91, within the small hydrophilic intercysteine loop, and residues 95TVRGLG100 COOH-terminal to the loop were switched to hLH beta residues 94RRST97 and residues 101GGPKDH106, respectively. Substitution of hLH beta residues 94RRST97 in place of hFSH beta residues 88DSDS91 did not affect FSH receptor binding or activation but, remarkably, conferred LH receptor binding activity to the chimera. This chimera retained the ability to stimulate progesterone production in a Yl cell line which expresses human FSH receptor. Replacing hFSH beta residues 95TVRGLG100 with hLH beta residues 101GGPKDH106 diminished FSH receptor binding activity but did not confer LH receptor binding to the chimera. This study suggests that amino acids within hFSH beta residues 95TVRGLG100 are important for FSH binding specificity and that hLH beta residues 94RRST97 are involved in LH receptor binding specificity. Thus, although the hFSH beta and hLH beta subunits may fold similarly, the loci of receptor binding specificity are not entirely homologous.
3357	Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.	8084592	MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation.
3358	Nuclear redirection of a cytoplasmic helix-loop-helix protein via heterodimerization with a nuclear localizing partner.	7925642	The DNA-binding basic domain of helix-loop-helix transcriptional (HLH) factors in several instances also serves as a nuclear localization signal (NLS). Interestingly, some members of the HLH family of proteins lack a basic domain or any other recognizable NLS and yet still display efficient nuclear localization. To study this apparent paradox, we used the hematopoietic HLH protein SCL/tal as a model. Deletion of the basic domain converted SCL/tal from nuclear to a cytoplasmic protein. However, the basic domain deficient SCL/tal protein could be redirected to the nucleus by coexpression of E2-5, an SCL/tal-binding HLH protein with an intact basic domain. These results indicate that heterodimerization of HLH proteins may take place in the cytoplasm prior to nuclear localization and that nuclear localization for HLH complexes is a dominant process requiring only a single NLS per complex.
3359	The dimerization stability of the HLH-LZ transcription protein family is modulated by the leucine zippers: a CD and NMR study of TFEB and c-Myc.	7727380	In the HLH-LZ protein family, the helix-loop-helix DNA-binding dimerization domain is followed in the sequence by a leucine zipper motif. The precise function of this second dimerization domain is still unclear, since the HLH motif of a subset of this family has been shown to be necessary and sufficient for dimerization. However, deletion and mutagenesis studies of the leucine zipper in various HLH-LZ proteins have shown a clear influence of this motif on homo- and heterodimerization. In this paper, we present a structural characterization of synthetic peptides encompassing the leucine zipper sequences of c-Myc and TFEB, using circular dichroism, analytical ultracentrifugation, and nuclear magnetic resonance. We show that the different ability of the synthetic leucine zippers of c-Myc and TFEB to homodimerize at neutral pH reflects the different dimerization properties reported for the entire proteins. The TFEB protein is known to form homodimers. c-Myc, on the other hand, does not homodimerize in vivo, but is mostly found in heterodimeric complexes with Max, another protein of the HLH-LZ family. Accordingly, our results show that the TFEB peptide homodimerizes at neutral pH whereas the Myc peptide dimerizes to a comparable amount only at acidic pH and high ionic strength. Both synthetic peptides are far less stable than leucine zippers of the b-ZIP family. The relative stability of the two leucine zippers and the factors which stabilize the dimer formation are discussed.
3360	Regulation of G1 progression by E2A and Id helix-loop-helix proteins.	7925274	In NIH3T3 fibroblasts, the ubiquitous helix-loop-helix (HLH) protein E2A (E12/E47) and the myogenic HLH proteins MyoD, MRF4 and myogenin are growth-inhibitory, while two ubiquitous Id proteins lacking the basic region are not. The dimerization domain mediates inhibition. However, in addition to the HLH region, E2A contains two inhibitory regions over-lapping with the main transcriptional activation domains. The growth-suppressive activity of the intact E47 as well as MyoD was counteracted by the Id proteins. When E47 lacking the HLH domain was overexpressed, Id could no longer reverse growth inhibition. By increasing the amount of E47 with an inducible system or neutralizing the endogenous Id with microinjected anti-Id antibodies, withdrawal from the cell cycle occurred within hours before the G1-S transition point. The combined results suggest that the Id proteins are required for G1 progression. The antagonism between the E2A and Id proteins further suggests that both are involved in regulatory events prior to or near the restriction point in the G1 phase of the cell cycle.
3361	bHLH proteins encoded by the Enhancer of split complex of Drosophila negatively interfere with transcriptional activation mediated by proneural genes.	8078474	The Enhancer of split complex [E(SPL)-C] of Drosophila participates in the control of cell fate choice by uncommitted neuroectodermal cells in the embryo. It encodes seven proteins that belong to the basic helix-loop-helix (bHLH) family, six of which are expressed in very similar patterns in the neuroectoderm. Here we describe experiments aimed at unravelling the molecular basis of their function. We found that two products of the complex, HLH-M5 and ENHANCER OF SPLIT, are capable of binding as homo-and heterodimers to a sequence in the promoters of the Enhancer of split and achaete genes, called the N-box, which differs slightly from the consensus binding site (the E-box) for other bHLH proteins. In transient expression assays in cell culture, both proteins were found to attenuate the transcriptional activation mediated by the proneural bHLH proteins LETHAL OF SCUTE and DAUGHTERLESS at the Enhancer of split promoter.
3362	c-Myc represses transcription in vivo by a novel mechanism dependent on the initiator element and Myc box II.	8076602	We show that c-Myc, in addition to activating transcription through E-box Myc binding sites (Ems), also represses transcription by a mechanism dependent on initiator (Inr) elements of the basal promoters of susceptible genes. Repression was first observed as a component of c-Myc biphasic regulation of the adenovirus-2 major late promoter (MLP), which contains both Inr and Ems sequences. Two differentiation-specific genes containing Inr, the C/EBP alpha and albumin genes, are repressed through their basal promoters by c-Myc, but are activated by the related B-HLH-LZ factor, USF. Repression requires both the B-HLH-LZ and Myc box II (MBII) domains. Significantly, a MBII deletion mutant which is deficient in repression, but transactivates normally, fails to cooperate with an activated ras gene to transform primary fibroblasts. Thus Myc-dependent transactivation is insufficient for Ras cooperation and the novel transcription repression function is implicated in Ras cooperation as well as the suppression of Inr-dependent genes.
3363	GATA-and SP1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene.	8058326	The tal-1 gene, which is frequently activated in human T cell acute leukemias (T-ALLs), codes for a protein of the basic helix-loop-helix family (b-HLH) and potentially a transcription factor. In human and murine hematopoiesis tal-1 is expressed during the differentiation of the erythroid, megakaryocytic and mastocytic cell lineages. The expression of tal-1 appears to be comodulated with that of the transcription factor GATA-1 gene, suggesting that the GATA-1 protein may regulate the tal-1 gene activity in these hematopoietic lineages. To get further insights into the molecular mechanisms that control tal-1 expression, we have isolated 5' sequences of the murine gene and compared them to their human counterparts. The 5' flanking sequences from the two genes show several regions of high homology. The alignment of both sequences enabled us to predict that similarly, to the human, the mouse gene contains two alternative first exons (Ia and Ib). Remarkably, in both species, the proximal region of the tissue-specific exon Ia (i.e. gene segment -122 to +1) contains two GATA-motifs (at -65 and -33) and one SP-1 consensus binding site (-59). Mobility shift assays demonstrate that GATA proteins are able to interact with both GATA-motifs in a sequence specific fashion, but with different efficiencies. Moreover transfection studies show that the GATA-1 protein directly mediates tal-1 transcription by interacting with the -122/+1 fragment, defined as a minimal promoter in erythroid cells. Mutagenesis of the promoter establishes that the -33 GATA-binding site present in this fragment is critical for tal-1 expression in erythroid cells, but by itself does not lead to full promoter activity. Indeed, further mutations show that the second -65 GATA-binding site and the binding motif for SP1 (-59) significantly contribute to the overall activity of the proximal tal-1 promoter. Altogether, our data provide evidence that GATA-1 cooperates with the transcription factor SP1 to mediate the erythroid-specific expression of the tal-1 gene.
3364	Association of USF and c-Myc with a helix-loop-helix-consensus motif in the core promoter of the murine type II beta regulatory subunit gene of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase.	7838149	Previous studies showed that the core promoter of the mouse cAMP-dependent protein kinase regulatory subunit type II beta (RII beta) gene was composed of two functional elements. One element was GC rich and bound the Sp1 transcription factor. The second element contained a helix-loop-helix (HLH)-motif. Each element conferred transcriptional activity when inserted upstream of a reporter gene, chloramphenicol acetyltransferase and transfected into mouse NB2a neuroblastoma cells and Chinese hamster ovary (CHO) cells. The core promoter was further characterized by mutational analysis using electrophoretic mobility shift assays and by transfection into CHO and NB2a cells. Electrophoretic mobility shift assays showed that the HLH-consensus motif, CACGTG, present in the RII beta gene bound nuclear factors present in NB2a and CHO cells. Mutations in the HLH-core motif decreased the binding of these factors and reduced the transcriptional activity of constructs containing the chloramphenicol acetyltransferase reporter when transfected into these cells. The results showed that the central nucleotides as well as the adjacent bases were important for the interaction with the nuclear binding factors. UV cross-linking, Southwestern blot analysis, and interference of the mobility shift patterns by specific antisera directed against USF and c-Myc indicated that both of these transcription factors were forming complexes with the HLH-consensus motif. The results suggest that RII beta transcription may be regulated, in part, by USF and c-Myc in NB2a and CHO cells.
3365	PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter.	8070412	Expression of the low-affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type-specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis-acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3' purine tract (-50 to -39) within the MRR binds the macrophage and B cell-specific factor, PU.1, and a second E box-like element, termed MyE, upstream of the PU.1 box (-88 to -78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid-specific activity of the Fc gamma RIIIA promoter.
3366	Radioligand binding assay of progesterone receptors in the primate corpus luteum after in vivo treatment with the 3 beta-hydroxysteroid dehydrogenase inhibitor, trilostane.	8045986	We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3 beta-hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (+/- SE) serum levels of P from 97.8 +/- 16 to 2.7 +/- 1.3 nmol/L within 18 h (P < 0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H] R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (Kd) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 +/- 0.09 and 0.06 +/- 0.02 fmol/micrograms DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.
3367	Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix (HLH)-type transcription factors.	8045940	To elucidate regulatory mechanism(s) underlying differentiation of osteoblasts, we examined involvement of helix-loop-helix (HLH)-type transcription factors in osteoblast-specific expression of a phenotypic marker gene which encodes osteocalcin, a major noncollagenous bone matrix protein, exclusively expressed in osteoblasts. Overexpression of a dominant negative HLH protein, Id-1, decreased the activity of the 1.1-kb osteocalcin gene promoter cotransfected into rat osteoblastic osteosarcoma ROS17/2.8 cells. Analysis of deletion mutants revealed that a 264-bp fragment of osteocalcin promoter (-198 to +66) was sufficient for the Id-1-dependent suppression. Furthermore, the activity of the same promoter fragment (-198 to +66) was enhanced when antisense Id-1 expression vector was cotransfected. This osteocalcin gene promoter region contains two sites of an E-box motif, a consensus binding site for HLH proteins, which we refer to as OCE1 (CACATG, at -102) and OCE2 (CAGCTG, at -149), respectively. Mutagenesis in OCE1 but not OCE2 led to greater than 50% reduction in transcriptional activity of the osteocalcin gene promoter. Electrophoresis mobility shift assay indicated that factors in nuclear extracts prepared from ROS17/2.8 cells bound to the 30-bp oligonucleotide probe containing the E-box motif of OCE1. This binding was competed out by OCE1 oligonucleotide but neither by OCmE1 oligonucleotide in which E-box motif was mutated nor by OCE2. The OCE1-binding activity in the nuclear extracts of ROS17/2.8 cells was reduced by 70% when bacterially expressed Id-1 protein was added to the reaction mixture, suggesting the involvement of HLH proteins in the DNA/protein complex formation. In contrast to the osteoblast-like cells, OCE1-binding activity in the nuclear extracts of C3H10T1/2 fibroblasts was very low. However, when these fibroblasts were treated with recombinant human bone morphogenetic protein-2 which induced expression of osteocalcin as well as other phenotypic markers of osteoblasts, OCE1-binding activity was increased approximately 40-fold, indicating that OCE1 would be involved in the tissue-specific expression of the osteocalcin gene. These findings indicated for the first time that osteoblast-specific gene transcription is regulated via the interaction between certain E-box binding transcription factor(s) in osteoblasts and the OCE1 sequence in the promoter region of the osteocalcin gene.
3368	Expression of the helix-loop-helix protein, Id, during branching morphogenesis in the kidney.	7967343	Id, a member of the helix-loop-helix protein family, is an inhibitor of transcriptional activation by basic-helix-loop-helix proteins. In the developing mouse kidney, Id mRNA was observed as early as 12.5 days post-coitum (dpc) specifically in the condensed mesenchyme surrounding the ureteric buds by in situ hybridization. At 14.5 dpc, Id mRNA was localized to the collecting tubules and developing glomeruli while the surrounding mesenchyme lacked Id hybridization. From birth to day 10 postnatal, Id mRNA is localized to the collecting tubules, immature glomeruli and renal pelvis. In the adult kidney, Id mRNA was detectable by Northern blot analysis but no cell type-specific localization was noted by in situ hybridization. These results indicate a role for HLH-bHLH proteins in the differentiation of the epithelial structures of the kidney.
3369	Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper protein. Molecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein.	7811391	The cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.
3370	On the role of the invariant glutamine at position 54 in the human choriogonadotropin beta subunit.	7531278	The twelve Cys and eight of the non-Cys residues are invariant in the glycoprotein hormone beta subunits from a variety of mammalian species. beta-Gin-54 of human lutropin (hLH) and choriogonadotropin (hCG) is one of these invariant amino acid residues. A single A-->G mutation in the LH beta gene of a patient presenting with hypogonadism resulted in the replacement of Gin-54 with Arg [1]. The authors also reported that an expressed mutant of hLH beta, with Arg replacing Gin-54, associated with the alpha subunit, but there was no demonstrable binding of the mutant hormone to receptor. We have replaced Gin-54 in hCG beta with Glu and with Lys using site-directed mutagenesis. The expression plasmids pRSV-hCG beta (wild-type and mutants) were transiently transfected into CHO cells containing a stably integrated gene for bovine alpha, and the media were analyzed for holoproteins, which were characterized in vitro using competitive binding and steroidogenic assays with MA-10 cells. hCG beta(Glu-54) bound to alpha almost as well as hCG beta wild-type, and the resulting heterodimer competed with [125l]hCG binding to the LH/CG receptor and stimulated progesterone production to the same extent as the wild-type control. However, the apparent potencies, as judged by ED50s, were less than those of the wild-type control, the effect being more pronounced in binding than in steroidogenesis. In contrast, hCG beta(Lys-54) associated very poorly with alpha. Our results suggest that while Gin-54 in hCG beta participates in receptor binding, its major function appears to involve alpha binding. Such dual functionality leads to interesting models for holoprotein formation and receptor binding.
3371	Leaf emergence of spring wheat receiving varying nitrogen supply at different stages of development.	19700456	We examined effects of nitrogen (N) supply on leaf emergence of spring wheat (Triticum aestivum L.) grown in sand with nutrient solution containing different N concentrations (9NO3: 1NH4). In expt 1, the cultivar 'Gamenya' received nutrient solution twice weekly containing a constant N supply ranging from 50 to 2400 microM N. In expts 2 and 3, cultivars 'Aroona' and 'Gamenya' were irrigated hourly with nutrient solution containing either low (L = 50 micrroM N) or high (H = 2000 microM N) N supply. In expt 2, the N supply to half of the plants receiving L and H was changed at the double ridge stage of apical development, producing plants receiving LL, LH, HL and HH. In expt 3, N supply was changed firstly when the main stem apex was vegetative (one to two leaves) and secondly when the main stem apex was at double ridge stage (four to five leaves), producing plants receiving LLL, LHL, HLH and HHH. Leaves on the main stem and primary tillers were counted. Rate of leaf emergence was estimated from regression of number of leaves against thermal time; the phyllochron was calculated as 1/ rate of emergence. Severely N-deficient plants (which had at least a 60 % reduction in shoot dry weight) had slower rates of leaf emergence (expt 1). Fluctuating N supply sometimes, but not always, changed the rate of leaf emergence (expts 2 and 3). The N supply before double ridge stage had bigger effects on the phyllochron than that afterwards (expt 3). The phyllocrons of the main stems were generally lower than those of tillers, with a greater difference between stems in N-deficient plants. Low N supply at the vegetative apex stage decreased the total number of leaves on the main stem, while low N supply after double ridge did not.
3372	Initiation of periovulatory events in primate follicles using recombinant and native human luteinizing hormone to mimic the midcycle gonadotropin surge.	8027245	The amplitude and duration of the midcycle LH surge required for periovulatory changes in the primate follicle are incompletely defined. We reported that short (4- to 14-h) LH surges were insufficient to induce periovulatory events after multiple follicular development in macaques. In contrast, an 18- to 24-h LH surge induced oocyte maturation plus granulosa cell luteinization, but did not support corpus luteum function. In this study, the periovulatory changes following LH surges of 48 h elicited using pituitary (pit) or recombinant (r) human (h) LH were compared to those after 24-h LH surge durations or after urinary hCG (u-hCG) treatment. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. On day 10, animals (n = 3-5/group) received 1) a single injection of u-hCG [79 +/- 3 micrograms RP-1 equivalents (equiv), im], 2) two injections of pit-hLH (91 +/- 4 micrograms RP-1 equiv, im), 3) one injection of r-hLH (21 +/- 1 micrograms RP-1 equiv, im), or 4) two injections of r-hLH (21 +/- 1 micrograms RP-1 equiv). Oocytes and granulosa cells were obtained via follicle aspiration 27 h after the initial LH or hCG injection. In all groups, serum estradiol rose to similar peak levels by day 10. Circulating LH-like bioactivity was elevated for more than 48 h after u-hCG. Peak serum LH bioactivities were proportional to the administered LH doses, as determined in the in vitro bioassay. Two injections of either r-hLH or pit-hLH elicited surge levels (> 100 ng/mL) of bioactive LH for 36-48 h, whereas one injection sustained surge levels for only 18-24 h. The proportions of oocytes resuming meiosis (68-76%) were similar in all groups. Immunocytochemical staining for progesterone receptor and in vitro progesterone production by granulosa cells in all LH-treated groups were comparable to those of cells form the hCG-treated group. Peak levels of progesterone in the luteal phase were comparable in monkeys treated with two doses of pit-hLH and r-hLH (18.5 +/- 10.4 vs. 8.1 +/- 1.5 ng/mL) and approached that in u-hCG treated monkeys (39.5 +/- 18.0 ng/mL). However, progesterone levels in animals treated once with r-hLH (3.4 +/- 1.5 ng/mL) were less (P < 0.05) than those in u-hCG-treated monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)
3373	Embryonic expression and function of the Drosophila helix-loop-helix gene, extramacrochaetae.	7947322	extramacrochaetae (emc) encodes a HLH protein that functions as a regulator of sensory organ precursor formation by virtue of its ability to form inactive heterodimers with the bHLH products of the achaete-scute complex (AS-C) and/or daughterless (da) genes. Although zygotic emc function is required for embryonic viability, its role in embryogenesis is not well understood. We describe here the distribution of emc transcripts during embryonic development. We also show that emc transcript is maternally contributed. Finally, we demonstrate that emc function is required for normal midgut and Malpighian tubule development. These results suggest that negative regulation by emc may be a common feature of developmental processes involving da, the AS-C and possibly other helix-loop-helix proteins.
3374	A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene.	7920706	The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5'-deleted promoters showed that a positive element involved in the response to wounding was located between -307 and -99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from -296 to -283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5' end to -289 with a disrupted Box 1 was fused to a reporter gene for beta-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2 (-289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from -296 to -283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix-loop-helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2(-529)/GUS.
3375	b/HLH without the zip.	7664056	NaN
3376	The E2A and tal-1 helix-loop-helix proteins associate in vivo and are modulated by Id proteins during interleukin 6-induced myeloid differentiation.	8016095	The immunoglobulin enhancer-binding proteins, E12 and E47, encoded by the E2A gene belong to the basic helix-loop-helix (bHLH) family of regulatory proteins and act as transcriptional activators. In addition to their critical role in B-lymphocyte development, the E12 and E47 proteins have been implicated in the induction of myogenesis as heterodimeric partners of myogenic bHLH proteins, MyoD and myogenin. Here we demonstrate that the E2A proteins form heterodimers with the bHLH oncoprotein tal-1 in myeloid and erythroid cells and that these heterodimers specifically bind to the CANNTG DNA motif. Heterodimerization with tal-1 represses transactivation by E47 and could function to prevent the expression of immunoglobulin genes in cells other than B lymphocytes. DNA binding by E2A-tal-1 heterodimers in the M1 mouse myeloid cell line is abrogated upon terminal macrophage differentiation induced by the cytokine interleukin 6. The loss of E2A-tal-1 DNA binding is correlated with elevated expression of mRNA encoding the dominant negative HLH proteins, Id1 and particularly Id2. Moreover, recombinant Id proteins inhibit the E2A-tal-1-specific DNA binding activity from undifferentiated M1 cells. These results suggest that E2A-tal-1 heterodimers may play a role in preventing terminal differentiation in the myeloid lineage and provide a possible explanation for oncogenic transformation induced by ectopic tal-1 expression in acute T-cell lymphoblastic leukemias.
3377	DNA-protein interactions in the Caenorhabditis elegans embryo: oocyte and embryonic factors that bind to the promoter of the gut-specific ges-1 gene.	8200477	We describe an experimental system in which to investigate DNA-protein interactions in the early Caenorhabditis elegans embryo. A homogeneous population of developmentally blocked mid-proliferation stage embryos can be produced by exposure to the deoxynucleotide analog fluorodeoxyuridine. These blocked embryos remain viable for days and express a number of biochemical markers of early differentiation, for example, gut granules, the gut esterase ges-1, and two regulatory genes, mab-5 and hlh-1. Using the techniques of gel mobility shift and DNase I footprinting, we show that nuclear extracts prepared from these embryos contain factors that bind to the 5'-promoter sequences of the C. elegans gut-specific ges-1 gene. In particular, we examine a putative gut "activator" region, which was previously identified by deletion-transformation analysis and which contains two copies of a consensus GATA-factor binding sequence. Factors that bind to double-stranded oligonucleotides containing the ges-1 GATA sequences are present predominantly in nuclear extracts of embryos but are found neither in cytoplasmic nor in nuclear extracts of unfertilized oocytes. Two proteins, of 43 and 60 kDa, can be uv-crosslinked to double-stranded oligonucleotides containing the ges-1 GATA sequences. The sizes of these proteins correspond to the sizes expected for the elt-1 protein and for the skn-1 protein, two regulatory factors present in early C. elegans embryos and possible candidates for ges-1 control. However, we show that homozygous deficiency embryos (mDf7/mDf7 embryos and eDf19/eDf19 embryos, both of which lack the elt-1 gene, and nDf41/nDf41 embryos, which have no skn-1 gene), still express the ges-1 esterase. We conclude that neither the elt-1 gene nor the skn-1 gene is necessary zygotically for ges-1 expression. We suggest that neither the elt-1 protein nor the skn-1 protein interacts directly with the ges-1 gene and that the observed binding proteins must correspond to products of other genes. More generally, the present experimental system should allow the biochemical study of any gene expressed during early C. elegans embryogenesis.
3378	A novel basic helix-loop-helix protein is expressed in muscle attachment sites of the Drosophila epidermis.	8196652	We have found that a novel basic helix-loop-helix (bHLH) protein is expressed almost exclusively in the epidermal attachments sites for the somatic muscles of Drosophila melanogaster. A Drosophila cDNA library was screened with radioactively labeled E12 protein, which can dimerize with many HLH proteins. One clone that emerged from this screen encoded a previously unknown protein of 360 amino acids, named delilah, that contains both basic and HLH domains, similar to a group of cellular transcription factors implicated in cell type determination. Delilah protein formed heterodimers with E12 that bind to the muscle creatine kinase promoter. In situ hybridization with the delilah cDNA localized the expression of the gene to a subset of cells in the epidermis which form a distinct pattern involving both the segmental boundaries and intrasegmental clusters. This pattern was coincident with the known sites of attachment of the somatic muscles to tendon cells in the epidermis. delilah expression persists in snail mutant embryos which lack mesoderm, indicating that expression of the gene was not induced by attachment of the underlying muscles. The similarity of this gene to other bHLH genes suggests that it plays an important role in the differentiation of epidermal cells into muscle attachment sites.
3379	Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys.	8194632	To test the hypothesis that steroids locally modulate and may be required for normal follicular function and gametogenesis in primates, the effects of steroid reduction during gonadotropin-stimulated folliculogenesis was studied in rhesus monkeys.Animals received human FSH (hFSH; days 1 to 6) and hFSH+human LH (hLH; day 7) to promote multiple follicular growth, and then received hCG (day 8) for ovulatory maturation. Four animals received trilostane (3 beta-hydroxysteroid dehydrogenase inhibitor) on days 1 to 8 or no inhibitor (controls; n = 4). Follicles were aspirated 34 hours after hCG.Follicular growth, serum E2, P, and pregnenolone, oocyte nuclear maturity, and IVF.Trilostane markedly reduced E2 to levels as low as 7% of controls throughout the follicular phase. Pregnenolone was 66-fold greater during trilostane treatment relative to controls. In both groups, P was at baseline during follicular stimulation but was reduced for 72 hours after hCG in trilostane-treated animals. Despite E2 suppression, follicular growth and oocyte nuclear maturity were unaltered by trilostane. Trilostane hindered the fertilizability of metaphase II oocytes (15%) in three of four animals compared with controls (65%). Metaphase I oocytes that required > 8 hours to complete meiosis in vitro failed to fertilize in the same three of four receiving trilostane relative to controls (31%).Follicular growth and oocyte meiosis did not require high or increasing E2 levels. Levels of follicular products other than E2 may be of value in determining the progress of ovarian stimulation protocols. However, the acquisition of oocyte competence for fertilization may require steroids.
3380	Expressed sequence tags (EST) identify genes preferentially expressed in catfish chemosensory tissues.	8090068	Expressed sequence tags (ESTs) for the catfish (Ictalurus punctatus) were identified and characterized by shotgun sequencing coupled to Northern analysis. We have identified and characterized a number of cDNA clones from a catfish olfactory mucosal library that show differential tissue expression including several that are enriched in chemosensory tissue. Among the novel cDNA clones studied were an olfactory specific beta-tubulin and a novel member of the S-100 family of calcium-binding proteins that is highly expressed in barbel, olfactory mucosa and gill, but not in brain. Several clones of low abundance mRNAs were also identified, including one manifesting a basic-helix-loop-helix (b-HLH) motif that is typical of many transcription factors. Additional cDNA clones whose mRNAs are differentially expressed, but are of unknown function, were also obtained. These results demonstrate the case with which novel gene products enriched in chemosensory tissues can be identified.
3381	c-Myc does not require max for transcriptional activity in PC-12 cells.	8087425	The c-Myc proto-oncogene is a basic helix-loop-helix leucine zipper (b/HLH/LZ) protein that participates in cellular growth and differentiation. The expression of c-Myc mRNA is rapidly induced by nerve growth factor (NGF) and epidermal growth factor (EGF) in PC-12 pheochromocytoma cells. In most cell types, c-Myc forms a sequence-specific DNA binding complex with the stable, constitutively expressed Max. This complex can function as a transcriptional regulator. We show here that the expression of Max mRNA or protein was not detected in PC-12 cells. Nevertheless, treatment of PC-12 cells with NGF and serum caused an increase in the expression of the c-Myc protein and the transcription of a reporter gene linked to the Myc/Max DNA binding site. Transcription from the same reporter gene is stimulated by over-expression of c-Myc. These results suggest that c-Myc protein functions as a transcriptional regulator in PC-12 cells despite the lack of Max protein. Therefore, Myc/Max complexes may not be an absolute requirement for Myc-dependent gene expression.
3382	LH- and chorionic gonadotrophin-stimulated progesterone release in vitro by intact luteal tissue of the marmoset monkey (Callithrix jacchus).	8071639	The application of an in vitro microdialysis system (MDS) for studies on the gonadotrophic control of luteal progesterone secretion in the marmoset monkey is described. Luteal tissue collected from a total of six animals (9 +/- 1 days after ovulation) was perfused with Ringer solution (without and with lipoprotein, 0.6 microgram/ml). The tissue was exposed to repeated applications of human LH (hLH) and human chorionic gonadotrophin (hCG) (1, 10 and 100 IU/ml) each of 60 min duration. Perfusate was collected in 15-min fractions and assayed for progesterone content. Results showed that addition of lipoproteins to the Ringer solution had a marked effect of progesterone secretion in terms of maintaining stable baseline levels and improving reproducibility of gonadotrophin-induced responses. Progesterone secretion was significantly stimulated by both gonadotrophins at each dose tested. Maximal elevations were obtained with 10 IU/ml and there were no apparent differences in responses to hLH and hCG in terms of either magnitude or duration. This study indicates that MDS provides a useful in vitro approach for studying the gonadotrophic control of the corpus luteum in non-human primates. The results did not demonstrate disparate actions of hLH and hCG in their ability to stimulate luteal progesterone secretion.
3383	The Caenorhabditis elegans MYOD homologue HLH-1 is essential for proper muscle function and complete morphogenesis.	8050369	A family of muscle-specific helix-loop-helix transcription factors (myoD, myogenin, myf-5 and MRF4) has been implicated in the control of vertebrate skeletal myogenesis. Searches for homologues of this family in Caenorhabditis elegans identified a single family member, hlh-1, which is expressed in striated muscles and their clonal precursors. We have isolated a null allele of hlh-1 following chemical mutagenesis. Animals homozygous for the null mutation produce contractile body-wall muscles, although muscle contractions are weak and coordination is defective. In addition to the evident muscle defects, mutant animals fail to complete embryonic elongation and die as larvae or young adults. Ultrastructural analysis of the mutant muscle reveals an apparently normal local lattice of thick and thin filaments, with more global defects in sarcomere organization and muscle cell placement. Mosaic studies using the point mutation and an extrachromosomal transgene indicate that the requirement for hlh-1 is fully zygotic, with no maternal hlh-1 requirement for either muscle development or viability.
3384	Involvement of interferon-gamma and macrophage colony-stimulating factor in pathogenesis of haemophagocytic lymphohistiocytosis in adults.	7947264	We investigated the role of monocyte/macrophage-activating cytokines in pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in 21 adult patients. Sera from patients with active HLH contained extremely high levels of macrophage colony-stimulating factor (M-CSF) and of interferon-gamma (IFN-gamma). These levels returned to almost normal during remission. Neither interleukin-4 nor granulocyte/macrophage colony-stimulating factor could be detected. Active HLH sera also contained high concentrations of inflammatory monokines, such as interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha). Serum concentrations of soluble CD8 and soluble interleukin-2 receptor were extremely high during active HLH, and returned to virtually normal levels during remission. Circulating CD2+ T-cells obtained from patients with active HLH spontaneously secreted M-CSF and IFN-gamma in vitro, whereas circulating monocytes did not produce detectable levels of both M-CSF and IFN-gamma, but produced high levels of IL-6 and TNF-alpha. These findings suggest that IFN-gamma and M-CSF at least partly from T-cells, such as CD8+ T-cells, might contribute to activation of monocytes or histiocytes, resulting in the up-regulated monokine production and haemophagocytosis in HLH.
3385	The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein.	7926730	Cell growth and differentiation are usually antagonistic. Proteins of the basic helix-loop-helix (bHLH) family bind DNA and play important roles in the differentiation of specific cell types. Id proteins heterodimerize with bHLH transcription factors, blocking their activation of lineage-specific gene expression and thereby inhibiting cellular differentiation. To examine the effect of Id-2 on cell proliferation, we overexpressed Id-2 in the human osteosarcoma cell line U2OS. Id-2 expression in U2OS reduced the serum requirement for growth and stimulated cellular proliferation by shortening the doubling time and increasing the percentage of cells in S phase. We demonstrated that Id-2 expression was able to reverse the inhibition of cellular proliferation and the block in cell cycle progression mediated by the product of the retinoblastoma tumor suppressor gene pRB. This effect was not associated with changes in the state of pRb phosphorylation in transfected cells. In vitro, unphosphorylated pRb from cell lysates specifically bound Id-2 but was not able to bind a mutated form of Id-2 lacking the HLH domain that also did not antagonize the growth arrest by pRb. In vitro-synthesized pRb containing mutations within the E1A/large T-binding pocket did not bind Id-2. However, wild-type pRb was able to bind to a region of Id-2 corresponding to only the HLH domain. In vivo, a physical association between Id-2 and pRb was seen in cross-linked extracts from SAOS-2 cells transfected with Id-2 and pRb. Our data identify a role for Id-2 in the regulation of cellular proliferation and suggest that the interaction between Id-2 and pRB is a molecular pathway over which synchronous changes in growth and differentiation are mediated in vivo.
3386	Purification of E. coli-synthesized Pan proteins and development of a Pan-specific monoclonal antibody.	7523277	The helix-loop-helix (HLH) transcription factors, Pan-1 (E47) and Pan-2 (E12), are produced by the mechanism of alternative transcript splicing. Pan-1 and Pan-2 were expressed in Escherichia coli, and a purification scheme was developed. Purified Pan-2 was used to immunize Smith-Webster mice and a hybridoma was generated that produced a monoclonal antibody (Yae) that specifically recognized both native and denatured Pan-1 and Pan-2. Deletion mapping and sequence transfer studies have localized the determinant recognized by the Yae antibody to the region 195-208 of Pan-2. This region is conserved in Pan-1 and Pan-2. The Yae antibody recognized in vitro-synthesized ITF-1, a third E2A (Pan) gene product also produced by the mechanism of alternative RNA splicing, but did not recognize the related HLH proteins, ITF-2, REB alpha, or REB beta. By Western blot assay of pancreatic acinar cells, the Yae antibody detected a single protein species of 72 kD that comigrated with in vitro-synthesized Pan-1 and Pan-2.
3387	Functional expression or recombinant human luteinizing hormone/human choriogonadotropin receptor.	8198582	The functional capacity of the recombinant human LH/hCG receptor was tested on the basis of gonadotropin stimulation of cAMP production by stable transfections of Chinese hamster ovary (CHO) cells. A CHO cell line expressed with the hLH/hCG receptor cDNA covering the entire amino acid coding region revealed the presence of LH/hCG binding site (Kd: 1.45 x 10(-10)M) on the plasma membrane. Treatment of transfected cells(CHO-LH/hCGR) with hCG induced dose-dependent increases in intracellular cAMP production, indicating that the expressed human LH/hCG receptor functionally couples with endogenous adenylyl cyclase. Although hCG induced dose-dependent increases in cAMP production, rat and bovine LH and human FSH did not alter cAMP levels compared to control values. Northern blot analysis with a cRNA probe derived from human LH/hCG receptor cDNA indicated the presence of three LH/hCG receptor mRNA transcripts(5.4, 3.6 and 2.4 kilobases) in RNA prepared from human ovary. Preincubation of CHO-LH/hCGR cells with hCG for 16h decreased the subsequent cAMP production caused by a 30min pulse of hCG stimulation. These results indicate that desensitization of the adenylyl cyclase response to hCG stimulation occurs in CHO-LH/hCGR cells. Therefore, this cell line provides a tool with which to pursue detailed studies on the molecular basis of LH/hCG induced desensitization.
3388	Eutopic production of human chorionic gonadotropin beta (hCG beta) and luteinizing hormone beta (hLH beta) in the human testis.	7513655	The classical pregnancy and tumor marker hCG has long been considered to be only accidentally expressed ectopically, e.g. by tumors. The biological functions of low levels of hCG beta, hCG alpha and holo-hCG in the sera of nonpregnant healthy individuals remained unclear. Immunological analyses by our ultrasensitive time-resolved fluoroimmunoassays revealed a concentration gradient from < 5 pg hCG beta/ml in cubital vein serum versus up to 480 pg hCG beta/ml in the corresponding benign testicular hydrocele fluids. Moreover, hCG beta and its cognate molecule luteinizing hormone beta (LH beta) were present in cytosolic extracts of normal human testes. Both hCG beta and hLH beta are eutopically produced as proven by RT-PCR and subsequent Southern and dot blot analyses. Thus, the view of a purely systemic hormonal function of hLH, and of hCG during pregnancy needs a reassessement as hCG beta and hLH beta are synthesized in the human testis and autocrine/paracrine actions seem to be likely.
3389	Immunochemical mapping of human lutropin: II. Characterization of two monoclonal antipeptide antibodies reacting with the native beta-subunit.	9397933	To investigate the epitopes present on the beta-subunit of the human lutropin (hLHbeta) and their topographical relationship at the surface of the molecule, we produced two monoclonal antipeptide antibodies, designated LHP03 and LHP04, capable of binding to the radiolabeled 125I-hLHbeta and directed to the 43-52 and 110-117 regions of the hLHbeta, respectively. Analysis of the accessibility of the epitopes on hLH and on the beta-subunit of human chorionic gonadotropin (hCGbeta), equine LH (eLHbeta) and ovine LH (oLHbeta) indicated that: (i) LHP03 binds to both the free hLHbeta subunit and dimeric hLH whereas LHP04 binds preferentially to the free hLHbeta, (ii) LHP03 recognizes weakly the hCGbeta and oLHbeta in comparison to hLHbeta and (iii) LHP04 binds oLHbeta as well as hLHbeta but does not bind to hCGbeta and eLHbeta. The topographical relationship of epitopes recognized by LHP03 and monoclonal antibodies recognizing dimer specific epitopes on hLH allowed us to localize discontinuous antigenic sites that overlaps or are located outside the hHLbeta(43-52) region. Together, our results demonstrated that the hHLbeta(43-52) portion is accessible on both the free hLHbeta subunit and hLH whereas the COOH-terminal portion, hHLbeta(110-117), is probably buried at the alpha/beta interface of the hormone.
3390	Immunochemical mapping of human lutropin: I. Delineation of a conformational antigenic determinant.	9397932	Lutropin (LH), follitropin (FSH), thyrotropin (TSH) and choriogonadotropin (CG) are assembled of two non-covalently alpha (alpha) and beta (beta) subunits. We studied the discontinuous antigenic regions recognized by a monoclonal anti-hLH antibody designated as LH05 which binds to hLH, hCG and hTSH and does not cross-react with either the free subunits or hFSH. LH05 and an antibody designated HT13, recognizing an epitope partly comprizing the alpha64-76 region, did not bind simultaneously to hCG. Furthermore, LH05 was unable to combine with an anti-peptide antibody (LHP03) directed to residues 43-52 of hLHbeta. Thus, LH05 recognizes an epitope partly overlapping with those recognized by HT13 and LHP03. Using various hybrid molecules, we showed that the human alpha-subunit plays a critical role in the assembly of the epitope that, in contrast, contains amino acid residues conserved in the various beta-subunit of several species. Together, our results suggest that the amino acids Leu49-Pro50, Tyr59-Arg60 and Leu86-Ser87 in the hLHbeta and the alpha64-76 region are probably included in the epitope recognized by LH05 which appeared to be not accessible on the CG/LH receptor.
3391	The HLH domain of a zebrafish HE12 homologue can partially substitute for functions of the HLH domain of Drosophila DAUGHTERLESS.	7918099	We have identified a zebrafish homologue of the human E12 protein, which we have called ZFE12. It shows a high degree of identity to HE12 throughout its entire sequence, particularly in the bHLH domain (93%); amino acid sequence identity of the bHLH domain of ZFE12 to Drosophila DAUGHTERLESS is also very high (75%). During early embryogenesis, expression of ZfE12 is widespread. Following gastrulation, ZfE12 transcripts can be detected in all embryonic tissues with the exception of the notochord, although zones with relatively higher densities of transcripts are present in the developing brain and in the somites. To assay biological activities associated with the ZFE12 protein, two P-element constructs were made, each carrying a Drosophila daughterless gene that had been modified by replacing either the HLH domain or the entire C-terminus including the bHLH domain, by the equivalent domains of ZfE12. These constructs were used to transform flies and tested for their ability to rescue the daughterless mutant phenotype. Complete rescue of the neural phenotype and of the embryonic lethality was obtained. However, the daughterless function could not be completely restored. Although fertile flies transheterozygous for a hypomorphic and an amorphic mutation and carrying the construct encoding the zebrafish HLH domain emerged, they lacked various types of sensory organs on head and thorax and showed slight wing defects. Mutants carrying the second construct occasionally reached pupal stages but died subsequently. These data demonstrate that the HLH domain of ZFE12 can carry out most, but not all, functions performed by the corresponding region of the DAUGHTERLESS protein in Drosophila.
3392	Genomic organization, alternative polyadenylation, and chromosomal localization of Grg, a mouse gene related to the groucho transcript of the Drosophila Enhancer of split complex.	7916324	The Grg gene encodes a 197-amino-acid protein homologous to the amino-terminal domain of the product of the groucho gene of the Drosophila Enhancer of split complex. We describe here the genomic organization of the mouse Grg gene. It spans approximately 7 kb on chromosome 10 and consists of seven exons. The 3' region of the Grg gene contains two functional polyadenylation sites that give rise to two transcripts that are differentially expressed among adult mouse tissues. The promoter region is very GC rich and lacks TATA box and "initiator" sequences. Primer extension analysis and ribonuclease protection assays show that Grg has a major transcription start site situated down-stream of putative binding motifs for the transcription factors Sp1, E2A, and PuF.
3393	Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro.	8011332	The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 micrograms/ml human follicle-stimulating hormone (hFSH), (2) 10 micrograms/ml human luteinizing hormone (hLH), (3) 1.0 microgram/ml hFSH and 10 micrograms/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 +/- 1.7 hr; hLH: 10.1 +/- 1.8 hr; both gonadotropins: 8.8 +/- 1.1 hr) when compared to controls (17.4 +/- 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 +/- 1.2 hr; hLH: 35.1 +/- 1.4 hr; both gonadotropins: 35.2 +/- 1.3 hr; no gonadotropins: 34.1 +/- 1.2 hr). The mean interval to extrusion of PB1 was 34.1 +/- 0.6 hr.(ABSTRACT TRUNCATED AT 250 WORDS)
3394	Interleukin-1 beta, interleukin-6, and growth hormone levels in human follicular fluid.	7711382	To investigate possible relationships of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and growth hormone (GH) with biochemical variables in human follicular fluid (FF) and selected in vitro fertilization (IVF) parameters.A total of 67 FF samples (n = 67 patients undergoing oocyte retrieval for IVF) was evaluated. IL-1 beta, IL-6, GH, hLH, FSH, PRL, hCG, testosterone, total protein, fibrinogen, sialic acid, alpha 1-antitrypsin, plasminogen levels, and spectrophotometric absorbance at 458 nm were analyzed for selected FF. IL-6 and GH levels of serum and FF samples were also compared (n = 23).Immunoreactive levels of IL-1 beta, IL-6, and GH were detected in all FF samples. A positive correlation existed for IL-6 (r = 0.5069, P = 0.0161) when serum-to-FF levels were compared (concentration ratio, 1:1.857). Smaller-volume follicles (< 4 ml) were associated with high IL-1 beta levels (P = 0.0229), and an additional tendency of IL-1 beta to decrease with increasing embryo cleavage and scoring was observed. With the exception of a weak positive correlation between follicular IL-1 beta and testosterone levels (r = 0.3128, P = 0.025), no other relationship with biochemical variables or IVF parameters (etiology, e.g., endometriosis) could be implicated.Substantially higher IL-6 levels occurred in FF compared to serum, thus supporting intrafollicular production. Interleukin-1 beta, IL-6, and GH levels in FF are, however, unsuitable markers for in vitro fertilization outcome.
3395	daughterless is essential for neuronal precursor differentiation but not for initiation of neuronal precursor formation in Drosophila embryo.	7600969	The first steps of neuronal precursor formation require several genes that encode transcription regulators with the helix-loop-helix (HLH) motif, including the proneural genes of the achaete-scute complex AS-C (achaete (ac), scute (sc) and lethal of scute (l'sc)) and daughterless (da). The da protein dimerizes with AS-C products in vitro to form DNA-binding proteins. Previous studies have shown that the AS-C genes are expressed initially in discrete clusters of ectodermal cells (the proneural clusters) and then more strongly in the neuronal precursors that arise from these clusters and delaminate from the epidermal layer. In this paper, we studied the distribution of da protein with an antibody raised against Da. We found that Da is ubiquitously but non-uniformly distributed. Within the ectodermal layer, its level is neither elevated (as in the case of AS-C genes) nor reduced (as in the case of emc product) in the proneural cluster. It is, however, at higher levels in many neuronal precursors. We further studied the requirement of da in neuronal precursor development by using a variety of markers for neuronal precursors. Our results reveal the existence of at least two stages in neuronal precursor formation. da is not required for the initial appearance of nascent neuronal precursors but is required for these cells to express multiple neuronal precursor genes and to produce the normal number of neurons.
3396	Neuroectodermal transcription of the Drosophila neurogenic genes E(spl) and HLH-m5 is regulated by proneural genes.	7600959	The Enhancer of split gene complex (E(SPL)-C) of Drosophila comprises seven genes encoding bHLH proteins, which are required by neuroectodermal cells for epidermal development. Using promoter and gene fusions with the lacZ gene, we determined the location of cis-acting sequences necessary for normal expression of two of the genes of the E(SPL)-C, E(spl) and HLH-m5. About 0.46 kb of E(spl) and 1.9 kb of HLH-m5 upstream sequences are necessary to reproduce the normal transcription pattern of these genes. The gene products of achaete, scute and lethal of scute, together with that of ventral nervous system condensation defective, act synergistically to specify the neuroectodermal E(spl) and HLH-m5 expression domains. Negative cross- and autoregulatory interactions of the E(SPL)-C on E(spl) contribute, directly or indirectly, to this regulation. Interactions involve DNA binding, since mutagenesis of binding sites for bHLH proteins in the E(spl) promoter abolishes neuroectodermal expression and activates ectopic expression in neuroblasts. A model for activation and repression of E(spl) in the neuroectoderm and neuroblasts, respectively, is proposed.
3397	Activation of helix-loop-helix proteins Id1, Id2 and Id3 during neural differentiation.	7908517	Id is a nuclear factor containing a helix-loop-helix (HLH) motif. It has been reported that Id functions as an inhibitor of cell differentiation and its gene expression is down-regulated during cell differentiation. We have characterized three Id-related cDNAs, Id1, Id2 and Id3, isolated from nerve growth factor (NGF)-stimulated PC12 cells. Structural analysis revealed that all three Id's contain putative phosphorylation sites for cyclic AMP-dependent kinase, casein kinase II and protein kinase C near and inside the HLH motifs. In contradiction to the previous reports, Northern blot analysis revealed that NGF induces rapid and transient increase of all three Id gene transcriptions. Furthermore, in situ hybridization of rat embryo showed that all three Id genes are highly expressed in neural precursors rather than differentiated neural cells. These results indicate that Id family members may function as immediate-early gene products, and that the expression of the Id family may play an important role in the early stage of neural differentiation.
3398	The expression pattern of Id4, a novel dominant negative helix-loop-helix protein, is distinct from Id1, Id2 and Id3.	8139914	Molecular interaction between transcription factors containing an basic-helix-loop-helix (bHLH) domain is known to regulate differentiation in several cellular systems including myogenesis, neurogenesis and haematopoiesis. DNA-binding activity of the bHLH proteins is mediated via the basic region and is dependent upon formation of homo- and/or heterodimers of these transcription factors. Dominant negative (dn) HLH proteins (Id1, Id2, Id3 and emc) also contain the HLH-dimerization domain but lack the DNA-binding basic region. Formation of heterodimers between dnHLH and bHLH proteins abolishes the DNA-binding activity of the latter. Concordantly, it was shown that the dnHLH protein Id1 inhibits differentiation of muscle and myeloid cells in vitro. Therefore, it was postulated that dnHLH proteins serve as general antagonists of cell differentiation. We have isolated and characterized a novel mouse dnHLH gene, designated Id4. The Id4 protein contains a HLH domain highly conserved among the dnHLH proteins from mouse and drosophila. Outside of the HLH domain, three additional short regions of the dnHLH proteins show some degree of homology. DNA-binding of E47 homo- as well as E47/MyoD heterodimers is inhibited by Id4. Transcription of the Id4 gene results in three RNA molecules of 3.7, 2.0 and 1.7 kb which are presumably a result of differential splicing and/or alternatively used polyadenylation sites within the 3' untranslated region. During embryogenesis, Id4 expression is up-regulated between day 9.5 and 13.5 of gestation. The highest expression in adult tissues was detected in testis, brain and kidney. Comparison of the expression patterns of the four mouse dnHLH genes revealed that Id4 expression differs from the more restricted expression of Id2 as well as from the widespread expression of Id1 and Id3.
3399	Functional characterization of the INO2 gene of Saccharomyces cerevisiae. A positive regulator of phospholipid biosynthesis.	8125958	The INO2 locus encodes a novel product showing structural similarity to the basic helix-loop-helix (b-HLH) family of regulatory proteins (Nikoloff, D.M., McGraw, P., and Henry, S.A. (1992) Nucleic Acids Res. 20, 3253). The ino2 mutants exhibit pleiotropic defects in phospholipid metabolism including inability to derepress the biosynthetic enzyme inositol-1-phosphate synthase. Localization of mutations in ino2 strains has demonstrated that the b-HLH domain is required for biological activity and is sensitive to perturbation, thereby establishing a correlation between the structure and function of Ino2p. Defects in the b-HLH domain of Ino2p resulted in reduced DNA binding activity. In addition, the absence of a specific DNA-protein complex correlated with a reduction or loss of INO1 transcription. Studies using Ino2p-specific antibody revealed that Ino2p participates in the formation of specific DNA-protein complexes. Ino2p-dependent binding activity overlapped with a region of the INO1 promoter that contains two potential HLH consensus binding sites. Furthermore, Ino2p showed single base pair discrimination in a putative binding site, establishing a relationship between Ino2p and its target binding site.
3400	Decreased number and size and the defective function of testicular macrophages in long-term hypophysectomized rats are reversed by treatment with human gonadotrophins.	8182367	Macrophages are a common cell type in the testicular interstitium of the rat and are morphologically and functionally related to Leydig cells. We investigated the number of macrophages and Leydig cells in long-term (24 weeks) hypophysectomized (LTHX) or sham-operated rats. LTHX rats showed a 76% decrease in the number of macrophages, whereas the number of Leydig cells was only slightly decreased (by 18%). The profile areas of both macrophages and Leydig cells were very much decreased (46% and 66% respectively). Sham-operated and LTHX rats were treated with vehicle or human FSH and LH (hFSH/hLH; 75 IU/kg body weight per day) for 1 week. This treatment induced a 286% increase in the number of macrophages and a 32% increase in the number of Leydig cells in LTHX rats. The profile areas of macrophages and Leydig cells were also increased (212% and 184% respectively). About 80% of macrophages showed vacuolization of the cytoplasm. Gonadotrophin treatment did not induce changes in cell numbers in sham-operated animals but about 30% of macrophages showed large cytoplasmic vacuoles. Vehicle- or hormone-treated LTHX rats were given a single injection of ethylene dimethane sulphonate (EDS) and killed 72 h later. Leydig cells were absent from the testicular interstitium of sham-operated rats but there were large numbers of dead Leydig cells (about 40% of the pre-existing population) in the testicular interstitium of LTHX rats 3 days after EDS treatment. Complete clearance of the testicular interstitium from EDS-killed Leydig cells was found in LTHX rats treated with hFSH/hLH.(ABSTRACT TRUNCATED AT 250 WORDS)
3401	Measurement of gonadotropins in dried blood spots.	8131281	We describe direct immunofluorometric assays for luteinizing hormone (hLH) and follicle-stimulating hormone (hFSH) in fingerstick blood spots dried on filter paper, based on modifications of commercially available kits. Determinations are made from 2.5-mm-diameter discs (3 microL of dried blood) punched out from blood spot standards and samples. Sample dose detection limits of the assays (IU/L) are 0.26 for LH and 0.13 for FSH, with mean interassay CVs of 11.6% (LH) and 7.8% (FSH) at low concentrations. Analytical recoveries of added hormone averaged 100% for LH and 95% for FSH. Clinical studies showed that values for blood spots (x) and directly assayed plasma (y) are highly correlated, so that results from blood spots can be converted directly to plasma equivalents, as follows: yLH = 0.07 + 1.90 xLH, and yFSH = 0.424 + 2.207 xFSH. These gonadotropins are stable in blood spots for at least a year under refrigeration; LH for at least 8 weeks and FSH 6 weeks at 22 degrees C; and both hormones for a week at 37 degrees C. These methods thus allow self-sampling, serial sampling, and mailing of specimens.
3402	A versatile expression vector for the in vitro study of protein-protein interactions: characterization of E47 mutant proteins.	8108132	Several mutants of the E47 protein, a member of the family of basic/helix-loop-helix (b-HLH) transcriptional regulators, were examined for their ability to homo- and heterodimerize with the protein product of the T-cell oncogene tal-1/SCL. For this purpose, a novel bacterial expression system was developed in which proteins are expressed as fusions appended to glutathione-S-transferase via a thrombin cleavage site and either one or four protein kinase recognition sites embedded in a glycine-rich domain. Since the interaction domain can be purified away from the glutathione-S-transferase moiety and the radioactive label is located in a flexible N-terminal tag, protein folding should occur normally. Our studies with E47 proteins prepared in this system indicate that the ratio between E47 homodimers and E47/tal-1 heterodimers can dramatically shift upon subtle mutations in the loop region and the second helix of the E47 protein. This unexpected results suggests a novel mechanism to alter the equilibrium between different transactivating protein complexes of the b-HLH class.
3403	Identification of amino-acid polymorphism within the leucine zipper motif of mouse transcription factor A1.	8112613	Mouse transcription factor A1 (A1) is a mouse homologue of human E47, a ubiquitously expressed DNA-binding protein which contains a basic region, helix-loop-helix (HLH) and leucine zipper (LZ) motifs [Walker et al., Nucleic Acids Res. 18 (1990) 1159-1166]. Analyses of the nucleotide (nt) sequences of A1 cDNAs isolated from various mouse strains revealed amino acid (aa) polymorphism in the highly conserved region within the LZ motif. Interestingly, the location and pattern of aa deletions are identical to those previously described for the aa polymorphism within the human counterpart, E47 (E12) [Kamps et al., Cell 60 (1990) 547-555].
3404	The molecular basis of skeletal muscle differentiation.	8202645	In recent years, significant advances have been made in understanding the molecular mechanisms involved in the regulation of skeletal-muscle differentiation. This review focuses on the role of the MyoD family of myogenic transcription factors that includes MyoD, myf-5, myogenin, and MRF4 (herculin or myf-6) in myogenesis. Members of this family share sequence homology for the basic-helix-loop-helix (bHLH) regulatory motif. The basic domain is required for DNA binding, whereas the HLH domain is required for dimerization. The bHLH motif confers both properties of transcriptional activation of muscle specific genes and inhibition of cell growth through collaboration with the E2A gene products (E12 and E47) and the retinoblastoma gene product (pRB). The functions of the MyoD family can be suppressed through inhibition of their expression or activity by various factors. These include peptide growth factors (FGF and TGF-beta), immediate early gene products (Fos, Jun and Myc), other oncogene products (Ras, Src), the Id protein, and innervation. The use of gene-knockout animal models has shown that there is some degree of functional redundancy in that inactivation of either MyoD or myf-5 has no effect on muscle development, whereas inactivation of both genes results in an absolute lack of muscle cells. In contrast, the inactivation of myogenin alone results in mice with gross deficiency of mature muscle.
3405	The presence of a human chorionic gonadotropin-like protein and its binding site in Saccharomyces cerevisiae.	8168461	In this study, we characterize from Saccharomyces cerevisiae: 1) a protein that has immunological similarities to human chorionic gonadotropin (hCG), and 2) a binding site for this hCG-like protein which also binds hCG and human luteinizing hormone (hLH). Saccharomyces cerevisiae chorionic gonadotropin-like protein (ScCGLP) was purified in several steps. This protein when analyzed by SDS-PAGE, under nondenaturing conditions, produced two bands, one at 110-kDa, and another at 57.5-kDa. Under denaturing conditions, only the 57.5-kDa band appeared. This 57.5-kDa band also reacted in a Western blot, using a polyclonal antibody directed against hCG. Purified ScCGLP reacted in the following hCG immunoassays: 1) polyclonal rabbit anti-hCG equilibrium assay, 2) carboxyl-tail hCG equilibrium assay, 3) two equilibrium assays using monoclonal antibodies, and 4) free alpha-chain subunit equilibrium assay using a monoclonal antibody. Characterization of hCG/hLH binding sites in Saccharomyces cerevisiae was performed, and the ability of the ScCGLP to displace I125-hCG was also shown. Human CG and hLH were able to compete for I125-hCG binding to Saccharomyces cerevisiae blastospores (Kds of approximately 10(-8) M), while ScCGLP competed with higher affinity (Kd = 9.41 x 10(-10) M). The hCG-like immunoactivity was also present in Saccharomyces growth media, as well as in all brands of commercial beer studied.
3406	Characterization and purification of the chorionic gonadotropin-like protein binding site in Candida albicans.	8168460	Recent studies from our laboratory have shown that human chorionic gonadotropin (hCG), human luteinizing hormone (hLH), and CG-like proteins from Xanthomonas maltophilia (xCG) and Candida albicans (CaCGLP) induce Candida albicans transition from blastospores to hyphal forms. Xanthomonas maltophilia CG-like protein (xCG), hCG, and hLH also bind to Candida albicans blastospores with a high-affinity nM Kd, indicating that these substances can control Candida albicans pathogenicity. The work reported herein describes the purification of the binding site for these CG-like proteins from Candida albicans. The purification developed involved alcohol extraction followed by affinity chromatography. The product obtained was a protein of 64-69 kDa, as analyzed by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), Western blot and Sephadex G-100 column chromatography. This binding site reacted in a Western blot with both 125I-hCG and 125I-CaCGLP. Purified CaCGLP was able to displace specifically 125I-hCG bound to Candida albicans blastospores. Scatchard plot analysis showed that the Kd of this reaction was of high affinity in the nM range, and also indicated the presence of one single binding site. These results lead us to conclude: 1) Candida albicans possesses a binding site which is able to bind hCG, hLH and CG-like proteins from Xanthomonas maltophilia and Candida albicans; 2) This binding site is a hydrophobic protein of approximately 64 kDa and; 3) We postulate that CaCGLP is the natural ligand for this binding site and this system is used to control Candida albicans transition and, therefore, pathogenicity.
3407	Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.	8294468	Three complete cDNA clones encoding Id-related helix-loop-helix (HLH) proteins lacking a basic region were isolated from a pcD2 cDNA expression library prepared from TIG-3 human diploid fibroblasts (HDF). Of these cDNAs (Id-1H, Id-1H', and Id-2H), two (Id-1H and Id-1H') appeared to be derived by alternative RNA splicing. Id-1H and Id-2H seem to be human homologues of mouse Id-1 and Id-2, respectively, and have potential to encode 154 and 135 amino acid proteins. The Id-1H and Id-2H mRNAs were barely detectable in quiescent early passage HDF; serum coordinately induced both mRNAs, with two peaks of expression, in early and late in G1. Antisense oligomers complementary to Id-1H and Id-2H mRNA prevented early passage HDF from entering the S phase of the cell cycle. The treatment of serum-stimulated early passage cells with the antisense Id-1H oligomer completely abolished Id-1H. In senescent cells, serum barely induced the Id-1H and Id-2H mRNAs, although the levels of c-myc expression induced were similar in early passage and senescent cells. The expression levels of these Id genes vary among immortal human cell lines. Both genes were overexpressed in VA4 SV40-transformed lung fibroblasts and EJ-1 bladder carcinoma cells, while these genes were expressed at a very low level in SVts8 cells derived from SV40 tsA-transformed TIG-3 cells. SVts8 cells may acquire some function redundant to Id proteins. HT1080 fibrosarcoma cells expressed the Id-1H gene but not the Id-2H gene, suggesting these Id genes may subserve redundant functions.
3408	Differential-effects of max proteins (long and short) on growth and differentiation of murine erythroleukemia-cells.	21607333	The myc gene product belongs to a family of basic, Helix-Loop-Helix, Leucine Zipper (b-HLH-LZ) proteins involved in cell growth, differentiation and tumorigenesis. The Max protein dimerizes with Myc to form a complex which binds to DNA and promotes transcription of target genes. Max exists in two major forms, Max-9 (long) and Max (short), which differ by 9 amino acids just amino to the basic region. We compared the in vivo behavior of the two forms in stable transfectants of long and short wild type (Lwt, Swt) and basic region mutants (Lbm, Sbm). While both Lwt-max and Swt-max clones exhibit delays in cell growth and differentiation, these delays are far more pronounced in the Lwt-max clones. In contrast, no difference is noted between Lbm-Max and Sbm-Max in their observed ability to delay growth, to accelerate HMBA-induced differentiation and to induce spontaneous differentiation. We suggest that a difference in affinity for DNA underlies the differential activities of Lwt- and Swt-Max.
3409	Search for antibodies for immunoenzymatic assay of human lutropin.	8572906	Five peptides corresponding to human lutropin (hLH) subunit fragments were synthesized by a solid phase method and their physicochemical characteristics are presented. Antibodies induced in rabbits with 3 peptides of beta hLH fragments coupled to thyrotropin did not bind hLH and human chorionic gonadotropin (hCG). Also 2 synthetic peptides of alpha hLH fragments did not react with rabbit anti-hLH, anti-hCG and anti-alpha hCG antibodies. Using 2 monoclonal anti-alpha hCG and anti-beta hLH antibodies a sensitive sandwich ELISA technique was elaborated. Using this technique stability of hLH was investigated. The ELISA was also applied for assays of urine hLH in normal and ovulation days.
3410	Structure and function of the b/HLH/Z domain of USF.	8306960	The basic/helix-loop-helix/leucine zipper (b/HLH/Z) transcription factor upstream stimulatory factor (USF) and its isolated DNA binding domain undergo a random coil to alpha-helix folding transition on recognizing their cognate DNA. The USF b/HLH cocrystal structure resembles the structure of the b/HLH/Z domain of the homologous protein Max and reveals (i) that the truncated, b/HLH DNA binding domain homodimerizes, forming a parallel, left-handed four-helix bundle, and (ii) that the basic region becomes alpha-helical on binding to the major groove of the DNA sequence CACGTG. Hydrodynamic measurements show that the b/HLH/Z DNA binding domain of USF exists as a bivalent homotetramer. This tetramer forms at the USF physiological intranuclear concentration, and depends on the integrity of the leucine zipper motif. The ability to bind simultaneously to two independent sites suggests a role in DNA looping for the b/HLH/Z and Myc-related families of eukaryotic transcription factors.
3411	c-jun inhibits transcriptional activation by the insulin enhancer, and the insulin control element is the target of control.	8264634	Selective transcription of the insulin gene in pancreatic beta cells is regulated by its enhancer, located between nucleotides -340 and -91 relative to the transcription start site. One of the principal control elements within the enhancer is found between nucleotides -100 and -91 (GCCATCTGCT, referred to as the insulin control element [ICE]) and is regulated by both positive- and negative-acting transcription factors in the helix-loop-helix (HLH) family. It was previously shown that the c-jun proto-oncogene can repress insulin gene transcription. We have found that c-jun inhibits ICE-stimulated transcription. Inhibition of ICE-directed transcription is mediated by sequences within the carboxy-terminal region of the protein. These c-jun sequences span an activation domain and the basic leucine zipper DNA binding-dimerization region of the protein. Both regions of c-jun are conserved within the other members of the jun family: junB and junD. These proteins also suppress ICE-mediated transcription. The jun proteins do not appear to inhibit insulin gene transcription by binding directly to the ICE. c-jun and junB also block the trans-activation potential of two skeletal muscle-specific HLH proteins, MyoD and myogenin. These results suggests that the jun proteins may be common transcription control factors used in skeletal muscle and pancreatic beta cells to regulate HLH-mediated activity. We discuss the possible significance of these observations to insulin gene transcription in pancreatic beta cells.
3412	Immunosuppression: preliminary results of alternative maintenance therapy for familial hemophagocytic lymphohistocytosis (FHL).	8127256	Hemophagocytic lymphohistiocytosis (HLH) describes a group of disorders with similar clinical features that are associated with a very high mortality rate. Patients with HLH, and particularly the infantile form referred to as familial hemophagocytic lymphohistiocytosis (FHL), are often treated with multiple courses of epipodophyllotoxins, such as etoposide, for prolonged periods of time. Because of the concern regarding the risk of epipodophyllotoxin-induced acute myelogenous leukemia (AML) we have explored the use of immunosuppression as maintenance therapy for patients with FHL while they await the only known definitive treatment, i.e., bone marrow transplantation (BMT). We report 2 infants with FHL who had significant central nervous system involvement at diagnosis. Both were initially treated with etoposide, methotrexate, and glucocorticosteroids. Once clinical improvement was achieved these patients were successfully maintained in clinical remission of FHL on daily cyclosporine A (CSA) and glucocorticosteroids along with intermittent intrathecal methotrexate for 5 months until appropriate unrelated donors could be identified for BMT.
3413	[Immunoassay and measurement of the biological activity of serum hLH: results obtained of a multicenter study].	7872518	The discrepancy between results obtained using different anti-hLH antibodies available on the market led us (in the context of a contract with the CNAMTS) to compare the results of hLH immunoassays (eight different reagents) with those of hLH bioassays based on the measurement of the steroidal activity of this hormone on mouse, rat or pig testicular preparations (five methodologies). Twenty-nine samples of serum or human serum pools were tested. The values resulting from these tests reveal differences linked to the nature of the antibodies or to the methodological conditions used for the testicular preparations. Some antibodies seem unable to identify some hLH forms which, however, can be detected by biological methods.
3414	[Human luteinizing hormone (hLH)].	7638098	Heterodimeric composition and amino acid sequence of hLH is reviewed. The hormone isoforms and methods of their assay are summarized. Releasing of the hormone, its control by synthetic antagonists and agonists and their significance for therapy are described. Structure and function of hLH receptor are presented.
3415	Expression and differential regulation of Id1, a dominant negative regulator of basic helix-loop-helix transcription factors, in glomerular mesangial cells.	8284227	Id is a family of dominant negative helix-loop-helix (HLH) proteins that block cell-specific transcription mediated by basic HLH (bHLH) transcription mediated by basic HLH (bHLH) transcription factors. We have analyzed Id1 expression in mesangial cells as a first step towards understanding the putative role of bHLH transcription factors in cell type-specific gene expression in the kidney. Glomerular mesangial cells expressed an abundant 1.1 kb mRNA transcript for Id1, but in contrast to other cell types Id1 mRNA was expressed in both randomly cycling cells and in serum-deprived, quiescent cultures. When quiescent mesangial cells were treated with serum to re-enter G1, Id1 mRNA levels were rapidly (2-4 h) and transiently down-regulated. Down-regulation of Id1 mRNA following addition of serum to mesangial cells was cell type-specific and contrasted with induction of Id1 by serum in BHK-21 and 3T3 fibroblasts. Down-regulation of Id1 mRNA correlated with mitogenesis and occurred when quiescent cells were treated with growth factors that activate G protein-coupled receptors and receptor protein tyrosine kinases but not with a non-mitogenic cAMP analog. Down-regulation of Id1 by growth factors required de novo protein synthesis, suggesting that a labile protein was involved. Appearance of E-box DNA binding activity in mesangial cell extracts followed down-regulation of Id1 message. Steady state Id1 mRNA levels and E-box DNA binding activity were not tightly correlated, suggesting complex regulation of Id1 activity. mRNA transcripts for E2A gene products were also expressed in mesangial cells, but these cells failed to express mRNAs for MyoA/MyoD-related genes. Collectively, these data demonstrate that Id1 is expressed in renal mesangial cells and suggest that bHLH complexes might be important for transcriptional regulation in the kidney. In addition, the observation that Id1 mRNA is transiently down-regulated by serum in mesangial cells suggests that Id1 gene expression is more complicated than previously appreciated and is tightly regulated in a cell-specific manner.
3416	A rat homolog of the Drosophila enhancer of split (groucho) locus lacking WD-40 repeats.	8245004	In Drosophila, neurogenic loci function in defining cellular fate by interpreting the identity of other cells in the immediate environment. To begin studies of mammalian homologs of these genes, we have isolated two rat homologs of the neurogenic locus Enhancer of split. The protein encoded by the Drosophila Enhancer of split locus is complex and contains five distinct regions based on amino acid composition. One region contains six WD-40 repeats, which were first described in the beta subunit of the heterotrimeric guanine nucleotide-binding protein. One of the rat cDNAs we isolated, R-esp1, encodes a novel form that lacks the WD-40 repeating units. Data are presented demonstrating that the R-esp1 cDNA is a full-length clone encoding an expressed 24-kDa protein. Antibodies raised against this protein stain the nucleus of both PC-12 and GH3 cells. The second clone, R-esp2, encodes a full-length homolog containing WD-40 repeats. The hydrodynamic properties of in vitro translated R-esp1 and R-esp2 proteins indicate that they do not stably self-associate or form heterodimers. A model is presented for the possible role of the R-esp1 protein in the negative regulation of Enhancer of split proteins containing WD-40 repeats.
3417	HLH proteins, fly neurogenesis, and vertebrate myogenesis.	8252617	NaN
3418	Human myometrial smooth muscle cells are novel targets of direct regulation by human chorionic gonadotropin.	8286597	Human myometrium contains receptors for hCG/human LH (hLH). This suggested the possibility that hCG and hLH might regulate human myometrium, which has not previously been considered a direct target of gonadotropin regulation. To investigate such a possibility, highly pure and viable smooth muscle cells were isolated from nonpregnant human myometrium and cultured as monolayers. The cells contained hCG/LH receptor mRNA transcripts and a 50-kDa immunoreactive protein that can bind 125I-hCG in a ligand-specific manner. The presence of hCG during culture resulted in a significant increase of myometrial smooth muscle cell density. The hCG effect was time- and concentration-dependent and was mimicked by hLH but not by human FSH or human FSH or human thyroid-stimulating hormone. Human CG also greatly increased the size of a subpopulation of myometrial smooth muscle cells without affecting their chromosomal ploidy. Antibodies to hCG/LH receptors and hCG blocked hCG effects. Human prolactin and growth hormone, which do not bind to hCG/LH receptors, also increased the myometrial smooth muscle cell density. A protein kinase A inhibitor (H-89) blocked hCG response whereas calphostin (a protein kinase C inhibitor) and lavendustin A (a tyrosine kinase inhibitor) had no effect on hCG response, suggesting that a cAMP/protein kinase A signaling mechanism is involved in hCG action. Eicosanoids from cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism are probably not involved, because the inhibitors of these enzymes had no effect on hCG response. While progesterone and estradiol could not mimic or modify hCG action, epidermal growth factor did mimic hCG in increasing myometrial smooth muscle cell density.(ABSTRACT TRUNCATED AT 250 WORDS)
3419	Dimerization specificity of myogenic helix-loop-helix DNA-binding factors directed by nonconserved hydrophilic residues.	8253390	The myogenic regulatory factor MyoD dimerizes with other positive and negative regulatory factors through a conserved region called the helix-loop-helix (HLH) domain. Using a non-DNA-binding MyoD mutant with a normal HLH domain as a dimerization competitor in gel mobility shift assays in conjunction with various MyoD HLH mutants, nonhydrophobic amino acids were identified in the HLH domain that contribute to dimerization specificity with E12. The assay detected subtle differences in dimerization activity among the mutant MyoD proteins that correlated with their ability to activate transcription in vivo, but this correlation was not apparent in the absence of competitor. The identification of such nonhydrophobic residues enabled us to predict the differences in dimerization affinity among the four vertebrate myogenic factors with E12. The experiments confirmed the prediction. Furthermore, a high-affinity homodimerizing analog of MyoD was designed by a single substitution at one of these residue positions. These experimental results were strengthened when they were analyzed in terms of the crystal structure for the Max bHLHZip domain homodimer. This analysis has allowed us to identify those residues that form charged residue pairs between the two HLH domains of MyoD and E12 and determine the dimerization specificity of the bHLH proteins.
3420	The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.	8247002	Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.
3421	Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretions by the fetal rat testis in vitro.	8404683	The in vitro effect of FSH on testosterone secretion by the fetal rat testis was studied. Testes were cultured in the presence or absence of either commercial human (h) FSH (Metrodine; 200 mIU/ml) or recombinant hFSH (200 mIU/ml) for 3 days and with 100 ng/ml ovine LH during the last 4 h of culture. To avoid a stimulatory effect by the 0.4% LH that contaminates Metrodine, the cultures were performed in the presence of a monoclonal anti-hLH beta antibody and with a concentration of Metrodine that had no short term stimulatory effect on testosterone production by the fetal testes in vitro. Metrodine treatment had a positive long term effect on both basal and LH-stimulated testosterone secretion by fetal testes explanted on days 18.5, 20.5, and 22.5 postconception, which was abolished by the addition of a monoclonal anti-hFSH beta antibody. LH-free recombinant FSH also augmented basal and LH-stimulated testosterone secretion of testes explanted on days 13.5, 14.5, and 18.5 postconception. The positive effect of recombinant hFSH appeared during the second day of treatment with day 14.5 and 18.5 testes and on the third day of treatment with day 13.5 testes. As it is widely accepted that FSH receptors are exclusively localized on Sertoli cells, these results suggest that on or before day 15.5 of fetal life, 1) Sertoli cells are able to respond to FSH, 2) Sertoli cells can produce factors that are able to act on Leydig cell function, and 3) Leydig cells are sensitive to FSH-induced Sertoli cell factors. In conclusion, this study points out a potential paracrine control of fetal Leydig cell function and/or differentiation by fetal Sertoli cells as soon as fetal Leydig cells differentiate.
3422	Neuronal expression of regulatory helix-loop-helix factor Id2 gene in mouse.	8224536	Id-like helix-loop-helix (HLH) proteins, which lack a basic DNA binding domain, have been suggested to serve as general inhibitors of differentiation. We present data that Id2 is expressed in specific cell types during neurogenesis and in the adult. At early stages of neurogenesis, Id2 is expressed in the ventricular zone of neuroepithelium. After the first neuronal populations are born, the expression of Id2 is down regulated in neuroepithelial cells and continues to be high in Purkinje cells of the cerebellum, in mitral cells of the olfactory bulb, and in layers 2, 3, and 5 of the cerebral cortex. In neuronally differentiating cell lines, the Id2 expression is up regulated (PCC7), down regulated (NG108), or unchanged (N18) during differentiation. Analyses of promoter sequences of the Id2 gene identify the region which is responsible for the down regulation of transcription during neuronal differentiation. Our data indicate that Id2 has different functions in different cell types during neurogenesis.
3423	Mode of action of prostaglandin F2 alpha in human luteinized granulosa cells: role of protein kinase C.	8143907	It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.
3424	Pit-1 messenger ribonucleic acid is differentially expressed in human pituitary adenomas.	8077322	Thirty-four human pituitary adenomas were examined for the presence of Pit-1 mRNA via Northern analysis. All tumors that were immunoreactive with either human (h) PRL or hGH antiserum contained Pit-1 mRNA (8 of 8) as did a small percentage of alpha-subunit only (1 of 3) and null tumors (1 of 9). Two tumors that contained TSH beta mRNA by RNAse protection assay also contained Pit-1 mRNA. Tumors that were immunoreactive with antiserum for hACTH, hFSH beta, or hLH beta without containing PRL, GH, or TSH beta were uniformly Pit-1 negative (0 of 14). Although RNA from the majority of Pit-1-positive tumors (10 of 11) contained primarily a 2.4-kilobase (kb) Pit-1 mRNA transcript, 1 tumor appeared to contain a 1.6-kb mRNA. Analysis of human pituitary autopsy specimens revealed no 1.6-kb Pit-1 mRNA, indicating that this variant may be tumor specific. The wide variety of serum PRL, GH, and insulin-like growth factor-I levels in patients with Pit-1-positive tumors suggests that although Pit-1 is necessary for PRL and GH production, it is not uniformly associated with hormone production, as determined by either immunohistochemistry or elevated serum levels. The expression of Pit-1 mRNA in a small fraction of null and alpha-subunit only tumors indicates that there may be a previously unsuspected diversity of origin within these particular subsets of adenomas.
3425	Serum glycoprotein hormones and their free alpha-subunit in a healthy elderly population selected according to the SENIEUR protocol. Analyses with ultrasensitive time resolved fluoroimmunoassays.	7510812	The SENIEUR protocol was elaborated by a working party of European Community's Concerted Action Programme on Aging (EURAGE) to define strict admission criteria for 'healthy' elderly subjects and young controls for immunogerontological studies. This protocol, which is based on case history, laboratory values and drug consumption, intends to limit the influence of underlying disease and/or medication in order to allow analyses of the aging process per se. In a group of 38 male and 37 female individuals we determined the impact of age and classification according to the SENIEUR protocol on luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) and free glycoprotein hormone alpha-subunit serum values. Analyses were performed by a set of ultrasensitive time-resolved immunofluorometric assays (IFMA) using our own panel of monoclonal antibodies (MCA). HLH and hFSH, but also hCG and free alpha serum levels increased highly significantly with age in the female population (P < 0.001). In males hFSH, hLH hCG and the free alpha-subunit increased with age. However, only the rise of hFSH and of Free alpha was statistically significant (P < 0.01). The influence of the SENIEUR status on the respective hormone serum levels was determined using two factor analysis of variance, which revealed no statistically significant difference (P > 0.01) between SENIEUR and NON-SENIEUR individuals for all four analytes in both sexes. We conclude that the age related increase of hLH, hFSH, hCG and free alpha is an intrinsic age-dependent phenomen and is not modified by or due to underlying disease or medication as demonstrated by analyses of SENIEUR individuals. Since SENIEUR and NON-SENIEUR individuals had comparable hormone values, a randomly chosen, 'apparently healthy' population seems to be sufficient for physiological studies on serum GPH levels. Lastly, these age related hormonal changes in an extremely well defined healthy population underline the need for age adjusted 'normal' hormone values as elaborated in this communication.
3426	Retinoic acid repression of cell-specific helix-loop-helix-octamer activation of the calcitonin/calcitonin gene-related peptide enhancer.	8413210	We have investigated the mechanism underlying repression of calcitonin/calcitonin gene-related peptide (CT/CGRP) gene expression by retinoic acid. Retinoic acid treatment of the CA77 thyroid C-cell line decreased CT/CGRP promoter activity two- to threefold, which correlates well with the decrease in calcitonin and CGRP mRNA levels. Repression is mediated through the nuclear retinoic acid receptors (RAR) on the basis of the retinoid specificity, the sensitivity of repression (half-maximal repression at 0.2 nM), and the additional repression caused by cotransfection of an alpha-RAR expression vector. The sequences required for retinoic acid repression were localized to an 18-bp element containing cell-specific enhancer activity. The enhancer binds helix-loop-helix (HLH) and octamer transcription factors that act synergistically to activate transcription. Retinoic acid repression requires both these factors since mutations in either motif resulted in the loss of repression. Furthermore, repression was observed only in cell lines containing enhancer activity. We have used electrophoretic mobility shift assays to show that repression does not involve direct DNA binding of RAR or RAR-retinoid X receptor heterodimers. Instead, repression appears to involve interactions with the stimulatory enhancer factors. Following retinoic acid treatment, there was a specific decrease in an enhancer complex containing both HLH and octamer proteins. Formation of the HLH-octamer complex was also specifically blocked by the addition of exogenous RAR-retinoid X receptor protein. These results demonstrate that RAR can repress CT/CGRP gene transcription by interfering with combinatorial activation by cell-specific HLH and octamer proteins.
3427	Expression of the helix-loop-helix factor Id during mouse embryonic development.	8405673	Expression and regulation of the helix-loop-helix (HLH) protein Id in cardiac cells and its negative effect on cardiac gene expression indicate that Id may play a role in cardiac development. To investigate this issue further, we have performed in situ analysis of Id mRNA in developing mouse embryos from Embryonic Days 7 to 15.5 and in neonatal heart. Consistent with its postulated role as an inhibitor of differentiation, Id mRNA was expressed in specific populations of proliferating mesenchymal and epithelial tissues, with its expression declining as differentiation progressed. In addition, patterns of Id mRNA hybridization overlapped considerably with those of two other developmentally important genes, twist and hox 7/7.1, of the HLH and homeobox family, respectively. Id mRNA hybridization was not observed in early epithelial somites or in the myotomal compartment of the somite, suggesting that Id is unlikely to play an inhibitory role in these early myogenic precursors. Id mRNA was expressed in specific regions of the developing nervous system. In the heart, Id mRNA was expressed at high levels in developing cardiac cushions and ridges of the outflow tract and continued to be expressed at high levels in valvular tissue of the neonatal heart.
3428	Ovarian thecal/interstitial androgen synthesis is enhanced by a follicle-stimulating hormone-stimulated paracrine mechanism.	8404591	To obtain direct evidence for FSH-stimulated paracrine signaling in the ovary, 21-day-old intact or hypophysectomized female Wistar rats received four sc injections of recombinant human FSH (rhFSH; total dose, 16-72 IU) at 12-h intervals. Ovaries were removed 48 h after the first injection to extract total RNA for Northern analysis of 17-hydroxylase/C17-20-lyase (cytochrome P450c17 alpha) mRNA or to isolate thecal/interstitial cells for assessment of basal and hLH-responsive androgen synthesis in vitro. In situ hybridization with a 35S-labeled cytochrome P450c17 alpha cRNA probe confirmed that expression of the cytochrome P450c17 alpha gene was specific to thecal/interstitial cells. The approximately 2.0-kilobase P450c17 alpha mRNA signal in ovarian total RNA from intact animals was increased approximately 5-fold by treatment with rhFSH (total dose, 72 IU) or PMSG (15 IU). This effect was shown to be dose dependent, with a approximately 2-fold increase in response to 16 IU (total dose) rhFSH. P450c17 alpha mRNA levels in isolated granulosa and thecal/interstitial cell total RNA from intact animals were compared to establish which was the principal cellular site of P450c17 alpha mRNA expression. The P450c17 alpha mRNA signal was undetectable in control granulosa cells and only barely discernible after treatment with 72 IU (total dose) rhFSH. In contrast, P450c17 alpha mRNA was abundant in control thecal/interstitial mRNA, and its level was increased 4- to 6-fold by treatment with rhFSH. Treatment of hypophysectomized animals with rhFSH did not consistently alter ovarian P450c17 alpha mRNA levels. During culture for 48 h in serum-free medium, basal androgen (androstenedione plus androsterone) production by thecal/interstitial cells from intact animals was unaffected by treatment with rhFSH in vivo, but hLH-stimulated androgen production by these cells was enhanced approximately 2-fold. Neither basal nor hLH-responsive androgen production by thecal/interstitial cells from hypophysectomized animals was altered by previous treatment with rhFSH in vivo. Treatment of thecal/interstitial cell cultures from both intact and hypophysectomized animals with inhibin (0.1-30 ng/ml), a putative granulosa-derived paracrine factor, did not measurably affect basal androgen synthesis, but potently enhanced LH-responsive androgen synthesis in vitro. Similarly, treatment of thecal/interstitial cell cultures with conditioned medium from FSH-treated granulosa cell cultures significantly enhanced LH-responsive, but not basal, androgen production. We conclude that treatment of pituitary-intact rats with "pure" FSH modulates thecal/interstitial cell androgen synthesis. Granulosa cells, but not thecal cells, possess FSH receptors, and thecal/interstitial cells are the principal ovarian sites of P450c17 alpha expression.(ABSTRACT TRUNCATED AT 400 WORDS)
3429	Activation of gene transcription by the amino terminus of the N-Myc protein does not require association with the protein encoded by the retinoblastoma suppressor gene RB1.	8378092	N-Myc encodes a nuclear phosphoprotein that contains a basic region (BR), a helix-loop-helix (HLH) and a leucine zipper (Zip). These motifs are hallmarks of certain transcription factors. In pursuit of the question if N-Myc can activate transcription, we have employed an experimental model involving the yeast transcription factor Gal4. We have first generated fusion proteins containing the Gal4 DNA-binding domain joined to portions of N-Myc. Subsequently we have analysed if chimeric proteins can transactivate the transcription of a reporter under the control of Gal4 binding sites. Here we show that the amino terminal portion of N-Myc activates transcription. Activation maps to a domain highly conserved among Myc-proteins and to other non-conserved sequences, suggesting functional redundancy. Previous studies had documented in vitro association of the RB1 protein with N-Myc (Rustig et al., 1991). We here confirm this observation and identify the region encompassing the transactivation domain as responsible for RB1 binding. Analyses of N-Myc transactivation in retinoblastoma cell line WERI lacking a function RB1 protein gave results similar to those with cell lines having an intact RB1 protein, showing that RB1 protein is not required for transactivation by N-Myc. The present findings leave open the question if deregulated expression of N-Myc contributes to tumorigenesis by transcriptional activation of as yet unidentified target genes or by functionally inactivating the protein encoded by the tumor suppressor gene RB1, or by a combination of both.
3430	Activation of the thyrotropin (TSH) receptor by human chorionic gonadotropin and luteinizing hormone in Chinese hamster ovary cells expressing functional human TSH receptors.	7691861	It is well known that peptide heterogeneity exists in the hCG-beta subunit in pregnancy and in patients with trophoblastic diseases. To elucidate the differences in thyrotropic activity of hCG molecules, we examined cAMP accumulation and TSH receptor binding of intact hCG, hLH, and a recombinant hCG that lacked the C-terminal extension on the beta-subunit, hCG (alpha wt/beta delta T), using Chinese hamster ovary (CHO) cells transfected with hTSH receptors. hLH, which shares 85% sequence identity with the hCG-beta molecule except for the C-terminal amino acid residue extension of the hCG-beta subunit, bound to the TSH receptor and stimulated adenylate cyclase about 10 times more potently than hCG on a molar basis. This was consistent with the result that cAMP stimulation by mutant hCG (alpha wt/beta delta T) was greater than intact hCG. hLH also increased iodide uptake and thymidine incorporation in FRTL-5 rat thyroid cells more potently than intact hCG. These results demonstrate that hLH is a more potent TSH than hCG and that the C-terminal extension of the hCG beta-subunit can interfere with hCG interaction with the hTSH receptor. hCG lacking the C-terminal extension of the beta-subunit occurs in the mixture of heterogeneous hCG molecular forms of pregnancy and trophoblastic diseases and may contribute to the hyperthyroidism in patients with hydatidiform mole, choriocarcinoma, and hyperemesis gravidarum.
3431	HLH forced dimers: tethering MyoD to E47 generates a dominant positive myogenic factor insulated from negative regulation by Id.	7691411	Basic-helix-loop-helix (bHLH) class transcription factors bind DNA as hetero- and homodimers. In murine myogenic cells the HLH network includes multiple members of the E protein, MyoD, and Id families; changes in the network characterize muscle determination and differentiation and have been proposed as causal for these developmental transitions. To test the importance of HLH partner choice in these cellular decisions, we have designed a strategy in which the identity of a bHLH dimer is specified by joining two monomers via a flexible polypeptide linker. A MyoD-E47 polyprotein avidly bound the same DNA targets as its unlinked counterpart, but, unlike intermolecular dimers that are very sensitive to inhibition by Id, MyoD-E47 was resistant to Id challenge. In cells MyoD-E47 acted as a dominant positive myogenic factor, capable of initiating myogenic determination and also substantially bypassing negative regulation of differentiation by serum growth factors.
3432	Potential role of helix-loop-helix proteins in cardiac gene expression.	8394227	Because helix-loop-helix (HLH) transcription factors appear to play an important role in mesodermal development, we have investigated the potential role of these factors in cardiac gene expression. HLH proteins interact with DNA at consensus "E-box" sites and may be tissue specific or more widely expressed. We have examined cardiac cells for expression and regulation of widely expressed factors Pan1/Pan2 and the inhibitor of differentiation (Id) by RNase protection analysis. The effect of MyoD, Id, and Pan1/Pan2 expression on skeletal and cardiac promoters in cardiac cells was examined by transient cotransfection studies. Our results indicate that neonatal ventricular cells are a functional HLH environment, because MyoD can activate a skeletal muscle-specific promoter in these cells. MyoD, however, has no effect on the expression of several genes that are expressed in cardiac cells. In addition, Id may be an early response gene for signal transduction in cardiac cells, because increases in Id mRNA occurred within 30 minutes of stimulation with serum or phenylephrine. Activities of three cardiac promoter elements in primary ventricular myocytes were not downregulated by Id. Surprisingly, expression of Pan1 and Pan2 exhibited a strong negative effect on cardiac expression of the myosin light chain-2 promoter.
3433	Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila.	8370521	The dorsal (dl) morphogen has been implicated in the establishment of the embryonic mesoderm, neuroectoderm, and dorsal ectoderm in Drosophila. Here we show that the simultaneous reduction in the levels of dl and any one of several helix-loop-helix (HLH) proteins results in severe disruptions in the formation of mesoderm and neuroectoderm. Certain triple heterozygous combinations essentially lack mesoderm as a result of a block in ventral furrow formation during gastrulation. HLH proteins that have been implicated previously in sex determination and neurogenesis (daughterless, achaete, and scute) are shown to be required for the formation of these embryonic tissues. Evidence is also presented that dl-HLH interactions involve the direct physical association of these proteins in solution mediated by the rel and HLH domains. We discuss the striking parallels in mesoderm formation and sex determination.
3434	A bZIP protein, sisterless-a, collaborates with bHLH transcription factors early in Drosophila development to determine sex.	8370520	Sexual identity in Drosophila is determined by zygotic X-chromosome dose. Two potent indicators of X-chromosome dose are sisterless-a (sis-a) and sisterless-b (sis-b). Genetic analysis has shown that a diplo-X dose of these genes activates their regulatory target, the feminizing switch gene Sex-lethal (Sxl), whereas a haplo-X dose leaves Sxl inactive. sis-b encodes a transcriptional activator of the bHLH family that dimerizes with several other HLH proteins required for the proper assessment of X dose. Here, we report that sis-a encodes a bZIP protein homolog that functions in all somatic nuclei to activate Sxl transcription. In contrast with other elements of the sex-determination signal, the functioning of this transcription factor in somatic cells may be specific to X-chromosome counting. Using in situ hybridization, we determined the time course of sis-a, sis-b, and Sxl transcription during the first few hours after fertilization. The pattern of sis-a RNA accumulation is very similar to that for sis-b, with a peak in nuclear cycle 12 at about the time of onset of Sxl transcription. Considered in the context of other studies, these results suggest that the ability to distinguish one X from two is attributable to combinatorial interactions between bZIP and bHLH proteins and their target, Sxl, as well as to positive and negative interactions with maternally supplied and zygotically produced proteins.
3435	The E2A gene product contains two separable and functionally distinct transcription activation domains.	8367464	The E2A gene encodes transcription factors of the helix-loop-helix (HLH) family which are implicated in cell-specific transcriptional control in several cell lineages, including pancreatic beta cells. In the present work, we show by deletion mapping of both the E2A protein itself and the Gal4-E2A fusion protein that the protein contains at least two distinct activation domains. One domain (located between amino acids 1 and 153) functions efficiently in a variety of mammalian cell lines. The second domain (located between amino acids 369 and 485) functions preferentially in pancreatic beta cell lines. The latter domain shows a pattern of heptad repeats of leucine residues characteristics of "leucine zipper" transcription factors; site-directed mutagenesis of leucines within this repeat led to substantial reductions in activity. The selective properties of this activation domain may contribute to cell-specific transcription directed by the E2A gene.
3436	Isolation and characterization of human LH isoforms.	8277226	Thirty-nine human LH (hLH) isoforms were chromatographically separated from human pituitary extracts using a mild purification procedure which consisted of preparative isoelectric focusing, high-performance ion-exchange chromatography and immobilized metal-affinity chromatography. Twenty of these hLH isoforms were characterized by LH radioreceptor assay, SDS-PAGE and amino acid analysis, and 17 were shown to be highly purified (> 90% pure). The specific activities of these hLH isoforms ranged from 1980 to 38,650 IU/mg protein in terms of the 2nd IS for human pituitary LH, based on protein content as determined by amino acid analysis. hFSH and hTSH content were < 0.5% and < 7.8% respectively. The purity was assessed by silver staining on SDS-PAGE. Under non-reducing conditions, a single band of apparent molecular mass 23.5-24.5 kDa was observed, whereas under reducing conditions the isoforms migrated as two distinct bands, 21.1-22.4 kDa and 18.0-20.5 kDa, probably corresponding to the alpha and beta subunits of hLH respectively. The remaining three less pure isoform preparations (70-90% pure) contained additional bands of 16 kDa and 26.3 kDa under non-reducing conditions. All isoforms showed a low molecular mass band(s) of 11-14 kDa which was < 7% of stained material as assessed by densitometry. Amino acid composition of the 17 hLH isoforms was similar to the published cDNA composition of hLH. Further fractionation of one hLH isoform (hLH IIc) on reversed-phase high-performance liquid chromatography yielded four peaks identified by N-terminal sequencing as two alpha and two beta hLH subunits identical to their cDNA-derived N-terminal sequences. No additional sequences indicative of internal clipping of hLH were observed. The two pairs of alpha and beta subunits probably represent two separate hLH isoforms in this preparation. It was concluded that a mild purification procedure with high recoveries for the isolation of intact hLH isoforms has been developed, and 17 isoforms of high purity suitable for further biological and physicochemical characterization have been isolated. These isoform preparations are free of other contaminating proteins, but may still contain multiple hLH-related species.
3437	Structure and significance of human luteinizing hormone-beta core fragment purified from human pituitary extracts.	7689962	A fragment of the hCG beta-subunit is present in high concentrations in the urine of pregnant women and the urine from individuals with ovarian or other cancers. The utility of immunoreactive measurement of this fragment to monitor therapy of such cancers is compromised, however, because high concentrations of the molecule are also detected in the urine of healthy postmenopausal women. It has been suggested that the latter observations may be due to a cross-reacting human (h) LH beta core fragment, presumably of pituitary origin, but no such fragment had ever been isolated. We have now isolated a hLH beta core fragment from a pituitary tissue extract. Its structure is exactly analogous to that of the hCG beta core fragment. The finding of a discrete hLH beta core fragment in a tissue extract suggests that it may be produced within pituitary tissue, rather than by a peripheral degradation process. We have also found that the same immunoaffinity method used to extract the hLH beta core fragment from the pituitary extract purified several protein fragments from postmenopausal urine, none of which was related to hLH or hCG. The availability of the pituitary hLH beta core fragment may allow development of assays that distinguish it from its hCG analog.
3438	Definition of the DNA-binding site repertoire for the Drosophila transcription factor SNAIL.	8371971	The Drosophila gene snail (sna) which encodes a zinc finger protein is essential for dorsal-ventral pattern formation in the developing embryo. We have defined a repertoire of SNAIL (SNA) binding sites using recombinant SNA proteins to select specific binding sequences from a pool of random sequence nucleotides. The bound sequences which were selected by multiple rounds of gel retardation and amplification by the polymerase chain reaction (PCR) were subsequently cloned and sequenced. The consensus sequence, 5'G/A A/t G/A A CAGGTG C/t A C 3', with a highly conserved core of 6 bases, CAGGTG, shares no significant homology with known binding sequences of other Drosophila zinc finger proteins. However, the CAGGTG core is identical to the core motif of aHLH (helix-loop-helix) binding sites. The strongest SNA binding is obtained with sequences containing this core motif whereas reduced binding is seen for sequences with canonical CANNTG HLH motifs. Interestingly, SNA binding is detected in the promoter region of the snail gene. Transient expression in co-transfection experiments using a SNA binding element (SBE) linked to a heterologous promoter indicates that SNA has the ability to function as a transcription activator.
3439	Molecular characterization of HES-2, a mammalian helix-loop-helix factor structurally related to Drosophila hairy and Enhancer of split.	8354270	Drosophila hairy (h) plays a crucial role in early development as a pair-rule segmentation gene. h and its structurally related gene Enhancer of split [E(spl)] are also required for normal sensory neurogenesis in late development. To analyze the molecular mechanisms of mammalian development, we recently characterized three rat helix-loop-helix (HLH) factors that show structural homology to the Drosophila h and E(spl) gene products, and found that rat factors exhibit distinct spatiotemporal expression patterns and act as a negative regulator. Here, we report the molecular characterization of another member of this family, designated HES-2. Rat HES-2 protein has a basic HLH domain homologous to h and E(spl) as well as the carboxy-terminal Trp-Arg-Pro-Trp sequence conserved among this family. The HES-2 mRNA is present as early as embryonic day 9.5 and is detected in a variety of tissues of both embryos and adults. DNase-I-footprinting analyses indicate that HES-2 binds to all E box sequences (CANNTG) we tested as well as to the N-box sequences (CACNAG). Further studies of gel-mobility-shift assays show that HES-2 has a higher affinity for the E box than for the N box. Transient transfection analyses suggest that HES-2 decreases the transcription originating from the promoters containing either the E box or the N box. These results indicate that HES-2 acts as a negative regulator through interaction with both E-box and N-box sequences.
3440	The daughterless gene product in Drosophila is a nuclear protein that is broadly expressed throughout the organism during development.	8217842	The daughterless (da) gene in Drosophila functions in the regulation of at least three significant developmental pathways: sex determination, neurogenesis and oogenesis. As a member of the helix-loop-helix (HLH) family of DNA binding proteins, the da gene product appears to act as a transcription factor. Based on the genetic and molecular characterization of da, it has been proposed that the da protein (Da) functions as a generic member of this family, serving throughout development as a necessary binding partner for an assortment of other HLH proteins. As a result of temporally and/or spatially restricted expression, these binding partners would provide some regulatory specificity to the functional transcription complex. In order to participate in this way in the regulation of multiple genes, Da must be expressed in numerous times and places during development. Using anti-Da antibodies, we validate two predictions of this scenario of Da function: (1) Da protein is not only nuclear localized, but also associated with chromosomes in vivo; and (2) Da protein is widely distributed, both spatially and temporally, throughout development. With regard to the essential role of maternal da+ in progeny sex determination, little, if any, Da protein is synthesized in the maternal germline. This suggests that the female-specific germline function of da+ is provided to the zygote as maternally synthesized RNA that becomes translated early in embryogenesis.
3441	Cloning and chromosome mapping of the human Mel-18 gene which encodes a DNA-binding protein with a new 'RING-finger' motif.	8325509	It has recently been reported that there exists a new 'RING-finger' protein family among the zinc-finger (Zf) proteins. Previously, we had isolated the mouse Mel-18 cDNA (mMel-18) encoding the nuclear RING-finger protein that exhibits an ability to bind to a nonspecific DNA column. Here, we have isolated and characterized the human Mel-18 cDNA (hMel-18) using the mMel-18 cDNA as a probe. The deduced hMel-18 protein contains 344 amino acids (38 kDa) with a RING-finger motif, a helix-loop-helix (HLH)-like structure and a Pro/Ser-rich region. The hMel-18 gene is conserved among vertebrates. Its mRNA is highly expressed in placenta, lung and kidney, but the level is low in liver, pancreas and skeletal muscle. Using in situ hybridization, we mapped hMel-18 to band q22 of chromosome 12. It is possible that the Mel-18/bmi-1 gene family represents a mammalian homologue of the Drosophila polycomb gene group.
3442	Regulation of the calcitonin/calcitonin gene-related peptide gene by cell-specific synergy between helix-loop-helix and octamer-binding transcription factors.	8340417	The calcitonin/calcitonin gene-related peptide (CGRP) gene is transcribed in thyroid C-cells and a subset of neurons. We have localized sequences required for cell-specific enhancement of calcitonin/CGRP transcription in rat thyroid C-cell lines. An 18-base pair element approximately 1 kilobase pair upstream of the transcriptional start site stimulated expression of a luciferase reporter gene 50-fold in 44-2C C-cells. There was less than 2-fold stimulation in HeLa and Rat-1 cells, which do not express the endogenous calcitonin/CGRP gene. The enhancer contains potential binding sites for helix-loop-helix (HLH) and octamer transcription factors based on sequence homologies. The functional significance of these sites was shown by point mutations in the HLH and octamer motifs and by separation of the two motifs, all of which decreased enhancer activity greater than 10-fold. The involvement of HLH proteins was further shown by co-expression of the mammalian achaete-scute homologue-1 HLH protein, which activated the enhancer severalfold in HeLa cells. Electrophoretic mobility shift analyses revealed several DNA-protein complexes containing HLH and octamer-binding proteins. One octamer-binding complex (OB1) most likely contains the ubiquitous Oct-1 protein, whereas a second complex (OB2) was cell-specific. In contrast to OB1, OB2 had lower affinity for a consensus octamer motif, and its DNA binding was not affected by addition of antiserum that recognizes Oct-1 and Oct-2 proteins. In addition, we observed a large complex that appears to contain both an HLH protein and OB2. These results demonstrate that calcitonin/CGRP enhancer activity is controlled by a cell-specific synergistic activation involving HLH and octamer-binding factors.
3443	Separable regulatory elements governing myogenin transcription in mouse embryogenesis.	8392225	Expression of the myogenic helix-loop-helix (HLH) protein myogenin in muscle cell precursors within somites and limb buds is among the earliest events associated with myogenic lineage determination in vertebrates. Mutations in the myogenin promoter that abolish binding sites for myogenic HLH proteins or myocyte enhancer factor-2 (MEF-2) suppressed transcription of a linked lacZ transgene in subsets of myogenic precursors in mouse embryos. These results suggest that myogenic HLH proteins and MEF-2 participate in separable regulatory circuits leading to myogenin transcription and provide evidence for positional regulation of myogenic regulators in the embryo.
3444	Gene-regulatory properties of Myc helix-loop-helix/leucine zipper mutants: Max-dependent DNA binding and transcriptional activation in yeast correlates with transforming capacity.	8510929	Max is a basic helix-loop-helix/leucine zipper (bHLH/LZ) protein that forms sequence-specific DNA-binding complexes with the c-Myc oncoprotein (Myc). Using Saccharomyces cerevisiae, we have shown that the Max bHLH/LZ domain enables Myc to activate transcription through CACGTG and CACATG sequences in vivo, and that the number and context of such sites determines the level of activation. In addition, we have used yeast to investigate the role of the Myc helix-loop-helix (HLH) and leucine zipper (LZ) motifs in mediating Max-dependent DNA-binding and transcriptional activation in vivo using HLH/LZ mutants generated by site-directed mutagenesis. The results show that, while both motifs are essential for Myc to activate transcription, helix 2 of the HLH together with the contiguous LZ suffice to mediate complex formation with Max, whilst helix 1 is essential for sequence-specific DNA binding of Myc-Max complexes. Furthermore, the ability of Myc HLH/LZ mutants to bind DNA and activate transcription in collaboration with Max correlates closely with their neoplastic transforming activity in higher eukaryotic cells.
3445	Development of an LH receptor assay capable of measuring serum LH/CG in a wide variety of species.	8410832	The development of a radioreceptor assay (RRA) that can measure serum LH in a variety of species and CG in sera and urine of pregnant women and monkeys is reported. Using sheep luteal membrane as the receptor source and 125I-labeled hLH/hCG as the tracer, dose-response (displacement) curves were obtained using hLH or hCG as standard. The addition of LH-free serum (200 microliters per tube) had no affect on the standard displacement curve. The assay is simple, requires less than 90 min to complete and provides reproducible results. The sensitivity of the assay was 0.6 ng hLH per tube and the intra- and interassay variations were 9.6 and 9.8, respectively. Sera obtained from male and female bonnet monkeys (Macaca radiata) and monkey pituitary extract showed parallelism to the standard curve. The concentrations of LH measured correlated with the physiological status of the animals. Sera of rats, rabbits, hamsters, guinea-pigs, sheep and humans showed parallelism to the hLH standard curve indicating the viability of the RRA to measure serum LH of different species. Since the receptors recognize LH and CG, detection of pregnancy in monkeys and women was possible using this assay. The sensitivity of the assay of hCG was 8.7 miu per tube. This RRA could be a convenient alternative to the Leydig cell bioassay for obtaining the LH bioactivity profile of sera and biological fluids.
3446	Purification of a high molecular weight follicle-stimulating hormone receptor-binding inhibitor from human follicular fluid.	8325939	In a previous study we reported the presence in human follicular fluid (hFF) of a FSH receptor-binding inhibitor (hFSH-BI) with FSH agonist activity, which was immunologically similar to FSH but could be distinguished from FSH on the basis of its greater stability in acid. We have now purified hFF-derived hFSH-BI after molecular sieving on Sephracyl S-100 ion exchange chromatography using Diethyl-aminoethyl-cellulose followed by polyacrylamide gel electrophoresis (PAGE). The purified hFSH-BI had a potency approximately 12,000-fold greater than that of dialyzed hFF, based on its ability to inhibit the binding of [125I]hFSH to its membrane receptor. The purified hFSH-BI also had FSH agonist activity, stimulating estradiol synthesis in cultured rat Sertoli cells. Upon sodium dodecyl sulfate (SDS)-PAGE, hFSH-BI migrated as two bands of almost identical mobility, with an estimated mol wt of 57,000, compared with 30,000 for pituitary FSH run simultaneously. A monoclonal antibody to hFSH that also recognizes hFSH-BI was used for Western blot analysis of the SDS-PAGE fraction. The Western blot confirmed the detection of two bands with very similar mobilities and estimated mol wt of 57,000, which were clearly distinguishable from that of immunologically reactive hFSH run in parallel. The hFSH-BI bands showed similar profiles upon cyanogen bromide cleavage and had indistinguishable amino acid compositions. The amino acid composition of hFSH-BI was clearly distinct from those of hFSH, hLH, hCG, and the alpha-subunit of human inhibin. Our studies confirm the presence in hFF of a unique agonist protein which is of potential importance in the regulation of gonadal function.
3447	Spectrophotometric analysis of human follicular fluid with regard to in vitro fertilization (IVF) parameters, follicular protein, and hormone content.	8003881	Our purpose was to investigate possible relationships with spectrophotometric absorbance (458-nm region) and biochemical variables in follicular fluid (FF) as well as in vitro fertilization (IVF) outcome.This study included 227 normal ovulatory women undergoing oocyte retrieval for IVF. Blood-uncontaminated fluid samples, identified by spectrophotometry, were investigated. Spectrophotometric absorbance of FF at 458 nm (n = 426), as well as hLH, FSH, PRL, hCG, testosterone, sialic acid, alpha 1-antitrypsin and plasminogen of selected fluids, was analyzed.Small-volume follicles (< or = 2 ml) were associated with higher absorbance profiles (P < 0.05), when compared to volumes greater than 2 ml. Our data suggest that the presence or absence of an oocyte, the potential of an oocyte to fertilize or cleave, failed to show any relationship with maximum FF absorbance at 458 nm. Maximum absorbances were significantly lower in FF from patients who subsequently became clinically pregnant (P = 0.039). No correlation between FF absorbances and biochemical parameters (P > 0.15) were established.Absorbance of clear FF at 458 nm should not be viewed as the single parameter to predict oocyte development in vitro.
3448	E2A and E2-2 are subunits of B-cell-specific E2-box DNA-binding proteins.	8497267	A class of helix-loop-helix (HLH) proteins, including E2A (E12 and E47), E2-2, and HEB, that bind in vitro to DNA sequences present in the immunoglobulin (Ig) enhancers has recently been identified. E12, E47, E2-2, and HEB are each present in B cells. The presence of many different HLH proteins raises the question of which of the HLH proteins actually binds the Ig enhancer elements in B cells. Using monoclonal antibodies specific for both E2A and E2-2, we show that both E2-2 and E2A polypeptides are present in B-cell-specific Ig enhancer-binding complexes. E2-box-binding complexes in pre-B cells contain both E2-2 and E2A HLH subunits, whereas in mature B cells only E2A gene products are present. We show that the difference in E2-box-binding complexes in pre-B and mature B cells may be caused by differential expression of E2A and E2-2.
3449	Regulation of the proneural gene achaete by helix-loop-helix proteins.	8497266	The Achaete (Ac) protein, a transcriptional regulator of the basic-helix-loop-helix (bHLH) type, confers upon ectodermal cells the ability to become neural precursors. Its temporally and spatially regulated expression, together with that of the related Scute (Sc) protein, helps define the pattern of Drosophila melanogaster sensory organs. We have examined the transcriptional control of the ac gene and shown, using in vivo assays, that several E-boxes, putative interacting sites for bHLH proteins, present in the ac promoter are most important for ac regulation. They most likely mediate ac self-stimulation and sc trans-activation. We also demonstrate that ac transcription is negatively regulated in vivo by the gene extramacrochaetae (emc) in a manner dependent on Ac and Sc products. emc encodes an HLH protein that lacks the basic region and presumably antagonizes Ac and Sc function by sequestering these proteins in complexes unable to interact with DNA. Our results strongly support the model of negative regulation of emc on ac and sc transcription through titration of their products. As currently thought, this seems accomplished by heterodimerization via the HLH domain, because an amino acid substitution in this region abolishes the emc antagonistic effect both in vitro and in vivo.
3450	Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain.	84795348479531	The three-dimensional structure of the basic/helix-loop-helix/leucine zipper domain of the transcription factor Max complexed with DNA has been determined by X-ray crystallography at 2.9 A resolution. Max binds as a dimer to its recognition sequence CACGTG by direct contacts between the alpha-helical basic region and the major groove. This symmetric homodimer, a new protein fold, is a parallel, left-handed, four-helix bundle, with each monomer containing two alpha-helical segments separated by a loop. The two alpha-helical segments are composed of the basic region plus helix 1 and helix 2 plus the leucine repeat, respectively. As in GCN4, the leucine repeat forms a parallel coiled coil.
3451	cAMP-dependent protein kinase represses myogenic differentiation and the activity of the muscle-specific helix-loop-helix transcription factors Myf-5 and MyoD.	8387507	Myf-5 and MyoD are members of a family of muscle-specific basic helix-loop-helix (bHLH) proteins that are fundamental for myogenic cell differentiation and transcriptional activation of muscle-specific genes. Here we report that elevated levels of the intracellular signaling molecule cAMP and overexpression of cAMP-dependent protein kinase (PKA) inhibit myogenic differentiation. PKA represses the transcriptional activation of muscle-specific genes by the myogenic regulators Myf-5 and MyoD. The repression is directed at the basic HLH domain and is mediated through the E-box DNA consensus motif to which these proteins bind. However, phosphorylation of Myf-5 and MyoD by PKA in vitro does not affect their ability to bind to DNA. PKA specifically inhibits the activity of myogenic bHLH proteins, but not of other HLH proteins, such as the ubiquitously expressed E2A gene products E12 and E47 (E2-5). Our results demonstrate that PKA mediates the cAMP-induced inhibition of muscle cell differentiation by repressing the activity of Myf-5 and MyoD. The inhibition by PKA occurs post-translationally and presumably affects the transactivation process at a step following DNA-binding. The regulation of Myf-5 and MyoD function by a cAMP-dependent pathway may partly explain how external signals generated by serum and certain peptide growth factors can be transduced to the nucleus and inhibit dominant-acting factors that are responsible for myoblast differentiation.
3452	Luteinizing hormone pulse characteristics in early pubertal boys are the same whether measured by radioimmuno- or immunofluorometric assay.	8496308	We tested the hypothesis that the improved sensitivity of immunofluorometric (IFMA) assays will lead to an increase in the number of detectable LH pulses compared to RIA in early pubertal boys, in whom LH secretion is low. To test this hypothesis we determined plasma LH concentrations in six pubertal boys (bone age, 12-16 yr) by IFMA and compared the results to RIA data reported previously. Each boy was given an infusion of saline, followed 1 week later by an infusion of testosterone (T; 960 nmol/h) for 33 h starting at 1000 h. Starting at 1200 h, blood was obtained every 15 min for LH determinations (RIA and IFMA) and every 30 min for T measurements. At the end of both studies, responses to GnRH (250 ng/kg) were assessed. The assay sensitivities for LH by RIA and IFMA (Delfia hLH Spec Pharmacia Diagnostics ENI, Columbia, MA) were 1.0 and 0.05 IU/L, respectively. LH pulses were identified by three independent pulse detection programs: Detect, Cluster, and Kushler-Brown. The correlation for LH values as measured by RIA and IFMA was highly significant (r = 0.81). There was a poor correlation between LH values determined by IFMA and RIA when LH values within 4 times the SD of each assay sensitivity were compared (r = 0.08; P = NS). T infusion suppressed LH pulse frequency by 40% and 66%, as determined by RIA and IFMA, respectively (P = NS). Using the Detect program, during the complete study in all 6 boys, 117 pulses of LH were identified by RIA and 93 by IFMA (79% ratio of detection IFMA/RIA). During saline infusion there were 73 vs. 69 LH pulses (94%), while during T infusion there were 24 vs. 44 LH pulses (55%), as detected by IFMA vs. RIA, respectively. Administration of naloxone did not accelerate LH pulse frequency during T infusion, as determined by either method. Changes in pituitary responses to exogenous GnRH also showed similar trends of augmentation by T infusion by both methods. We conclude that the use of IFMA does not lead to the anticipated increase in the detectability of LH pulsatility. Actually, fewer LH pulses were identified by IFMA in this group of boys. We speculate that this is due to the increased specificity of the IFMA assay. More significant was the finding that the physiological interpretation of the effects of T and naloxone on LH pulse frequency and responses to GnRH did not change whether LH was measured by RIA or IFMA.
3453	Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain.	8477671	LH from anterior pituitary and hCG from placenta bind to a common receptor in gonadal and nongonadal reproductive tissues. There have been numerous examples suggesting that the brain may also contain hCG/LH receptors, yet there has been no evidence for their existence so far. We now demonstrate by reverse transcription-nested polymerase chain reaction and northern blotting that the rat brain contains hCG/LH receptor mRNA. A major receptor transcript of 2.6 kilobases and minor transcripts of 1.8 and 4.4 kilobases were found. Western immunoblotting, ligand blotting, and covalent receptor cross-linking studies have shown that rat brain also contains an 80-kilodalton receptor protein that can bind hCG and hLH, but not hFSH. Rat testis used as a positive control showed a higher abundance of multiple transcripts and an 80-kilodalton receptor protein that can bind [125I]hCG. Rat liver used as a negative control did not contain any receptor transcripts or protein. In situ hybridization, dot blotting, immunocytochemistry, and topical autoradiography have revealed that hCG/LH receptors are present in rat hippocampus; dentate gyrus; hypothalamus; cerebellum; choroid plexus; ependymal cells of third, fourth, and lateral ventricles; cortex; brainstem; bovine hypothalamus; and human area postrema. These novel findings could potentially explain numerous previous observations and suggest new possibilities concerning the regulation of brain functions by hCG and LH.
3454	Bioactivity of human growth hormone in serum: validation of an in vitro bioassay.	8477657	GH, in clinical practice, is determined by RIA, but RIA estimates may not accurately reflect serum GH bioactivity. The available measures of GH bioactivity lack either sensitivity, specificity, or a physiologically relevant end point. The objective of this research was to develop a physiologically relevant GH bioassay which would not only measure the bioactivity of purified GH preparations, but would also have sufficient sensitivity to measure GH bioactivity in human serum. The method consisted of incubating murine 3T3-F442A adipocytes in serum-free medium containing BSA, 14C-glucose, and increasing concentrations of GH or test materials for 24 h, followed by measurement of conversion of glucose to lipid. Interference by nonspecific serum factors was reduced by the addition of 10 micrograms/liter insulin, 25 nM dexamethasone, and 37 nM estradiol to the medium. In the presence of 10 micrograms/liter insulin, 50 micrograms/liter insulin-like growth factor-1 did not alter the ability of GH to suppress lipid accumulation. Epinephrine and glucagon could suppress lipid accumulation but only at concentrations greatly in excess of the physiological range in serum. Twenty two thousand dalton hGH produced dose-dependent suppression of lipid accumulation which was linear between 0.625 and 10 micrograms/liter (r = 0.926; P = 0.0001) with a half-maximal response of 3.0 +/- 0.2 micrograms/liter (n = six experiments). The intra- and interassay coefficients of variation were 7% and 19%, respectively. The assay was specific for GH since addition of human PRL produced suppression of lipid accumulation only at concentrations where contamination of the preparation by GH became a significant factor. ACTH also suppressed lipid accumulation but only at doses of 1000 micrograms/liter or greater. Human placental lactogen and hLH, hFSH, and hTSH did not cross-react with GH in this assay. Addition of human serum did not alter the slope of ED50 of the GH dose-response curve. Pools of serum from prepubertal and pubertal boys and girls, subjects treated with arginine or insulin, a diabetic girl, and a boy with gigantism who had a serum GH content of 80 micrograms/liter by RIA and 40 micrograms/liter by bioassay, produced dose response curves parallel to that of the GH standard curve. Serum from patients with hypopituitarism did not produce significant suppression of lipid accumulation in any assay. Recovery of 5 micrograms/liter GH added to human serum was 94%. Twenty thousand dalton GH also suppressed lipid accumulation in this assay, but was 2-fold less potent than 22,000 dalton GH.(ABSTRACT TRUNCATED AT 400 WORDS)
3455	[CHL15--a new gene controlling the replication of chromosomes in saccharomycetes yeast: cloning, physical mapping, sequencing, and sequence analysis].	8316240	We have analyzed the CHL15 gene, earlier identified in a screen for yeast mutants with increased loss of chromosome III and artificial circular and linear chromosomes in mitosis. Mutations in the CHL15 gene lead to a 100-fold increase in the rate of chromosome III loss per cell division and a 200-fold increase in the rate of marker homozygosis on this chromosome by mitotic recombination. Analysis of segregation of artificial circular minichromosome and artificially generated nonessential marker chromosome fragment indicated that sister chromatid loss (1:0 segregation) is a main reason of chromosome destabilization in the chl15-1 mutant. A genomic clone of CHL15 was isolated and used to map its physical position on chromosome XVI. Nucleotide sequence analysis of CHL15 revealed a 2.8-kb open reading frame with a 105-kD predicted protein sequence. At the N-terminal region of the protein sequences potentially able to form DNA-binding domains defined as zinc-fingers were found. The C-terminal region of the predicted protein displayed a similarity to sequence of regulatory proteins known as the helix-loop-helix (HLH) proteins. Data on partial deletion analysis suggest that the HLH domain is essential for the function of the CHL15 gene product. Analysis of the upstream untranslated region of CHL15 revealed the presence of the hexamer element, ACGCGT (an MluI restriction site) controlling both the periodic expression and coordinate regulation of the DNA synthesis genes in budding yeast. Deletion in the RAD52 gene, the product of which is involved in double-strand break/recombination repair and replication, leads to a considerable decrease in the growth rate of the chl15 mutant. We suggest that CHL15 is a new DNA synthesis gene in the yeast Saccharomyces cerevisiae.
3456	Administration of an aromatase inhibitor during the late follicular phase of gonadotropin-treated cycles in rhesus monkeys: effects on follicle development, oocyte maturation, and subsequent luteal function.	8473415	Local modulation of follicular and gametogenic functions by ovarian androgens and estrogens in mammalian species has been proposed. This study examined the effects of elevated androgen/estrogen ratios during follicular maturation in vivo by inhibiting aromatase activity in rhesus monkeys. To obviate steroid feedback effects, gonadotropin-treated animals were used. Beginning at menses (day 1), animals received human (h) FSH (60 IU/day, im) on days 1-6, followed by hFSH plus hLH (60 IU/day, im) on days 7-9 to promote the growth of multiple follicles. Ovulatory maturation was induced by hCG (1000 IU, im) on day 10. On days 8-10, four animals received an aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD; 1-1.25 g, orally, twice/day), while five served as controls and received no further treatment. Within 8 h of ATD treatment, a 63% reduction in serum estradiol levels relative to control values was evident, which reached maximal suppression (84%) by day 10. A marked elevation (17-fold) in serum androstenedione and a lesser increase (2.6-fold) in serum testosterone occurred with aromatase inhibition, yielding androstenedione/estradiol (18.0) and testosterone/estradiol (1.9) ratios greater than those in controls (0.6 and 0.3, respectively). ATD treatment did not alter follicular diameters or the total number of follicles per animal (20 +/- 3) relative to control values (16 +/- 3). Of the total cohort classified, the proportion of oocytes collected at prophase I was greater (P < 0.05) after ATD treatment (31%) than in controls (11%). Completion of oocyte meiosis to metaphase II was retarded (P < 0.05) in ATD-treated (4%) compared to control (26%) animals. Furthermore, the in vitro fertilization rate of metaphase II oocytes from ATD-treated animals (9%) was reduced (P < 0.05) relative to that in controls (25%). While basal progesterone production by luteinizing granulosa cells in vitro was similar between groups, the addition of hCG in vitro enhanced progesterone secretion by cells from ATD-treated animals (3.1 +/- 0.3-fold over basal) to a greater extent (P = 0.05) than in controls (1.5 +/- 0.3-fold). Progesterone receptor was detected by immunocytochemistry in nuclei of luteinizing granulosa cells from ATD-treated animals as well as controls. Serum progesterone profiles and the length of the luteal phase were similar between groups. Thus, acute elevation of serum androgen/estrogen ratios in vivo during follicular maturation was detrimental to the gametogenic functions of the primate follicle, but did not alter follicular growth, events of early luteinization, or subsequent luteal function.(ABSTRACT TRUNCATED AT 400 WORDS)
3457	Identification of DNA-binding protein(s) in the developing heart.	8462740	An antiserum (anti-H2) directed at the second helix of the helix-loop-helix (HLH) protein MyoD1 reacts with a protein expressed during avian cardiac myocyte differentiation. Indirect immunohistochemical whole mount staining with anti-H2 detected a protein expressed in stage 11 hearts, but not in hearts of older embryos. At the cellular level, this staining is confined to the nucleus of cardiac cells suggesting that these proteins may have DNA-binding abilities. Several proteins were immunoprecipitated by anti-H2 from stage 11 heart tissue. Protein extracts from similarly staged hearts, when incubated with the muscle-specific enhancer sequence of muscle creatinine kinase (MCK), gave a stage-specific band shift in electromobility shift assays (EMSA), and these protein-DNA complexes were recognized and supershifted by anti-H2. Incubation with a MCK sequence containing a mutated E box did not produce a shift. The specific shift was present as early as stage 6, remained through stage 13, and disappeared by stage 17. These data suggest the presence of at least one protein that is transiently expressed in the differentiating cardiac myocyte, that is immunochemically reactive with an antiserum raised against the second helix of MyoD1, and that binds to a muscle-specific DNA enhancer sequence.
3458	DNA binding by N- and L-Myc proteins.	8455937	N- and L-Myc, like c-Myc, contain adjacent basic region (BR), helix-loop-helix (HLH) and leucine zipper (LZ) motifs, which characterize a family of DNA-binding proteins. We have used a polymerase chain reaction (PCR)-based binding site selection technique to demonstrate that the most highly preferred binding site for both N- and L-Myc fusion proteins contains a CACGTG motif, the core binding sequence previously identified for c-Myc. Further analysis identified other N-Myc binding sequences, including asymmetric sequences such as CAT-GTG. N-Myc, like c-Myc, preferentially forms heterodimeric DNA-binding complexes with Max protein. Mutational analyses of N-Myc basic region (BR), helix-loop-helix (HLH) and leucine zipper (LZ) regions revealed that all three regions are necessary for DNA binding by N-Myc-Max complexes, and that dimerization requires both HLH and LZ motifs, while BR sequences are needed only for DNA binding. Our findings support the notion that the LZ motif is a critical element in dimer formation by bHLH-LZ proteins.
3459	Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene.	8455616	Pro-opiomelanocortin (POMC)-expressing cells appear to be the first pituitary cells committed to hormone production. In this work, we have identified an element of the POMC promoter which confers cell-specific activity. This element did not exhibit any activity on its own and required at least one other element of the promoter to manifest its cell-specific activity. Fine mutagenesis of this element indicated that a CANNTG motif is responsible for activity. This E-box motif is typical of binding sites for helix-loop-helix (HLH) transcription factors; however, the POMC cell-specific E box cannot be replaced by other E boxes like the kappa E2 site of the immunoglobulin gene or a muscle-specific E box. Similar E boxes which are present in the insulin gene promoter were shown to contribute to the pancreatic specificity of the insulin promoter. However, E-box-binding proteins found in nuclear extracts from POMC-expressing AtT-20 cells and from insulin-expressing cells have different electrophoretic mobilities. The AtT-20 proteins were named CUTE (for corticotroph upstream transcription element-binding) proteins, and they were not found in any other cells. CUTE proteins have DNA-binding properties characteristic of HLH transcription factors. Overexpression of the dominant negative HLH protein Id or of the ubiquitous positive HLH factor rat Pan-2 decreased or augmented POMC promoter activity, respectively. These observations are consistent with the hypothesis that CUTE factors might be heterodimers. This hypothesis was further supported by antibody shift experiments and by abrogation of DNA binding in the presence of bacterially expressed Id protein. Thus, the cell-specific CUTE proteins and their binding site in the POMC promoter appear to be important determinants for cell specificity of this promoter. The requirement for HLH factors in POMC transcription also presents the possibility that these factors are involved in differentiation of pituitary cells, in analogy with the role of HLH factors in muscle development.
3460	ME1 and GE1: basic helix-loop-helix transcription factors expressed at high levels in the developing nervous system and in morphogenetically active regions.	8261111	Several class A basic helix-loop-helix (bHLH) transcription factors have been cloned from the developing mouse and chick nervous system. The cloned cDNAs (ME1, ME2, ME3, ME4, in the mouse and GE1, GE2 in the chick) have HLH coding regions highly homologous to other known class A bHLH genes. The genes corresponding to ME1 and GE1 are abundantly expressed during development of the central nervous system. ME1 and GE1 are expressed in proliferating neuroblasts and in cells at the initial stages of differentiation (for example in the external granule cell layer of the cerebellum and in the lateral region of the ventricular zone in the developing neural tube and cortex). They are also expressed at high levels in morphogenetically active regions such as limb buds, somites and mesonephric tubules. The expression of ME1 and GE1 decreases once cellular differentiation is over. Based on the expression of ME1 and GE1 in regions of active cellular proliferation and differentiation and on the known role of other bHLH factors in development, we suggest that ME1 and GE1 play important roles during development of the nervous system as well as in other organ systems.
3461	Interactions among vertebrate helix-loop-helix proteins in yeast using the two-hybrid system.	8383120	The helix-loop-helix (HLH) motif is contained in a rapidly growing family of transcription factors and has been shown to mediate dimerization among heterologous HLH-containing proteins. E12 is a widely expressed HLH protein that preferentially forms heterodimers with cell type-specific HLH proteins such as MyoD, myogenin, and the achaete-scute gene products. As a first step toward screening for novel cell type-specific partners of E12, we used a modification of the two-hybrid assay for detection of protein-protein interactions in vivo to determine whether dimerization of HLH proteins with E12 can occur in yeast. Using the GAL4 DNA-binding domain fused to the E12 HLH motif and the GAL4 transcription activation domain fused to MyoD, we show that E12 and MyoD can efficiently dimerize in yeast and reconstruct a hybrid transcription factor that activates reporter genes linked to the GAL4 DNA-binding site. The GAL4 DNA-binding domain fused to E12 was used to screen a mouse T-cell cDNA library in which the cDNA was fused to the GAL4 activation domain. Several cDNA clones encoding proteins that interact with E12 were isolated, one of which corresponded to the HLH protein Id-2. Given the ability of E12 to dimerize preferentially with cell type-specific HLH proteins, this strategy should be useful for cloning novel partners for E12 from a variety of cell types.
3462	Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.	8441414	The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.
3463	An immediate early human gene encodes an Id-like helix-loop-helix protein and is regulated by protein kinase C activation in diverse cell types.	8437843	Transcription factors characterized by the presence of a helix-loop-helix (HLH) domain play a central role in the regulation of cell growth/differentiation and tumorigenesis. We report here the cDNA sequence of a human early-response gene, designated HLH 1R21, encoding a 15-kDa HLH protein that lacks a basic, DNA-binding domain and which by a number of criteria appears to be the human homologue of mouse HLH 462. Like its murine counterpart, HLH 1R21 protein functions as an Id (inhibitor of DNA binding) transcription factor by inhibiting the binding of E2A-containing protein complexes to muscle creatine kinase E-box enhancer oligonucleotide in vitro. However HLH 1R21 does not inhibit the binding of HLH Max protein to a Max-binding oligonucleotide in vitro, indicating that it has limited promiscuity in its ability to antagonize the function of other HLH transcription factors. In addition, HLH 1R21 mRNA transcripts are regulated by phorbol ester treatment of a diverse range of human cell lines and, when overexpressed in mouse NIH3T3 cells, HLH 1R21 induces a morphologically transformed phenotype.
3464	A subunit interaction site in human luteinizing hormone: identification by photoaffinity cross-linking.	7679977	The sites of noncovalent association between the alpha- (common) and beta- (hormone-specific) subunits in the glycoprotein hormones [LH, human CG (hCG), FSH, and TSH] remain to be completely defined. This information is essential to efforts to map the three-dimensional structure of the hormones and to help explain the stability of the hormone heterodimer in the circulation. Among numerous approaches that have been employed to identify these sites of subunit interaction, chemical or photoaffinity cross-linking has the advantage of identifying the complementary sites of contact on the respective subunits. We have used the stable photoaffinity ligand, L-benzoylphenylalanine (Bpa), to evaluate subunit interaction by the hLH beta sequence (1-15). A peptide from this region of hCG beta has been shown previously to inhibit subunit association. The 3H-labeled Bpa was incorporated into the peptide at position 8 (Trp) during solid-phase synthesis. After incubation with alpha under long-wavelength (366 nm) ultraviolet light, a Bpa- (1-15)-labeled-alpha-fraction was isolated. Control experiments showed the binding to be covalent and specific to (1-15). Two other Bpa-labeled beta-fragments, the receptor-binding loop (38-57) and the (30-43) peptide containing the highly conserved CAGY sequence, did not cross-link. The site of contact in alpha was localized by peptide mapping and sequence analysis to the N-terminal fragment (18-33), most likely at Met-29 or Gly-30. Both the alpha- and beta-sites are adjacent to or overlapping receptor-binding regions. Subunit contact sites and receptor-binding segments may thus be oriented in close proximity to provide a multicomponent receptor-binding domain imparting full activity to the native hormone.
3465	IGH minisatellite suppression of USF-binding-site- and E mu-mediated transcriptional activation of the adenovirus major late promoter.	8451197	The 50bp repeat unit of the minisatellite within the DH-JH interval of the human immunoglobulin heavy chain locus binds a nuclear factor present in a wide variety of cell types. The binding site contains the myc/HLH motif, CACGTG, and represents a 15 of 17 base match for the USF/MLTF binding site adjacent to the adenovirus major late promoter (MLP). Unlike the USF/MLTF site, the IGH minisatellite possesses no enhancer activity. However, it can significantly suppress, in cis and in trans, USF-site-mediated transcriptional activation of the MLP. In murine myeloma cells, the IGH minisatellite can suppress, in trans, MLP activation by the murine heavy chain gene enhancer, E mu. These activities potentially represent a DNA-based form of squelching.
3466	High affinity DNA-binding Myc analogs: recognition by an alpha helix.	8431949	Myc and other basic-helix-loop-helix-leucine zipper (b-HLH-ZIP) proteins bind the sequence CACGTG. Exhaustive mutagenesis in the basic domain identified four amino acids critical for DNA binding with spacing suggestive of an alpha-helical face. Surprisingly, two highly conserved amino acids were nonessential for DNA binding. Circular dichroism demonstrated a DNA-induced alpha-helical transition. A series of analogs were constructed with multiple simultaneous alanine substitutions at nonessential positions and a critical lysine for arginine substitution. In this way 35-fold higher specific affinity for CACGTG was obtained as compared with the basic domain of c-Myc. These b-HLH-ZIP proteins appear to bind the same palindromic sequence and may compete for common sites in vivo. Additionally, a C-terminal basic region clamp motif was identified that was also identifiable in crystal structures from several different families of DNA-binding factors.
3467	Characterization of a human chorionic gonadotropin-like protein from Candida albicans.	8425486	Studies from our laboratory have demonstrated that human CG (hCG), human LH, (hLH), and an hCG-like protein extracted from Xanthomonas maltophilia were able to induce Candida albicans transition from the blastospore to the germ tube stage. In the present study, we describe the characterization of an hCG-like material extracted from Candida albicans blastospores (CaCGLP), which is potent in inducing transition and presumably represents the endogenous transition-inducing substance. This material was extracted from Candida albicans blastospores with glacial acetic acid and purified by affinity chromatography using a polyclonal rabbit anti-hCG antibody. The product obtained is a 68-kilodalton single band protein, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Under reduced conditions a protein smear is seen. Amino acid analysis showed a predominance of glycine (22%), followed by serine (12%), and glutamate (12%). This protein reacted in the following hCG immunoassays: 1) a polyclonal rabbit anti-hCG equilibrium assay, 2) a carboxyl-tail hCG equilibrium assay, 3) two hCG equilibrium assays using monoclonal antibodies (CG no. 4 and CG no. 9), 4) a free alpha-subunit equilibrium assay using a monoclonal antibody, and 5) an ultrasensitive immunoradiometric assay for hCG which does not cross-react with hLH, nor the free beta-subunit of hCG. The CaCGLP showed no reaction in a specific hLH immunoradiometric assay. When CaCGLP was tested in the transition assay, in the presence of 4% rat serum, it was found that this protein was 100 times more potent than hCG in producing Candida albicans transition. We conclude that Candida albicans produces a protein that has certain tertiary structure similarities to hCG and that this material is able to induce germ tube formation. We postulate that CaCGLP has an autocrine/paracrine effect in Candida albicans as a transition factor to control its own pathogenicity.
3468	Native and modified (acetylated) low density lipoprotein-supported steroidogenesis by macaque granulosa cells collected before and after the ovulatory stimulus: correlation with fluorescent lipoprotein uptake.	8425479	We recently reported that uptake of fluorescent-tagged low density lipoprotein (DiI-LDL) by macaque granulosa cells (GC) was greatly enhanced within 27 h of an ovulatory stimulus (hCG injection). The present study was designed to determine whether increased DiI-LDL uptake correlated with an increased capacity for LDL-supported steroidogenesis. We also tested whether modified [acetylated (ac)] LDL or high density lipoprotein (HDL), which are not ligands for the LDL receptor, supported progesterone (P) production. Beginning at menses, adult female rhesus macaques were treated with human (h) FSH and hLH for 9 days to promote development of multiple follicles. On day 10, monkeys were injected with hCG (1000 IU) or received no ovulatory stimulus. Large follicles were aspirated on day 10 (nonluteinized GC) or 27-34 h after hCG administration (luteinizing GC). GC (2 x 10(4)/0.2 ml) were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 plus insulin, transferrin, H2SeO3, and aprotinin, with 0-100 micrograms hLDL, ac-hLDL, or hHDL. P concentrations in medium were determined by RIA. LDL (1-25 micrograms/ml) dose-dependently increased (up to 15-fold; P < 0.05) basal and hCG-stimulated P production by luteinized GC on days 1-8 of culture. However, LDL (25 micrograms/ml) did not alter basal P production by nonluteinized GC and increased (2-fold; P < 0.05) hCG-stimulated P secretion only on days 4-8. Basal and hCG-stimulated P production by luteinized GC were initially (days 1-2) increased (up to 2-fold; P < 0.05), but later (days 6-8) suppressed (P < 0.05) in a dose-dependent manner by 1-100 micrograms ac-LDL/ml. Ac-LDL did not alter basal or hCG-stimulated P production by nonluteinized GC. HDL (1-100 micrograms/ml) did not alter P production by either luteinized or nonluteinized GC. The number of viable luteinized GC on day 8 was reduced (30-50%; P < 0.05) after exposure to 10 micrograms ac-LDL/ml or more, whereas only the highest dose (100 micrograms/ml) of LDL or HDL reduced cell survival. Ac-LDL did not alter the survival of nonluteinized GC in culture. Flow cytometric analyses using fluorescent-tagged lipoproteins (DiI-LDL/DiI-ac-LDL) demonstrated the uptake of both native and ac-LDL by luteinized GC. Uptake of DiI-LDL was competitively suppressed in a dose-dependent manner by unlabeled LDL, but not by ac-LDL. In contrast, DiI-ac-LDL uptake was competitively inhibited by both ac-LDL and LDL.(ABSTRACT TRUNCATED AT 400 WORDS)
3469	Deglycosylated human chorionic gonadotropin (hCG) antagonizes hCG stimulation of 3',5'-cyclic adenosine monophosphate accumulation through a noncompetitive interaction with recombinant human luteinizing hormone receptors.	8381073	In the rat, the antagonistic properties of deglycosylated (dg) gonadotropins in vitro are characterized by high affinity receptor binding but impaired ability to stimulate cAMP accumulation. In human, the functional role of N-linked sugars in human CG (hCG) action is unclear because of the unavailability of totally deglycosylated hCG and because of the difficulty involved in obtaining human gonadal tissues. We have recently prepared completely deglycosylated hCG using site-directed mutagenesis and expressed functional human LH (hLH) receptors using cloned complementary DNA. Since hLH receptor shows distinct ligand specificity from that of rat LH receptor, we examined binding kinetics and signal transduction of recombinant dg-hCG using recombinant hLH receptors. In embryonic human kidney cells (293) transfected with hLH receptor complementary DNA, 125I-hCG binding to its receptor was studied in the presence of varying amounts of unlabeled dg-hCG or wild type (WT)-hCG. Lineweaver-Burk analysis of the binding kinetics showed that the displacement of 125I-hCG by dg-hCG was noncompetitive whereas that seen for WT-hCG was competitive. The noncompetitive nature of dg-hCG binding was further confirmed using rat LH receptors present in testis membrane preparations. After preincubation of LH receptor-expressing 293 cells with WT-hCG, inclusion of 125I-hCG competitively displaced WT-hCG. In contrast, preincubation with dg-hCG prevented subsequent 125I-hCG binding to human LH receptor for at least 46 h. WT-hCG caused a dose-dependent increase in cAMP accumulation in the 293 cells with an ED50 of 10 ng/ml. However, dg-hCG was ineffective in inducing cAMP production with a maximal effect of only 12% of that stimulated by WT-hCG. In the presence of increasing doses of dg-hCG, stimulation of cAMP by WT-hCG was antagonized in a dose-dependent manner. In contrast, forskolin stimulation of cAMP was not antagonized by dg-hCG, indicating receptor-mediation of dg-hCG action. Similar to binding studies, preincubation with dg-hCG also dose-dependently blocked the subsequent stimulatory effect of WT-hCG on cAMP production. Thus, the noncompetitive binding of dg-hCG to hLH receptors and its antagonism of hCG stimulation of cAMP accumulation suggest that dg-hCG is an irreversible receptor blocker with unique antagonistic properties.
3470	Site-selected insertion of the transposon Tc1 into a Caenorhabditis elegans myosin light chain gene.	8380898	We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.
3471	Molecular homology among members of the R gene family in maize.	8220447	The R gene family determines the timing, distribution and amount of anthocyanin pigmentation in maize. This family comprises a set of regulatory genes, consisting of a cluster of several elements at the R locus, on chromosome 10, the Lc and Sn gene lying about two units R distal and B on chromosome 2. Each gene determines a tissue-specific pigmentation of different parts of the seed and plant. The proposed duplicated function of R, Sn, Lc and B loci is reflected in cDNA sequence similarity. In this paper an extensive analysis of the predicted proteins of the R, Sn, Lc and B genes together with a search for putative sites of post-translational modification is reported. A comparison with the prosite database discloses several N-glycosylation and phosphorylation sites, as well as the basic Helix-Loop-Helix (HLH) domain of transcriptional activators. Sn, Lc, and R-S show a high conservation of these sites, while B is more divergent. Analysis of the 5' leader of mRNA sequences discloses the presence of five ATG triplets with two upstream open reading frames (uORFs) of 38 and 15 amino acids and a loop structure indicating a possible mechanism of control at the translational level. It is conceivable that possible mechanisms acting at the translational and post-translational level could modulate the expression and the activation of these transcription factors. Northern analysis of various tissues of different R alleles highlights a strict correlation between pigment accumulation in different tissues and the expression of the regulatory and structural genes suggesting that the pattern of pigmentation relies on a mechanism of differential expression of the members of the R family. Analysis of the Sn promoter discloses the presence of several sequences resembling binding sites of known transcription factors (as GAGA and GT) that might be responsible for the spatial and light-induced expression of this gene. Two regions include a short sequence homologous to the consensus binding site of the B-HLH domain suggesting a self-regulatory control of the Sn gene.
3472	Partial nucleotide sequence of the Xanthomonas maltophilia chorionic gonadotropin-like receptor.	8427582	Xanthomonas maltophilia possesses a unique high-affinity binding site which binds human chorionic gonadotropin (hCG), but not human luteinizing hormone (hLH) or other glycoprotein hormones. We designed primers from the known nucleotide sequence of the human LH/CG receptor, spanning an area extending from transmembrane region 2 to transmembrane region 6. Genomic DNA extracted from Xanthomonas maltophilia was used to obtain a PCR amplified product using the above primers. The primary amplification product was cloned in a pCR11 TA cloning vector, and the partial nucleotide sequence of the gene determined. This determined sequence showed 73% identity with the human, as well as the rat LH/CG receptor. Comparison of the translated protein sequence with the human, rat and porcine receptor protein sequences showed a 52% similarity.
3473	Oncogenic activity of the c-Myc protein requires dimerization with Max.	8425220	c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs (b-HLH-LZ). Using a genetic approach, we demonstrate that binding to Max is essential for Myc transforming activity and that Myc homodimers are inactive. Mutants of Myc and Max that bind efficiently to each other but not to their wild-type partners were generated by either exchanging the HLH-LZ domains or reciprocally modifying LZ dimerization specificities. While transformation defective on their own, complementary mutants restore Myc transforming activity when coexpressed in cells. The HLH-LZ exchange mutants also have dominant negative activity on wild-type Myc function. In addition, wild-type max antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and Myc-Max dimers for common target DNA sites. Therefore, Max can function as both suppressor and activator of Myc. A general model for the role of Myc and Max in growth control is discussed.
3474	Detection and modulation in vivo of helix-loop-helix protein-protein interactions.	8380166	Studies are described that allow for the in vivo detection of helix-loop-helix (HLH) protein-protein interaction. The assay used requires HLH protein-protein interaction to reconstitute a functional GAL4 transcriptional activator, which in turn activates a reporter gene placed downstream of GAL4 DNA binding sequences. Using this assay, we are able to detect intracellular heterodimerization but not homodimerization of the MyoD, E12, and Id gene products. In addition, using this system we are unable to detect stable heterodimerization between MyoD and c-Jun. We also show that expression of activated rasH gene product does not inhibit and may stabilize HLH protein-protein interaction. This system may be of general utility in studying the modulation of transcription factor interactions.
3475	A functional analysis of the genes Enhancer of split and HLH-m5 during early neurogenesis in Drosophila melanogaster.	28305975	To assess the functional domains of the proteins encoded by E(spl) and HLH-m5, two genes of the Enhancer of split complex [E(SPL)-C] of Drosophila melanogaster, a number of variants have been made by in vitro mutagenesis, transformed into the germ line of the wild-type, and genetically combined with a chromosomal deletion lacking four of the genes of the E(SPL)-C. All constructs used attenuated the neurogenic phenotype associated with this deletion. However, constructs encoding proteins with truncated carboxy-termini exibited in all cases a higher activity than constructs encoding the full length version of the protein. Neutralization of the basic domain severely reduced, but did not completely abolish the rescuing activity of E(spl), while proteins in which a proline residue within the basic domain had been changed to either threonine or asparagine were slightly less efficient in their rescuing activity than the corresponding wild-type versions. We discuss the possible significance of these results for the function of the protein domains.
3476	Proposed structure for the DNA-binding domain of the helix-loop-helix family of eukaryotic gene regulatory proteins.	8433970	A modelled tertiary structure for the dimeric HLH domain of the E47 protein is presented. Structural information was obtained from the aligned sequences of > 40 members of the HLH family. The information was used to model each monomer as an alpha-helical hairpin, with knobs-into-holes packing of side-chains as found in antiparallel coiled-coil. The dimer forms a four-helix bundle with additional knobs-into-holes packing at the dimer interface. The size and electrostatic properties of core-forming residues are all accounted for in the model. The model does not violate any known properties of protein structure. The monomers are related by two-fold rotational symmetry, in agreement with the observed DNA-binding sites which are imperfect inverted repeats. The N-terminal basic region, in which DNA binding and base specificity reside, forms the first part of helix 1. A prediction based on the model structure is that the HLH domains do not bind to DNA in its B form but require a partially unwound conformation in order to enter the major groove.
3477	The B-HLH protein encoding the M-twist gene is located by in situ hybridization on murine chromosome 12.	8431638	NaN
3478	Both the helix-loop-helix and the leucine zipper motifs of c-Myc contribute to its dimerization specificity with Max.	8423990	The oncoprotein c-Myc contains two dimerization motifs- the helix-loop-helix (HLH) and the leucine zipper (LZ) - through which c-Myc specifically dimerizes with Max. We substituted regions of the c-Myc HLH and LZ motifs with the corresponding regions of structurally related proteins that do not interact with Max. Specific association of c-Myc with Max was dictated by helices 1 and 2 of the HLH motif and by the LZ. Within helix 2, residues 6 and 7 were important determinants of dimerization specificity. c-Myc and Max proteins with three amino acids inserted between their HLH and LZ motifs interacted efficiently, suggesting that the spacing between the motifs can be varied. Furthermore, alignment of the motifs at the primary sequence level was not obligatory, since Max with the three amino acid insertion could interact with wild-type c-Myc. These findings are consistent with our recently proposed model for the architecture of HLH/LZ domains.
3479	A rat gene with sequence homology to the Drosophila gene hairy is rapidly induced by growth factors known to influence neuronal differentiation.	8417318	Several genes encoding transcription factors with a helix-loop-helix (HLH) motif are involved in the early process of neural development in Drosophila spp. We report the isolation from the rat a homolog of one of these genes, called hairy. The rat-hairy-like (RHL) gene is expressed early during embryogenesis. In contrast to the restricted expression of hairy mRNA in Drosophila spp., however, the mRNA encoded by RHL is detectable in all tissues examined. Stimulation of PC12 pheochromocytoma cells by nerve growth factor, basis fibroblast growth factor, or epidermal growth factor or of Rat-1 fibroblasts by epidermal growth factor causes a rapid and transient induction of the RHL gene. Thus, RHL acts as an immediate-early gene that can potentially transduce growth factor signals during the development of the mammalian embryo.
3480	Biological activity of partially purified low molecular weight luteinizing hormone binding inhibitor in porcine follicular fluids.	8383399	We performed partial purification of low molecular weight luteinizing hormone binding inhibitor from porcine follicular fluids and examined its biological activities. Following ultrafiltration, gel filtration and anion exchange of the pooled porcine follicular fluids, low molecular weight fractions (500-10,000 MW) inhibited [125I]hLH binding to porcine granulosa cells in a dose-dependent manner. The binding inhibition kinetics study revealed that the luteinizing hormone binding inhibitor may indicate a non-competitive inhibition with [125I]hLH binding. In vitro bioassay using adult mouse testicular interstitial cells revealed that the partially purified luteinizing hormone binding inhibitor reduced ovine LH-stimulated testosterone and cAMP production in a dose-dependent manner, whereas the luteinizing hormone binding inhibitor did not affect basal production of testosterone and cAMP. The inhibitory activity was heat stable and did not disappear with activated charcoal adsorption. The results of the present study suggest that the luteinizing hormone binding inhibitor may play an important role as an ovarian non-steroidal regulator modulating the receptor binding of LH and LH-mediated steroidogenesis.
3481	A Schizosaccharomyces pombe gene that promotes sexual differentiation encodes a helix-loop-helix protein with homology to MyoD.	8381348	Nitrogen starvation of Schizosaccharomyces pombe induces a differentiated state in which haploid cells mate and sporulate. esc1+, a newly isolated S.pombe cDNA that promotes this sexual differentiation, encodes a putative transcription factor with a helix-loop-helix (HLH) motif similar to those of the human MyoD and Myf-5 myogenic differentiation inducers. Disruption of esc1+ in wild-type cells leads to a decrease in the efficiency of sexual conjugation, an early step in sexual differentiation. The disruption was also able partially to substitute for cAMP, an inhibitor of differentiation, to suppress the lethal, constitutive differentiation induced by the pat1 mutation. Conversely, overexpression of this cDNA conferred partial resistance to cAMP-mediated inhibition of differentiation. Transcription from this novel gene was induced early in response to nitrogen starvation and is largely independent of the ste11+ gene product, which is required for the differentiation-specific expression of other genes. Thus, this MyoD/Myf-5-like protein appears to promote sexual differentiation by modulating responses to decreases in cAMP, a part of the nitrogen starvation signal that induces differentiation.
3482	Regulation of muscle transcription by the MyoD family. The heart of the matter.	8380257	The two striated muscle cell types, skeletal and cardiac muscle, express overlapping sets of muscle-specific genes. Activation of muscle-specific transcription in skeletal muscle is controlled by the MyoD family of regulatory factors, which are expressed exclusively in skeletal muscle. Members of the MyoD family share homology within a basic helix-loop-helix (HLH) motif that mediates DNA binding and dimerization and form heterodimers with widely expressed HLH proteins, referred to as E proteins. Although many of the genes that are regulated by members of the MyoD family are also expressed in cardiac muscle, known members of the MyoD family have never been detected in cardiac muscle, suggesting that cardiac myocytes either express unique cell type-specific HLH proteins or rely on a distinct regulatory strategy for activation of cardiac muscle transcription. This review will summarize current knowledge of the mechanisms through which the MyoD family activates skeletal muscle transcription and will consider potential mechanisms that may regulate gene expression in the heart.
3483	In vitro effects of ethanol on gonadotropin-binding sites in porcine granulosa cells.	8307112	In this study, we examined the in vitro effects of ethanol on [125I]human follicle-stimulating hormone (hFSH) and [125I]human luteinizing hormone (hLH) binding to porcine granulosa cells (GCs). The GC suspensions and [125I]hFSH or [125I]hLH were incubated in the presence or absence of 1-50% (vol/vol) ethanol until steady state binding was attained for 2 h at 37 degrees C. The presence of 1-4% ethanol significantly enhanced the specific binding of hFSH to porcine GCs, with a maximal effect at 4% ethanol, however, had no effect on hLH binding. Higher concentration of ethanol (8-50%) significantly decreased both specific hFSH and hLH binding to porcine GCs. Scatchard analyses revealed that ethanol increased the number of FSH-binding sites without changing the affinity for FSH. The data suggest that porcine GCs contain a population of masked FSH-binding sites exposed by in vitro treatment with ethanol. Furthermore, the membrane fluidity may play an important role in the masking/unmasking mechanism of FSH-binding sites within the porcine GCs.
3484	X-ray crystallographic studies of eukaryotic transcription factors.	7956022	Our X-ray crystallographic work on eukaryotic transcription factors traverses the entire length of a typical class II nuclear gene promoter, including the TATA-box-binding protein, two b/HLH/Z promoter proximal binding factors (Max and USF), and an enhancer-binding factor (HNF-3 gamma). These high-resolution studies of specific protein/DNA interactions will be extended to include homologous proteins complexed with similar oligonucleotides and a systematic examination of the roles of individual amino acids and bases implicated in specific DNA binding. In the longer term, we will turn our attention to the myriad of protein/protein interactions occurring within TFIID and the PIC, and between the PIC and factors binding to promoter proximal and distal enhancer elements.
3485	FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains.	1335366	Myogenin belongs to a family of myogenic helix-loop-helix (HLH) proteins that activate muscle transcription through binding to a conserved DNA sequence associated with numerous muscle-specific genes. Fibroblast growth factor (FGF) inhibits myogenesis by inactivating myogenic HLH proteins. We show that activated protein kinase C (PKC) can substitute for FGF and inhibit transcriptional activity of myogenic HLH proteins. In transfected cells, FGF induces phosphorylation of a conserved site in the DNA-binding domain of myogenin. This site is phosphorylated by PKC in vivo and in vitro and mediates repression of the myogenic program through a loss in DNA binding activity. A myogenin mutant lacking the PKC phosphorylation site is not repressed by FGF, confirming this site as a molecular target for FGF-dependent repression of muscle transcription. These results establish a direct link between the signal transduction pathways that inhibit myogenesis and the transcription factors directly activating muscle-specific genes.
3486	Helix-loop-helix transcription factors E12 and E47 are not essential for skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, or neurogenesis.	1465450	E12 and E47 are two non-tissue-specific helix-loop-helix (HLH) transcription factors encoded by the E2A gene. Previous studies suggested that they are involved in regulation of differentiation in many tissue types including muscle, blood, and nerve through direct heterodimer interactions with tissue-specific HLH proteins. To gain further genetic insight into the functions of E12 and E47 during cell differentiation, we mutated both copies of the E2A gene in mouse embryonic stem (ES) cells and then tested the effect on differentiation in vitro. We find that the ES cells lacking functional E12 and E47 are capable of differentiating into both skeletal and cardiac muscle, erythrocytes, neurons, and cartilage that the same extent as wild-type cells. These results indicate that the E2A gene is not essential for differentiation of these cell types and suggest that redundant genes may control these developmental pathways.
3487	Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.	1464664	Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.
3488	Biphasic effect of Max on Myc cotransformation activity and dependence on amino- and carboxy-terminal Max functions.	1459463	In Ras cotransformation assays, Max exhibited a biphasic effect on Myc transformation activity. Cotransfection of low levels of Max expression plasmid stimulated Myc transformation activity, but cotransfection of high levels suppressed it. Mutations in the functionally undefined Max amino- and carboxy-terminal regions outside of the B/HLH/LZ motif partly separated these activities, suggesting various modes of Max regulation. We demonstrate that the Max protein is a nuclear protein in vivo and identify a carboxy-terminal region similar to nuclear localization signals whose integrity is necessary for efficient localization. Two mutants that delete amino- or carboxy-terminal consensus signals for casein kinase II (CKII) exhibited altered gel mobility and DNA-binding potential in vitro and showed modified transforming potential in the Ras cotransformation assay, suggesting that CKII or a CKII-related enzyme may regulate Max function in vivo. Our data suggest that both the ratio of Myc/Max hetero-oligomers to Max homo-oligomers and Max-specific regulation can contribute to determining the biological activity of Myc in vivo.
3489	Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split.	1340473	We report the molecular characterization of two novel rat helix-loop-helix (HLH) proteins, designated HES-1 and HES-3, that show structural homology to the Drosophila hairy and Enhancer of split [E(spl)] proteins, both of which are required for normal neurogenesis. HES-1 mRNA, expressed in various tissues of both embryos and adults, is present at a high level in the epithelial cells, including the embryonal neuroepithelial cells, as well as in the mesoderm-derived tissues such as the embryonal muscle. In contrast, HES-3 mRNA is produced exclusively in cerebellar Purkinje cells. HES-1 represses transcription by binding to the N box, which is a recognition sequence of E(spl) proteins. Interestingly, neither HES-1 nor HES-3 alone interacts efficiently with the E box, but each protein decreases the transcription induced by E-box-binding HLH activators such as E47. Furthermore, HES-1 also inhibits the functions of MyoD and MASH1 and effectively diminishes the myogenic conversion of C3H10T1/2 cells induced by MyoD. These results suggest that HES-1 may play an important role in mammalian development by negatively acting on the two different sequences while HES-3 acts as a repressor in a specific type of neurons.
3490	Spatial regulation of proneural gene activity: auto- and cross-activation of achaete is antagonized by extramacrochaetae.	1340471	The spatially restricted activities of achaete (ac) and scute (sc) are thought to define proneural clusters of potential sensory organ precursor cells in the imaginal discs of Drosophila. These genes encode transcriptional regulators of the basic helix-loop-helix (bHLH) class. We have found that direct, positive transcriptional autoregulation by the ac protein and cross-regulation by sc are essential for high-level expression of the ac promoter in the proneural cluster pattern and that autoactivation is important for the bristle-promoting function of the ac gene. These auto- and cross-regulatory activities are antagonized in a dose-dependent manner by the inhibitory HLH protein encoded by the extramacrochaetae (emc) gene. We have found that emc is expressed in the wing imaginal disc in a pattern strongly complementary to that of the proneural clusters. Our results indicate that emc plays an essential early role in defining territories of bristle-forming potential.
3491	Assaying glycoprotein hormones--the influence of glycosylation on immunoreactivity.	1283302	The epitopes of the human glycoprotein hormones (follicle-stimulating hormone [hFSH], luteinizing hormone [hLH], chorionic gonadotrophin [hCG], thyroid-stimulating hormone [hTSH] and erythropoietin [hEPO]) appear to consist only of peptide components. Their interactions with antibodies, however, are influenced by their bulky and often highly charged carbohydrate moieties. Thus, isoforms of these hormones (the majority of which are glycoforms) differ in their specific immunoreactivities as well as in their specific in vivo and in vitro bioactivities. This can create difficulties for the standardization of immunoassays as the isoform composition of a hormone depends both on its source and method of isolation.
3492	Enzyme-linked immunosorbent assay for rat and human luteinizing hormone.	1488099	A sensitive and specific enzyme-linked immunosorbent assay (ELISA) suitable for measuring rat and human luteinizing hormone (LH) is described. The LH-ELISA used anti-bLH beta subunit monoclonal antibody-coated plates, an antiserum against rLH or hLH and an antibody against rabbit IgG labelled with peroxidase. Using rLH-RP-3 or hLH WHO IRP 68/40 as standard diluted in assay buffer, the LH-ELISA had a sensitivity of 50 pg/ml and 0.78 mIU/ml, respectively. The LH-ELISA allowed accurate determination of LH in buffer, cell culture media, anterior pituitary extracts and sera and was highly specific for rat and human LH. The use of this ELISA offers improvements in convenience, economy, sensitivity and safety over comparable radioimmunoassay procedures.
3493	Definition of the transcriptional activation domain of recombinant 43-kilodalton USF.	1406684	The cellular transcription factor USF is involved in the regulation of both cellular and viral genes and consists of 43- and 44-kDa polypeptides which independently show site-specific DNA binding. Cloning of the corresponding cDNA revealed that the 43-kDa polypeptide (USF43) is a member of the basic (B)-helix-loop-helix (HLH)-leucine zipper (LZ) family of proteins and provided a means for its functional dissection. Initial structure-function studies revealed that the HLH and LZ regions are both important for USF43 oligomerization and DNA binding. The studies presented here have focused on the determination of domains that contribute to transcriptional activation in vitro and show that (i) both a small region close to the N terminus and a region between residues 93 and 156 contribute strongly to transcriptional activation, (ii) full activation depends on the presence of both domains, (iii) the B-HLH-LZ region has no intrinsic activation potential but DNA binding is absolutely required for transcriptional activation, and (iv) the B-HLH-LZ region can be replaced by the Gal4 DNA binding domain without loss of activation potential.
3494	Identification of restriction-fragment length polymorphisms for the human chorionic gonadotropin-beta/luteinizing hormone-beta gene cluster.	1385225	To determine whether restriction fragment length polymorphisms are present using a deoxyribonucleic acid (DNA) probe for human luteinizing hormone beta subunit (hLH-beta). If the gene for hLH-beta is polymorphic, genetic diagnosis of disorders of luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) production could become possible.Study of genomic DNA from controls with a variety of restriction enzymes to identify polymorphisms.Laboratories of the Department of Obstetrics and Gynecology, Department of Oral Biology, Medical College of Georgia, Augusta, Georgia.Unrelated control men and women seen in clinics at the Medical College of Georgia.Genomic DNA was extracted from patients and digested with eight different restriction enzymes for the study of the hLH-beta gene by Southern analysis.Fragment (band) sizes on radiographs from Southern blots were compared with those from molecular weight standards.Restriction fragment length polymorphisms were identified for four of the restriction enzymes, DraI, HincII, MboI, and KpnI. These polymorphisms may be useful in the diagnosis of disorders of hLH and hCG production.
3495	Sodium butyrate inhibits myogenesis by interfering with the transcriptional activation function of MyoD and myogenin.	1328872	Sodium butyrate reversibly inhibits muscle differentiation and blocks the expression of many muscle-specific genes in both proliferating myoblasts and differentiated myotubes. We investigated the role of the basic helix-loop-helix (bHLH) myogenic determinator proteins MyoD and myogenin in this inhibition. Our data suggest that both MyoD and myogenin are not able to function as transcriptional activators in the presence of butyrate, although both apparently retain the ability to bind DNA. Transcription of MyoD itself is extinguished in butyrate-treated myoblasts and myotubes, an effect that may be due to the inability of MyoD to autoactivate its own transcription. We present evidence that the HLH region of MyoD is essential for butyrate inhibition of MyoD. In contrast to MyoD and myogenin, butyrate does not inhibit the ubiquitous basic HLH protein E2-5 from functioning as a transcriptional activator.
3496	Contraceptive potential of an antiprogestin ZK 98.734: reversal of ZK 98.734--induced blockade of folliculogenesis with FSH and LH and their differential effects in bonnet monkeys.	1293045	Studies were undertaken in adult bonnet monkeys to investigate whether treatment with an antiprogestin ZK 98.734 at weekly intervals, starting from day one of menstrual cycle, could arrest ovulation and also to determine if ZK 98.734 induced blockade of ovulation could be reversed with gonadotropins. Adult animals have ovulatory menstrual cycles of normal duration were treated at weekly intervals with ZK 98.734 (25 mg/dose, sc, oil base) for 10 consecutive weeks and its effects on serum levels of estradiol, bioactive LH and progesterone, and endometrial histology were investigated. Following treatment with the antiprogestin they were treated with hMG or hFSH alone. Ovulation was blocked during treatment period in all the animals (n = 14). Typical follicular phase rise in estradiol levels was inhibited, mid cycle surge in the levels of bioactive LH was abolished and serum progesterone levels remained below 1 ng/ml throughout the treatment period. However, prolonged treatment had no significant effect on the basal levels of estradiol which were around 50 pg/ml. ZK 98.734 also had no significant effect on cortisol levels. In animals (n = 4) followed for recovery after the last dose, the treatment cycle length was increased to 117.8 + 6.8 days. In three animals the treatment cycles were anovulatory, whereas in one delayed ovulation with luteal insufficiency was observed. The endometrium had become atrophic. Treatment with hMG (Pergonal: 35 I.U. hLH and 35 I.U. hFSH) or hFSH (Metrodin, 35 I.U.) for 7 consecutive days initiated folliculogenesis and the animals ovulated either spontaneously or after a single im injection of hCG (100 I.U.) on day 8 in ZK 98.734 treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)
3497	Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death.	1423602	The Myf-5 gene, a member of the myogenic basic HLH factor family, has been inactivated in mice after homologous recombination in ES cells. Mice lacking Myf-5 were unable to breathe and died immediately after birth, owing to the absence of the major distal part of the ribs. Other skeletal abnormalities, except for complete ossification of the sternum, were not apparent. Histological examination of skeletal muscle from newborn mice revealed no morphological abnormalities. Northern blot analysis demonstrated normal levels of muscle-specific mRNAs including MyoD, myogenin, and Myf-6. However, the appearance of myotomal cells in early somites was delayed by several days. These results suggest that while Myf-5 plays a crucial role in the formation of lateral sclerotome derivatives, Myf-5 is dispensable for the development of skeletal muscle, perhaps because other members of the myogenic HLH family substitute for Myf-5 activity.
3498	Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development.	1330322	The myogenic basic HLH transcription factor family of genes, composed of MyoD, myogenin, Myf-5, and Myf-6, are thought to regulate skeletal muscle differentiation. To understand the role of MyoD in myogenesis, we have introduced a null mutation of MyoD into the germline of mice. Surprisingly, mice lacking MyoD are viable and fertile. Histological examination of skeletal muscle failed to reveal any morphological abnormalities in these mice. Furthermore, Northern analysis revealed normal levels of skeletal muscle-specific mRNAs. Significantly, Myf-5 mRNA levels are elevated in postnatal mutant mice. Normally, Myf-5 expression becomes markedly reduced at day 12 of gestation when MyoD mRNA first appears. This suggests that Myf-5 expression is repressed by MyoD. Our results indicate that MyoD is dispensable for skeletal muscle development in mice, revealing some degree of functional redundancy in the control of the skeletal myogenic developmental program.
3499	Molecular characterization of a rat negative regulator with a basic helix-loop-helix structure predominantly expressed in the developing nervous system.	1400497	We report here the cDNA cloning and characterization of a rat basic helix-loop-helix (HLH) factor, designated HES-5. This factor has a distant sequence homology to Drosophila hairy and Enhancer-of-split proteins, both of which are required for normal neurogenesis. DNase I footprinting analyses show that HES-5 binds to the sequence CACNAG (called N box), a recognition sequence of Enhancer-of-split proteins. Although HES-5 does not bind to the sequence CANNTG (called E box) recognized by other HLH factors, it attenuates the binding of E47, an HLH activator, to E box by forming a hetero-oligomer. In cotransfection analyses using NIH 3T3 cells, HES-5 significantly represses transcription originating from the promoter containing the N box sequences. Furthermore, HES-5 also partially inhibits the E47-induced expression from the promoter containing E boxes. Northern blot, RNase protection, and in situ hybridization analyses demonstrate that the HES-5 mRNA is specifically expressed in the nervous system. Prominent expression is observed in the ventricular zones of the embryonal brain vesicles and the outer nuclear layer of the neural retina. These results suggest that the negative regulator HES-5 may play an important role in neural development.
3500	Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD.	1327135	The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family. A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells. The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure. There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy. Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity. Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy. Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure. Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer. These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions. Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints. Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer. This topology is hypothesized to serve as a general model for other bHLH protein domains.
3501	Seven genes of the Enhancer of split complex of Drosophila melanogaster encode helix-loop-helix proteins.	1427040	Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells.
3502	The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells.	1427039	The Enhancer of split complex [E(spl)-C] of Drosophila melanogaster is located in the 96F region of the third chromosome and comprises at least seven structurally related genes, HLH-m delta, HLH-m gamma, HLH-m beta, HLH-m3, HLH-m5, HLH-m7 and E(spl). The functions of these genes are required during early neurogenesis to give neuroectodermal cells access to the epidermal pathway of development. Another gene in the 96F region, namely groucho, is also required for this process. However, groucho is not structurally related to, and appears to act independently of, the genes of the E(spl)-C; the possibility is discussed that groucho acts upstream to the E(spl)-C genes. Indirect evidence suggests that a neighboring transcription unit (m4) may also take part in the process. Of all these genes, only gro is essential; m4 is a dispensable gene, the deletion of which does not produce detectable morphogenetic abnormalities, and the genes of the E(spl)-C are to some extent redundant and can partially substitute for each other. This redundancy is probably due to the fact that the seven genes of the E(spl)-C encode highly conserved putative DNA-binding proteins of the bHLH family. The genes of the complex are interspersed among other genes which appear to be unrelated to the neuroepidermal lineage dichotomy.
3503	Mutational analysis of Max: role of basic, helix-loop-helix/leucine zipper domains in DNA binding, dimerization and regulation of Myc-mediated transcriptional activation.	1408152	The Max protein forms a heterodimeric complex with the Myc family of proteins and binds to DNA in a sequence-specific manner. We investigated the role of the helix-loop-helix (HLH), leucine zipper (LZ) and basic domains of Max in protein complex formation, DNA-binding activity and transcriptional regulation. We mutagenized the basic, HLH and LZ domains of Max and studied the ability of the normal and mutant proteins to bind to DNA as both homo- and heterodimers and their ability to heterodimerize with Myc. Helix-1 and helix-2 regions of Max were found to be critical for homodimer formation and subsequent DNA binding, while the LZ was essential for heterodimer formation. In transient transfection assays the Myc protein functioned as a transcriptional activator while Max protein repressed the trans-activation observed with Myc.
3504	Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max.	1406955	The c-myc protein (Myc) contains an amino-terminal transcriptional activation domain and a carboxy-terminal basic helix-loop-helix-leucine zipper (bHLH-Z) domain that directs dimerization of Myc with its partner, the max protein (Max), and promotes DNA binding to sites containing a CACGTG core consensus sequence. Despite these characteristics and the observation that Myc can modulate gene expression, a direct role for Myc or Max as transcription factors has never been demonstrated. Here we use Saccharomyces cerevisiae as an in vivo model system to show that the Myc protein is a sequence-specific transcriptional activator whose DNA binding is strictly dependent on dimerization with Max. Transactivation is mediated by the amino-terminal domain of Myc. We find that Max homodimers bind to the same DNA sequence as Myc+Max but that they fail to transactivate and thus can antagonize Myc+Max function. We also show that the Max HLH-Z domain has a higher affinity for the Myc HLH-Z domain than for itself, and suggest that the heterodimeric Myc+Max activator forms preferentially at equilibrium.
3505	Growth hormone and prolactin stimulate androgen receptor, insulin-like growth factor-I (IGF-I) and IGF-I receptor levels in the prostate of immature rats.	1360928	In this study we investigated the involvement of several different pituitary hormones on rat prostate development. 22-day-old Wistar rats, hypophysectomized (hypox) at 19 days of age were supplemented with highly purified human prolactin (hPRL), human luteinizing hormone (hLH), porcine follicle-stimulating hormone (pFSH), and bovine growth hormone (bGH) or with saline. Quantitative analysis of RNAs shows that treatment with either PRL or GH increases significantly steady-state mRNAs levels of the following genes in the prostate: androgen receptor (AR) (respectively 3.5- and 4.8-fold above hypox controls), IGF-I (5- and 2.7-fold), and IGF-I receptor (2.9- and 2.3-fold). LH and FSH, by contrast, have negative effects on these parameters. To test whether the enhancing effect of PRL and GH on AR-mRNA abundance was followed by increased content in the protein itself, binding assays were performed with the androgen agonist [3H]R1881 (131 and 153 fmol/mg protein while hypox controls contained 110 fmol/mg protein). In addition to the well-documented presence of prolactin receptors in prostatic tissues, we have further demonstrated, by means of nuclease S1 protection assays plus dot- and Northern-blot analyses, that a GH receptor mRNA is produced in the immature rat prostate. Moreover, we observed not only strong lactogenic but also purely somatogenic binding to be occurring in the immature prostates. Finally, we have studied IGF-I mRNA content in separated epithelial/stromal cell fractions and have concluded that IGF-I expression is principally located in the prostatic stroma. Taken together, these results suggest that PRL and GH are involved in regulating AR synthesis, at least partially by direct action on the organ. In this context IGF-I appears as a paracrine factor playing a role in epithelium/stroma interactions during prostatic development.
3506	[Generation of monoclonal antibodies (McAbs) against human luteinizing hormone (hLH) in a methylcellulose semi-solid medium].	1299544	McAbs against hLH were generated by a facile hybridoma technique according to the methods of Davis and Lee, et al. After routine immunization and cell fusion, the cells were plated in a semi-solid medium containing methylcellulose and HAT. Five to seven days later, visible clones were removed for subculture in DMEM liquid medium containing 15% FCS and then screened by ELISA. As a result, 12 hybrid cell lines secreting McAbs to the LH-B subunit were obtained. These Abs have been used in clinical diagnostic and immunohistochemical work, showing good results. The advantages of the semi-solid medium in preparing McAbs are discussed.
3507	deadpan, an essential pan-neural gene encoding an HLH protein, acts as a denominator in Drosophila sex determination.	1525829	In Drosophila, sex is determined by the X:A ratio. One major numerator element on the X chromosome is sisterless-b (sis-b), also called scute, which encodes an HLH-type transcription factor. We report here that an essential pan-neural gene, the autosomal HLH gene deadpan (dpn), acts as a denominator element. As revealed by dosage-dependent dominant interactions, males die with too high a ratio of sc+ to dpn+, caused by misexpression of Sex lethal (Sxl) in embryos, and females die with too low a ratio of sc+ to dpn+, because of altered embryonic Sxl expression. In addition, we found that the HLH gene extramacrochaetae (emc), like daughterless (da), is needed maternally for proper communication of the X:A ratio, thus supporting the idea that a set of HLH genes comprises a functional cassette that makes a sensitive and stable genetic switch used in both neural determination and sex determination.
3508	The Enhancer of split [E(spl)] locus of Drosophila encodes seven independent helix-loop-helix proteins.	1528887	The E(spl) locus is thought to participate in a cell interaction mechanism that controls the choice of many cell fates during Drosophila development, including the segregation of neural precursors. Previous studies have demonstrated that E(spl) is defined by two groups of closely related transcripts, (i) a cluster of three transcripts encoding proteins bearing a helix-loop-helix (HLH) motif and (ii) a single-copy gene encoding a nuclear protein containing repeated motifs first identified in the beta subunit of guanine nucleotide-binding proteins. Both groups interact genetically with the Notch locus, which codes for a transmembrane protein. We report the structure of four additional HLH-encoding genes that reside in the E(spl) complex and provide evidence that we have now identified all the remaining members of the E(spl) HLH cluster.
3509	Analysis of the oligomerization of myogenin and E2A products in vivo using a two-hybrid assay system.	1325437	Members of the helix-loop-helix (HLH) family of proteins bind DNA and activate transcription as homo- and heterodimers. Myogenin is a muscle-specific HLH protein that binds DNA in vitro as a heterodimer with several widely expressed HLH proteins, such as the E2A gene products E12 and E47. We describe a method for detection of protein-protein interactions among HLH proteins in vivo in which dimerization through the HLH motif reconstructs a hybrid transcription factor containing the DNA-binding domain of yeast GAL4 linked to one HLH motif and the activation domain of VP-16 linked to another. We have used this assay to investiagate whether myogenin forms homomeric or heteromeric complexes in vivo and to determine whether growth factors and oncogenes that inhibit myogenesis influence myogenin's ability to dimerize. The results show that myogenin heterodimerizes with E12 and E47 in vivo, but it does not homodimerize to a measurable extent. Peptide growth factors, as well as the immediate early gene products c-Jun, v-Fos, and c-Myc, inhibit the activity of myogenin through a mechanism independent of its association with E2A products.
3510	Immunochemical study of equine chorionic gonadotropin (eCG/PMSG): antigenic determinants on alpha- and beta-subunits.	1382612	In the present study we have established an immunochemical mapping of equine Chorionic Gonadotropin (eCG/PMSG) using three monoclonal antibodies (mAbs), namely the antibodies ECG01, E10 and D7, raised against the native hormone. These antibodies do not bind to reduced, alkylated hormone, suggesting that they recognize discontinuous rather than continuous epitopes. We have also assessed the reactivity of mAbs towards human CG, and ovine, porcine, equine and bovine LH and FSH. The antigenic determinant recognized by ECG01 is localized on the alpha-subunit of equine gonadotropins and of human CG and LH. The epitopes recognized by E10 and D7 mAbs appear to be very similar and are present on the beta-subunit of eCG and of LHs from all species tested, except hLH, as well as on porcine and equine FSHs. Attempts to specify the amino-acid residues involved in these epitopes suggest that ECG01 mAb might preferentially bind to residues around position 70 whereas the region around disulfide bridges Cys-88-Cys-90 might be involved in the epitopes recognized by D7 and E10 mAbs. Topographical relationships of epitopes show that ECG01 mAb never binds to eCG simultaneously with either D7 or E10 mAbs. Furthermore, simultaneous binding of D7 and E10 mAbs on eCG could not be achieved. Thus, these three epitopes appear to be closely located on the surface of eCG. Finally, ECG01 mAb inhibits eCG binding to LH and FSH receptors, suggesting that its antigenic site is closely related to hormone-receptor interaction site(s).
3511	Hlf, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17;19) acute lymphoblastic leukemia.	1516826	Oncogenic conversion of transcription factors by chromosomal translocations is implicated in leukemogenesis. We report that the t(17;19) in acute lymphoblastic leukemia produces a chimeric transcription factor consisting of the amino-terminal portion of HLH proteins E12/E47 (products of the E2A gene) fused to the basic DNA-binding and leucine zipper dimerization motifs of a novel hepatic protein called hepatic leukemia factor (Hlf). Hlf, which is not normally transcribed in lymphoid cells, belongs to the recently described PAR subfamily of basic leucine zipper (bZIP) proteins, which also includes Dbp and Tef/Vbp. Wild-type Hlf is able to bind DNA specifically as a homodimer or as a heterodimer with other PAR factors. Structural alterations of the E2a-Hlf fusion protein markedly impair its ability to bind DNA as a homodimer compared with wild-type Hlf. However, E2a-Hlf can bind DNA as a heterodimer with other PAR proteins, suggesting a novel mechanism for leukemogenic conversion of a bZIP transcription factor.
3512	SCL and related hemopoietic helix-loop-helix transcription factors.	1453013	The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus implying a role for SCL in erythroid differentiation events. However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL suggest it is also important in proliferation and self-renewal events in erythroid cells.
3513	Contraception.	1446424	The principle of vaccination for the purposes of fertility regulation is scientifically elegant and socially compelling. Factors such as economic production, convenience of use, relatively long-lasting but reversible protection, low failure rate and the avoidance of mechanical devices or exogenous hormones make this approach a potentially attractive option for family planning programmes in both developing and developed countries. The major efforts in research and development have involved the prospect of active immunization against specific antigens of sperm, oocyte, zygote and early embryo, and the pregnancy hormone human chorionic gonadotrophin (hCG). Several anti-hCG vaccines have entered clinical trials. They operate by preventing or interrupting pregnancy at the peri-implantation stage probably by neutralizing the luteotrophic effect of hCG. The most refined vaccine is one directed against the unique C-terminal peptide on the beta-subunit of hCG. This vaccine provokes antibodies that are specific for hCG and do not cross-react with human luteinizing hormone (hLH). Preclinical studies in baboons and data from a phase I human trial indicate that this method is free of side-effects and provides the promise of a duration of effectiveness of up to 12 months. Future research will optimize the anti-hCG approach, utilize new vaccine delivery systems and broaden the spectrum of target antigens of potential utility for contraceptive vaccines.Theoretically, a vaccine for fertility regulation is possible. Family planning programs in developing and developed countries probably will find this fertility regulation approach attractive because of acceptability of the vaccine principle, large scale production at relatively low cost, convenience of use, relatively long-lasting but reversible protection, low failure rate, and the avoidance of mechanical devices or exogenous hormones. Research and development efforts revolve around the likelihood of active immunization against specific antigens of sperm, oocyte, zygote, and early embryo and the pregnancy hormone human chorionic gonadotropin (hCG). Some anti-hCG vaccines are already being tested in clinical trials. These vaccines likely negate the luteotrophic effect of hCG at the peri-implantation stage, resulting in pregnancy prevention. The most advanced anti-hCG vaccine targets the unique C-terminal peptide on the beta-subunit of hCG. It stimulates hCG specific antibodies and does not cross-react with human luteinizing hormone. Baboon studies and phase I human trials show that this advanced vaccine does not cause side-effects and may be effective for up to 1 year. Future research centers on improving the anti-hCG approach, use of new vaccine delivery systems, and expanding the spectrum of target antigens of potential utility for contraceptive vaccines.
3514	Biochemical analyses of proteolytic nicking of the human glycoprotein hormone alpha-subunit and its effect on conformational epitopes.	1380433	Conformational features of two epitopes on the glycoprotein hormone alpha-subunit were investigated using two antihuman FSH (anti-hFSH) monoclonal antibodies (mAbs) 3A and 5F that recognize different epitopes and are specific for alpha-subunit. These mAbs were used to investigate whether the conformation of these epitopes was different in heterodimeric hFSH, hTSH, hLH, or hCG. Any differences in the mass of hormone in each preparation were accounted for by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis of all hormone preparations used in this study. Rabbit anti-hFSH alpha-(11-27) antipeptide antisera and [125I]protein-G were used in the Western blot analysis. Radioactivity associated with each band was determined and used to normalize the mass of alpha-subunit in each reference preparation used in the displacement assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also performed in order to examine the integrity of each of the hormone reference preparations. hTSH alpha and, to a lesser extent, hLH alpha preparations contained an internal nick in the polypeptide chain. RIA analysis performed using heterodimeric glycoprotein hormones as competitors revealed that an average 100-fold difference in the ED50 values for hFSH compared to the other glycoprotein hormones was seen with mAb 3A. Therefore, the conformation of 3A epitope appeared to be different in hFSH than in hTSH, hLH, or hCG. In comparison, the epitope recognized by mAb 5F only had an average 7-fold difference in reactivity (ED50 values) for hFSH compared to hTSH, hLH, and hCG. Likewise, competition assays using the respective alpha-subunits and mAb 5F revealed a pattern of competition similar to that observed with heterodimers, with an average 4-fold difference in the ED50 values for hFSH alpha compared to those for hTSH alpha, hLH alpha, and hCG alpha. Therefore, the conformation of the 5F epitope appears unaffected by association of alpha-subunit with beta-subunit. Accordingly, any differences in the conformation of the four alpha-subunits, as demonstrated by these small differences in ED50 values, appear to be inherent to each alpha-subunit. In fact, the 5F epitope appears to be quite rigid, since nicked alpha-subunit preparations could compete with [125I]hFSH for binding to 5F with comparable potency to non-nicked alpha-subunits. These findings support the concept that epitopes on heterodimeric hFSH alpha may have different conformational features. Some are specific for heterodimeric hFSH alpha, and we refer to these as conformationally active (flexible). Others are common to the four human glycoprotein hormone alpha-subunits, suggesting that they are conformationally constrained (rigid).
3515	Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor.	1325649	A cDNA encoding the murine Ah receptor (Ahb-1 allele for aromatic hydrocarbon responsiveness) has been isolated and characterized. Analysis of the deduced protein sequence revealed a region with similarity to the basic region/helix-loop-helix (BR/HLH) motif found in many transcription factors that undergo dimerization for function. In addition to the BR/HLH domain, the N-terminal domain of the Ah receptor has extensive sequence similarity to the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein and two regulatory proteins of Drosophila, Sim and Per. Photoaffinity labeling and peptide mapping studies indicate that the Ah receptor binds agonist at a domain that lies within this conserved N-terminal domain. The Ah receptor appears to be a ligand-activated transcription factor with a helix-loop-helix motif similar to those found in a variety of DNA-binding proteins, including Myc and MyoD.
3516	The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo.	1325394	rhomboid (rho) encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis. It is initially expressed before the completion of cellularization in lateral stripes within the presumptive neuroectoderm. Here, we present evidence that the maternal morphogen dorsal (dl) acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including twist (twi), to activate rho in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by snail (sna), which is also a direct target of the dl morphogen. A 300-bp region of the rho promoter (the NEE), which is sufficient for neuroectoderm expression, contains a cluster of dl and b-HLH activator sites that are closely linked to sna repressor sites. Mutations in these binding sites cause genetically predicted changes in the levels and limits of rho expression. In particular, the disruption of sna-binding sites causes a derepression of the pattern throughout ventral regions, providing evidence that sna is directly responsible for establishing the mesoderm/neuroectoderm boundary before gastrulation. The tight linkage of activator and repressor sites in the rho NEE is similar to the arrangement of binding sites observed in the even-skipped stripe 2 element, which is regulated by bicoid (bcd). This suggests that the dl and bcd morphogens use a similar mechanism to make stripes in the Drosophila embryo.
3517	Cellular localization of IGF-I and IGF-II mRNAs in immature hypophysectomized rat testis and epididymis after in vivo hormonal treatment.	1282380	IGF-I and II genes expression has been localized by in situ hybridization in testis and epididymis of immature hypophysectomized rats treated in vivo with either pFSH, hLH, bGH, hPRL or with saline. IGF-I mRNA expression was found in both Sertoli and Leydig cells after treatment with either FSH or LH. IGF-I mRNA was highly expressed in germ cells after FSH stimulation and to a lesser extent after GH or LH treatments. However, its expression was very low in hypophysectomized control or PRL treated rats. IGF-I mRNA was also expressed in stromal cells of epididymis after LH treatment and to a lesser extent after GH stimulation. In contrast, IGF-II mRNA expression was detected in all testicular cell types whatever the hormonal treatment (FSH, LH, GH, PRL). For each hormonal treatment testicular sections were examined after immunohistochemical staining with specific antisera against IGF-I and IGF-II. Both in situ hybridization and immunohistochemical data were examined in order to determine the testicular sites of synthesis of IGF-I and IGF-II.
3518	Subunit structure of cell-specific E box-binding proteins analyzed by quantitation of electrophoretic mobility shift.	1639803	Expression of insulin and immunoglobulin genes is dependent on the presence of E boxes (consensus sequence CAXXTG) within the enhancer regions. These sequences are recognized by cell-specific nuclear factors IEF1 (insulin enhancer factor 1) and LEF1 (lymphoid enhancer factor 1). Although IEF1 and LEF1 are distinct by several parameters, they are both recognized by antisera to the mouse helix-loop-helix (HLH) protein A1 (a homolog of the human protein E47, product of the E2A gene). This suggests that A1/E47 or a close relative is a component of both complexes. In order to further characterize the complexes, we have used in vitro translated DNA-binding proteins of known size to verify that electrophoretic mobility shift analysis can be used to estimate the molecular weight of DNA-binding proteins from both the HLH family and the leucine zipper family. Under the conditions used, migration is relatively insensitive to changes in protein charge. This analysis, in combination with mixing experiments between nuclear extracts and in vitro translated HLH proteins, indicates that IEF1 and LEF1 are dimeric complexes. IEF1 behaves as a complex of two proteins, one of which is 67 kDa and is recognized by antibodies to A1, and the second of which is 25 kDa. LEF1 on the other hand, appears to be a complex of two proteins of 67 kDa. The size of the 67-kDa subunits is consistent with that reported for the full-length E2A gene products. The 25-kDa subunit of IEF1 forms DNA-binding heterodimers with A1 but not MyoD and is present in a limited range of cell types, features characteristic of class B HLH proteins such as MyoD and achaete-scute. Taken together, the data support the idea that the E2A gene products are involved directly in regulation of insulin and immunoglobulin gene expression; regulation of the insulin gene apparently requires, in addition, the 25-kDa HLH protein (designated IESF1 for insulin enhancer-specific factor 1).
3519	Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins.	1644289	The helix-loop-helix (HLH) protein Id lacks the basic DNA-binding domain common to this class of proteins. In vitro experiments suggested that Id could associate tightly with two other HLH proteins encoded by the E2A gene, E12 and E47 (referred to here collectively as E proteins) and prevent their binding to a sequence present in the muscle creatine kinase (MCK) enhancer either as homo-oligomers or hetero-oligomers with MyoD. In this report we present evidence for the in vivo roles of Id and E proteins: (1) Id and E proteins co-fractionate and co-immunoprecipitate in whole-cell extracts prepared from myoblasts; (2) the loss of Id protein observed during the conversion of proliferating myoblasts into mature myotubes correlates with the formation of MyoD/E hetero-oligomeric complexes in whole-cell extracts (these complexes do not form when purified Id protein is added to the extracts); and (3) stable overexpression of Id mRNA and protein in the C2C12 muscle cell line inhibits differentiation in these cells 16 hr post-induction. The myotubes that do eventually form 48 hr post-induction have no detectable Id protein in the nucleus despite the persistence of exogenous Id mRNA. These data support a model in which Id can inhibit muscle cell differentiation by associating with E proteins and preventing them from forming active hetero-oligomeric complexes with the muscle determination gene products.
3520	Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles.	1639951	After stimulation of multiple follicular development, endogenous LH surges elicited by GnRH or GnRH agonist were of insufficient duration (4-14 h) to evoke oocyte maturation and luteinization in this species. In this study, periovulatory LH surge requirements were further titrated using hLH as the ovulatory stimulus. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. To induce ovulatory maturation on day 10, animals received: 1) hCG (1000 IU, im; n = 8); 2) highly-purified, urinary hLH (2542 IU, im; n = 4); or 3) hLH (2542 IU, im) followed by three injections of hLH (200 IU, im) at 8-h intervals (0800, 1600, 2400 h) daily during the luteal phase until menses (n = 3). Oocytes and luteinizing granulosa cells were obtained via follicle aspiration 27 h after the initial hLH or hCG injection. Estradiol and progesterone levels were measured in daily serum samples by RIA. Bioactive LH levels were determined at selected intervals within 36 h of the hLH ovulatory stimulus. Nuclear maturity of oocytes was evaluated as an indicator for reinitiation of meiosis. Luteinizing granulosa cells were processed for indirect immunocytochemistry using a monoclonal antibody to human progesterone receptor. In vitro progesterone production by luteinizing granulosa cells over 24 h was also assessed in the absence and presence of hCG. In all groups, serum estradiol rose to similar peak levels on day 10. After hLH, bioactive LH levels peaked (1262 +/- 79 ng/mL; mean +/- SEM) by 2-6 h, declined thereafter but remained above surge levels (100 ng/mL) for 18-24 h. Within 24 h of hLH injection, serum progesterone increased to 13 +/- 3 nmol/L, but returned to baseline in 1-6 days. In contrast, higher levels of progesterone were observed after hCG (114 +/- 51 nmol/L) and during luteal phase treatment with hLH (137 +/- 25 nmol/L) and the luteal phase was longer (11.5 +/- 0.4 and 14.3 +/- 0.7 days, respectively). Of the total cohort of oocytes aspirated, the proportion of oocytes resuming meiotic maturation (metaphase I plus metaphase II) was similar after hCG (76%) and hLH (74%). However, the proportion of oocytes maturing to metaphase II tended to be less (P = 0.08) after hLH (13%) than hCG (22%). Fertilization rates were similar between the two groups. Progesterone receptor was detected in nuclei of luteinizing granulosa cells from all animals receiving hCG, but only in some given hLH.(ABSTRACT TRUNCATED AT 400 WORDS)
3521	Inhibition of murine erythroleukemia cell differentiation by normal and partially deleted c-myc genes.	1452198	In our study of normal and partially deleted myc genes we found that N-myc, similarly to L-myc, can substitute for c-myc and inhibit MEL cell differentiation. All of the known putative functional domains of c-myc seem to be required for this inhibition. It is conceivable that c-myc inhibits differentiation by a mechanism that is related to its normal role in the cell, possibly by regulating transcription of genes involved in growth promotion. As was previously found for all of the other known activities of c-Myc, the HLH and LZ dimerization motifs are absolutely necessary for inhibition of MEL cell differentiation. Heterodimerization of Myc with Max or Max-like proteins could be a prerequisite for such inhibition. It is, therefore, of interest to study the regulation of max in MEL cells expressing normal and deregulated myc genes.
3522	The expression of human chorionic gonadotropin/luteinizing hormone receptors in human endometrial and myometrial blood vessels.	1379262	hCG/human LH (hLH) receptors have recently been found in human endometrial and myometrial cells and uterine vasculature. The present study was undertaken to further corroborate the immunocytochemical evidence for the presence of vascular receptors. Northern blot and in situ hybridization analyses have revealed that human uterus contains a major 4.3-kilobase and a minor 2.6-kilobase hCG/hLH receptor mRNA transcript and that these transcripts are present in part in endometrial and myometrial vascular smooth muscle cells and vascular endothelial cells. Immunoblot and immunocytochemical analyses have revealed that human uterus also contains a single immunoreactive receptor protein, and that this receptor protein in part is present in endometrial and myometrial vascular smooth muscle and vascular endothelium. The expression of receptor mRNA and/or immunoreactive receptor protein was higher in myometrial than in endometrial blood vessels, and higher in vessels of both uterine compartments from the secretory compared to proliferative phase, postmenopause, or pregnancy. The blood vessels in omentum, broad ligament, and parametrium did not immunostain for hCG/hLH receptors. A blood vessel seen traversing through parametrium immunostained for the receptor protein only after it entered the myometrium. The blood vessels in nontarget tissues did not immunostain, whereas those in some target tissues, but not all of them, immunostained for the receptor protein. In summary, the present study demonstrates for the first time that human endometrial and myometrial vascular smooth muscle and endothelium express hCG/hLH receptor mRNA and immunoreactive receptor protein. These findings suggest that hCG/hLH may directly regulate blood flow in human uterus and other target tissues. The reproductive state dependency of uterine vascular receptors suggests that these receptors are probably regulated by other reproductive hormones.
3523	MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer.	1360403	Myogenic helix-loop-helix (HLH) proteins, such as myogenin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are especially permissive to the actions of these myogenic regulators, many other cell types are refractory to myogenic conversion by them. Here we describe a novel homeodomain protein, MHox, that binds an A+T-rich element in the muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during embryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeobox genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding properties and pattern of MHox expression are unique among homeobox genes and suggest a role for MHox as a transcriptional regulator that participates in the establishment of diverse mesodermal cell types.
3524	Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C.	1323566	Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han-1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.
3525	Expression of recombinant human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts.	1322283	The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.
3526	Mutually exclusive expression of a helix-loop-helix gene and N-myc in human neuroblastomas and in normal development.	1628620	We have isolated a novel human gene encoding a helix-loop-helix (HLH) protein by molecularly cloning chromosome 1p36-specific CpG islands. The gene termed heir-1 was localized to the neuroblastoma consensus deletion at 1p36.2-p36.12. Its predicted protein is 95.8% identical to the mouse HLH462 protein and has clear homology to the mouse Id and Drosophila emc proteins. Heir-1 does not encode a basic DNA binding domain as found in basic HLH proteins. The gene is expressed specifically at high abundance in adult lung, kidney and adrenal medulla, but not in adult brain. Despite prominent heir-1 expression in adrenal medulla, which is a prime target for neuroblastomas, 10 out of 12 neuroblastoma-derived cell lines revealed very low levels of heir-1 mRNA. Low heir-1 expression was generally found in tumor cell lines with N-myc overexpression, whereas the two cell lines displaying high heir-1 levels did not overexpress N-myc. Mutually exclusive expression of both genes was also found by in situ hybridization in developing mouse tissues, particularly in the forebrain neuroectoderm. We conclude that heir-1 expression is reduced specifically in the majority of neuroblastomas and suggest an inverse correlation between heir-1 and N-myc expression in neuroblastoma tumors and in embryonic development.
3527	SCL gene in human tumors.	1625484	The SCL gene encodes a member of the helix-loop-helix (HLH) family of transcription factors and is reportedly involved in up to 25% of T-cell acute lymphoblastic leukemia (T-ALL). We have surveyed over 120 primary human tumors including melanomas, myeloid, and lymphoid leukemias, and other solid tumors without evidence of rearrangements involving SCL. These results are further supported by low level expression of SCL in these tumors (as assessed by a polymerase chain-reaction-based method). We conclude that rearrangement/translocation with subsequent activation of SCL occurs infrequently in myeloid leukemias and melanomas.
3528	Polyclonal antibodies against the polypeptide and carbohydrate epitopes of recombinant human choriogonadotropin beta-subunit.	1380928	In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081-4087) we reported the preparation and characterization of recombinant human choriogonadotropin beta subunit (hCG beta) using the baculovirus-insect cell expression system. The rhCG beta was found to contain high mannose type N-linked carbohydrates and 3-4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCG beta was similar to hCG beta in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCG beta was produced. The antiserum was found to be almost identical to anti-hCG beta in binding to hCG beta as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using 125I-hCG beta as a tracer. Further characterization of the anti-rhCG beta antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCG beta antisera pool as shown by dot blot and radioimmunoassays. The carbohydrate-specific antibodies were separated by affinity chromatography using an ovalbumin-glycopeptide-Sepharose column. The antibodies held on the ovalbumin affinity adsorbent were specific for the high mannose type carbohydrates such as those present in rhCG beta, rhCG and thyroglobulin and failed to react with transferrin, alpha 1-acid glycoprotein and hCG alpha, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCG beta also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCG beta since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCG beta-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCG beta and not in hCG beta. The eluate from the hCG beta-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCG beta and hCG beta. The high mannose type specific antibodies are potentially useful in differentiating between the high mannose and complex type of N-linked carbohydrates present in a glycoprotein. Also, the antibody could provide an effective reagent in studying the intracellular processing of the N-linked oligosaccharides.(ABSTRACT TRUNCATED AT 400 WORDS)
3529	Id expression during mouse development: a role in morphogenesis.	1361374	We have characterized the spatial and temporal pattern of Id transcription during mouse embryogenesis. The Id gene encodes a helix-loop-helix (HLH) protein which can heterodimerize with the ubiquitously expressed HLH protein products of the E2A gene, and prevent them from binding DNA either alone or as a heterodimer with tissue specific HLH transcription factors such as the muscle determination gene, MyoD1 (Benezra et al., 1990: Cell 61:49-59). Since Id has been shown to be down-regulated during induced differentiation in several cell lines, it has been postulated that Id plays a general inhibitory role in cell differentiation (Benezra et al., 1990). In situ analysis of Id mRNA expression in the mouse embryo was performed in order to determine whether the pattern of Id expression is consistent with this postulate. A detailed study throughout the entirety of mouse postimplantation development reveals that Id is expressed upon gastrulation at very high levels in almost all regions of the mouse embryo and expression declines as embryogenesis proceeds. In skeletal muscle, in which the inhibitory action of Id has been established in tissue culture models (Benezra et al., 1990), Id and the HLH myogenic factors are expressed in a mutually exclusive manner suggesting that myogenic precursors do not express both types of HLH gene products. In addition, Id colocalizes both spatially and temporally with Hox-7.1, a murine homeobox gene which is associated with regions of high cell proliferation and positional fate assignment.
3530	Repression of myogenin function by TGF-beta 1 is targeted at the basic helix-loop-helix motif and is independent of E2A products.	1317849	The muscle-specific helix-loop-helix (HLH) proteins myogenin, MyoD, myf5, and MRF4 form hetero-oligomers with ubiquitous HLH proteins encoded by the E2A gene and activate muscle transcription by binding to a DNA sequence known as an E-box (CANNTG). Transforming growth factor-beta (TGF-beta) can inhibit muscle differentiation by silencing the transcription-activating potential of myogenic HLH proteins without affecting their ability to bind DNA. We show that repression by TGF-beta is directed at the basic-HLH motif of myogenin and is independent of E2A products. Using a series of reporter genes as targets for trans-activation by myogenin, transcriptional repression by TGF-beta is also shown to map to the E-box motif and to not require heterologous DNA sequence elements. These results demonstrate that TGF-beta represses muscle-specific transcription through a post-translational mechanism that renders the basic-HLH regions of the myogenic regulators nonfunctional. The selective repression of myogenic HLH proteins by TGF-beta indicates that the TGF-beta signaling system can discriminate between different classes of HLH proteins and implies that myogenic HLH proteins activate muscle-specific transcription through a unique mechanism.
3531	The expression of human chorionic gonadotropin/human luteinizing hormone receptors in human gestational trophoblastic neoplasms.	1592864	Normal human placental trophoblasts have recently been shown to contain receptors for hCG/hLH. The present studies investigated the expression of these receptors in hyperplastic and anaplastic trophoblasts in gestational trophoblastic neoplasms. The results demonstrated that both hydatidiform moles and choriocarcinomas contained receptor messenger RNA (mRNA) and receptor protein. A variety of nontrophoblast tumors, on the other hand, contained neither receptor mRNA nor receptor protein. Choriocarcinomas contained more receptor mRNA and receptor protein than hydatidiform moles which in turn contained more than normal human placenta. Midluteal phase human corpus luteum contained more receptor mRNA than normal human placenta and about the same as choriocarcinomas. The hyperplastic and anaplastic trophoblasts in hydatidiform moles and choriocarcinomas contained more receptor immunostaining than the normal trophoblasts in the same tissue or those from normal placentas from about the same gestational age. The receptor immunostaining increased as the degree of trophoblast hyperplasia increased in hydatidiform moles. Anaplastic trophoblasts of choriocarcinomas contained a similar amount of receptor immunostaining as severely hyperplastic trophoblasts of hydatidiform moles. Invading anaplastic trophoblasts of choriocarcinoma contained greater amount of receptor immunostaining than the surrounding endometrial stromal and myometrial smooth muscle cells. In summary, this is the first study to our knowledge demonstrating the expression of hCG/hLH receptor gene in gestational trophoblastic neoplasms. The increased receptor expression in these neoplasms suggests that hCG, via its receptors, could play a fundamental and previously unsuspected autocrine role in the regulation of trophoblast transformation, growth, invasion, and high hCG secretion.
3532	Point mutations in the Drosophila hairy gene demonstrate in vivo requirements for basic, helix-loop-helix, and WRPW domains.	1588951	The Drosophila pair-rule gene, hairy (h), encodes a nuclear basic helix-loop-helix (bHLH) protein that regulates embryonic segmentation and adult bristle patterning. In both cases, the h protein behaves as a transcriptional repressor. In this study, we determined the molecular nature of 12 h alleles. One mutation maps within the HLH domain, consistent with h function requiring homodimerization or heterodimerization with other HLH proteins. A second mutation lies in the basic domain, suggesting that DNA binding is required for h activity. Several mutations show that the h C terminus, in particular the WRPW domain, is also required for h activity, perhaps by interacting with other proteins to mediate transcriptional repression. We show that the h protein in Drosophila virilis closely resembles that in D. melanogaster and includes completely conserved bHLH and WRPW domains.
3533	Structure of a receptor-binding fragment from human luteinizing hormone beta-subunit determined by [1H]- and [15N]nuclear magnetic resonance spectroscopy.	1495492	The structure of the glycoprotein hormones (LH, CG, FSH, and TSH) and their mechanism of receptor recognition are problems of long-standing interest and speculation. Here we describe the two-dimensional [1H]nuclear magnetic resonance ([1H]NMR) analysis of a linear peptide model for the intercysteine sequence (38-57) from the beta-subunit of human (h) LH. This sequence contains functional determinants for receptor binding and postreceptor activation and is predicted by computer-based modeling to fold as a compact minidomain containing a central amphipathic helix. To test this prediction, an Arg-extended disulfide-free (38-57) analog of enhanced solubility was prepared for complementary circular-dichroic and two-dimensional NMR studies. The linear peptide retains ovarian membrane receptor-binding activity. Although the peptide is not highly structured in aqueous solution, circular-dichroic analysis shows partial alpha-helix formation in a lipophilic medium (50% trifluoroethanol). Complete sequential assignment is obtained in 50% trifluoroethanol based on homonuclear and [15N]edited heteronuclear NMR methods. alpha-Helix-related (i,i + 3) connectivities are observed by nuclear-Overhauser effect spectroscopy that define an amphipathic alpha-helical segment (residues 41-48). Additional long range nuclear-Overhauser effects are observed in the C-terminal region that are consistent with beta-turns involving one or more proline residues; these may serve to reverse the direction of the peptide chain. A nuclear-Overhauser effect contact is identified between residues 38 and 55 at opposite ends of the linear sequence, suggesting that a loop configuration is significantly populated in this solvent system. These results, taken together, characterize elements of ordered structure in the 38-57 peptide, which appear to be distinguishing features of hLH (and the homologous region of hCG). We propose that the structure of this peptide provides a model for the structure of the corresponding region of native hLH in the hormone-receptor complex.
3534	Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos.	1589769	A facile method for isolating genes that encode interacting proteins has been developed with a polypeptide probe that contains an amino-terminal extension with recognition sites for a monoclonal antibody, a specific endopeptidase, and a site-specific protein kinase. This probe, containing the basic region-leucine zipper dimerization motif of c-Fos, was used to screen a complementary DNA library. A complementary DNA that encoded a member of the basic-helix-loop-helix-zipper (bHLH-Zip) family of proteins was isolated. The complementary DNA-encoded polypeptide FIP (Fos interacting protein) bound to oligonucleotide probes that contained DNA binding motifs for other HLH proteins. When cotransfected with c-Fos, FIP stimulated transcription of an AP-1-responsive promoter.
3535	Co-operativity of functional domains in the muscle-specific transcription factor Myf-5.	1582413	Myf-5 is a member of a family of muscle-specific transcription factors that activate myogenesis in 10T1/2 fibroblasts. Here we report the analysis of Myf-5 structural domains that are responsible for its biological activity. Site-directed mutagenesis revealed that two clusters of basic amino acids within a conserved basic region and two amphipathic helices within the adjacent HLH domain are essential for sequence-specific DNA binding and hetero-oligomerization, respectively. Transcriptional activation by Myf-5 requires two additional domains located in the amino- and carboxyl-termini. The two domains apparently co-operate since deletion of either one results in inactivation. Chimeric proteins between DNA binding domain of the yeast transcription factor GAL4 and the separate Myf-5 transactivator domains exhibit activity that is enhanced when both regions are combined. Dimerization of Myf-5 with the ubiquitously expressed bHLH protein E12 not only increases the affinity for DNA but also stimulates transactivation independently of DNA binding. The Myf-5 transactivator domains are dependent for activity on a specific amino acid sequence motif within the basic region when Myf-5 activity is mediated through the E-box DNA recognition sequence but not when DNA binding occurs through the GAL4 DNA binding domain. This demonstrates that muscle-specific transactivation by Myf-5 requires the collaboration of two activation domains and the DNA binding region in addition to sequence-specific DNA binding. Transcriptional activation and interaction with DNA are executed by separable domains; however, transactivation is influenced by the basic region in a manner distinguishable from DNA binding.
3536	Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins.	1577278	The B, R, C1, and Pl genes regulating the maize anthocyanin pigment biosynthetic pathway encode tissue-specific transcriptional activators. B and R are functionally duplicate genes that encode proteins with the basic-helix-loop-helix (b-HLH) motif found in Myc proteins. C1 and Pl encode functionally duplicate proteins with homology to the DNA-binding domain of Myb proteins. A member of the b-HLH family (B or R) and a member of the myb family (C1 or Pl) are both required for anthocyanin pigmentation. Transient assays in maize and yeast were used to analyze the functional domains of the B protein and its interaction with C1. The results of these studies demonstrate that the b-HLH domain of B and most of its carboxyl terminus can be deleted with only a partial loss of B-protein function. In contrast, relatively small deletions within the B amino-terminal-coding sequence resulted in no trans-activation. Analysis of fusion constructs encoding the DNA-binding domain of yeast GAL4 and portions of B failed to reveal a transcriptional activation domain in the B protein. However, an amino-terminal domain of B was found to recruit a transcriptional activation domain by an interaction with C1. Formation of this complex resulted in the activation of a synthetic promoter containing GAL4 recognition sites, demonstrating that this interaction does not require the normal target promoters for B and C1. B and C1 fusions with yeast GAL4 DNA-binding and transcriptional activation domains were also found to interact when synthesized and assayed in yeast. The domains responsible for this interaction map to a region that contains the Myb homologous repeats of the C1 protein and to the amino terminus of the B protein, which does not contain the b-HLH motif. These studies suggest that the regulation of the maize anthocyanin pigmentation pathway involves a direct interaction between members of two distinct classes of transcriptional activators.
3537	Inhibition of muscle differentiation by the adenovirus E1a protein: repression of the transcriptional activating function of the HLH protein Myf-5.	1315706	Myogenic differentiation can be inhibited by the adenovirus E1a protein in the rat L6 muscle cell line. The present investigation provides evidence that E1a interferes with the expression of myogenin and the activity of Myf-5, the two myogenic helix-loop-helix (HLH) proteins that are expressed in L6 muscle cells. In nuclei of E1a-expressing L6 cells, Myf-5 protein accumulates to normal or even elevated levels and shows no alterations of its ability to bind to the DNA-binding site (CANNTG). However, trans-activation of muscle-specific reporter genes by Myf-5 is strongly inhibited. The same inhibition by E1a can be shown for the other myogenic HLH proteins, MyoD, myogenin, and MRF4/Myf-6, that have been expressed in 10T1/2 fibroblasts. In contrast to the normal level of Myf-5 expression, synthesis of myogenin is entirely abolished in the differentiation-defective L6-E1a cells. Here, we demonstrate that the carboxy-terminal trans-activator domain and probably the basic-HLH (bHLH) region of Myf-5 constitute targets for the inhibition by E1a. The effect of E1a depends on its intact transforming regions but not on the transcriptional activator domain. Our data suggest that activation of myogenin gene expression and the establishment of the differentiated phenotype may require functional Myf-5. Expression of the Myf-5 gene, however, is apparently independent of auto- or cross-regulation by the myogenic HLH proteins.
3538	Body-wall muscle formation in Caenorhabditis elegans embryos that lack the MyoD homolog hlh-1.	1314423	The myoD family of DNA binding proteins has been implicated in the control of myogenesis in a variety of organisms. Searches for homologs in the nematode Caenorhabditis elegans yielded only one gene, designated hlh-1, expressed in body-wall muscle cells and their precursors. To assess the role of hlh-1 in C. elegans myogenesis, genetic deficiencies spanning the hlh-1 locus were isolated after gamma irradiation. Embryos homozygous for these deficiencies exhibited extensive body-wall muscle differentiation, including expression of several characteristic myofilament proteins and weak contracile behavior. Thus, zygotic hlh-1 expression was not required for body-wall muscle precursors to adopt muscle cell fates.
3539	SCL is coexpressed with GATA-1 in hemopoietic cells but is also expressed in developing brain.	1565464	The SCL gene encodes a putative transcription factor with a basic helix-loop-helix (B-HLH) motif and is known to be predominantly expressed in erythroid cells. Here we also demonstrate expression of SCL mRNA in normal mast cells, mast cell lines and megakaryocytic cell lines. SCL is therefore expressed in the same three lineages as GATA-1, a well-recognized hemopoietic transcription factor. SCL and GATA-1 mRNA were also co-expressed in interleukin 3-dependent primitive myeloid lines. In murine erythroleukemia (MEL) cells SCL and GATA-1 underwent coordinated biphasic modulation during hexamethylene bisacetamide (HMBA)-induced erythroid differentiation. The kinetics of SCL and GATA-1 mRNA expression was inversely correlated with changes in ID, a negative regulator of B-HLH proteins, and was distinct from changes in MYC, MYB and erythropoietin receptor transcripts. During myeloid differentiation of K562 cells, SCL and GATA-1 mRNA levels also underwent biphasic modulation. Thus SCL and GATA-1 are coordinately expressed in multiple hemopoietic lineages and coordinately regulated during induced erythroid and myeloid differentiation. In nonhemopoietic tissues SCL was only detected in adult and developing brain where GATA-1 is reportedly not expressed. In day 14.5 embryos analysed by in situ hybridization, SCL transcripts were detected in post-mitotic neurons in the metencephalon and roof of the mesencephalon. This suggests a previously unexpected role for SCL in neural differentiation.
3540	Anti-helix-loop-helix domain antibodies: discovery of autoantibodies that inhibit DNA binding activity of human centromere protein B (CENP-B).	1377670	Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromeric heterochromatin of human chromosomes. This protein was originally identified as the target antigen in autoimmune disease patients (often with scleroderma). In this study, we cloned a human CENP-B cDNA which was longer than the previously isolated one and expressed functional recombinant CENP-B in Escherichia coli. The DNA binding domain was finely located within the N-terminal 134-amino-acid residues covering a predicted helix-loop-helix (HLH) structure, by using a set of recombinant products with stepwise deletions from the C-terminus. From the analysis of their reactivity to anti-centromere sera from autoimmune disease patients, four epitopes were mapped on CENP-B antigen. In addition to two epitopes at the C-terminus, two were found on the HLH region at the N-terminus. In the analysis of the interaction between the antigen and autoantibodies, we found that the DNA binding activity of CENP-B was distorted by the attack of the anti-HLH domain antibodies in in vitro binding reactions. Our results suggest that the direct inhibition of the DNA binding activity by the autoantibodies might be involved in patients' autoimmune reactions in vivo.
3541	Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression.	1313772	Myogenin and MyoD belong to a family of muscle-specific helix-loop-helix (HLH) proteins that have the potential to activate muscle-specific genes in nonmyogenic cells. Peptide growth factors can block the ability of myogenin and MyoD to activate their target genes. Here, we show that the growth factor-inducible proto-oncogenes c-fos, c-jun, and junB mimic the effects of exogenous growth factors and suppress trans-activation of the muscle creatine kinase (MCK) enhancer by myogenin and MyoD. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, is an inefficient inhibitor of the trans-activating capacity of myogenin and MyoD. Transcriptional repression by Fos and Jun is specific to myogenic HLH proteins and is not observed with the widely expressed HLH protein E47, which recognizes the same DNA sequence. Repression of the MCK enhancer by Fos and Jun is targeted at the myogenin and MyoD DNA recognition sequence and can be mediated by the amino terminus of c-Jun. Comparison of several myogenin mutants for their responsiveness to Fos and Jun shows that repression is directed at the basic-HLH region. These results indicate that members of the Jun family can be distinguished on the basis of their effects on muscle-specific transcription and suggest there is cross talk between transcription factors that control myogenesis and those involved in cell proliferation.
3542	Inhibition of myeloid differentiation by the helix-loop-helix protein Id.	1372755	Id is a helix-loop-helix (HLH) protein that represses activity of several basic helix-loop-helix (bHLH) proteins involved in cell type--specific transcription and cell lineage commitment. The myeloid precursor cell line 32DC13(G) expressed Id messenger RNA, which was transiently decreased when cells were induced to terminally differentiate with granulocyte--colony-stimulating factor. Concomitant with the decrease of Id messenger RNA was the appearance in nuclear extracts of DNA binding proteins that recognized a canonical E-box motif, a DNA binding site for some bHLH proteins. Constitutive expression of an Id complementary DNA in 32DC13(G) cells blocked their ability to differentiate and to induce E-box-binding activity. These results suggest that Id and, hence, bHLH proteins function in the process of myeloid differentiation.
3543	Alternative multimeric structures affect myogenin DNA binding activity.	1312093	The native molecular weight of the basic helix-loop-helix (bHLH) proteins myogenin, MyoD, and E12 was calculated from their mobilities on sucrose gradients and molecular sieve chromatography. The muscle bHLH proteins associate to form a variety of higher order complexes, most of which are larger than dimers. Homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer whereas homotetramers and larger forms do not recognize this DNA sequence. The ubiquitous bHLH protein E12 forms monomers or homodimers with little evidence for higher order complexes. Mixtures of myogenin and E12 show some heterodimeric structures, but most of the myogenin remains in large complexes. This result using purified proteins is also obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin is present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein containing only the myogenin HLH region forms large homomeric complexes. A model is presented in which each helix associates with a different subunit to form chains or ring structures to explain these observations. The partition of myogenin in nuclear extracts into dimers that recognize known DNA sequences and higher order complexes that do not raises important new issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.
3544	NSCL-2: a basic domain helix-loop-helix gene expressed in early neurogenesis.	1633105	We have identified a new basic domain helix-loop-helix (bHLH) gene, NSCL-2, which was cloned because of its homology to the previously described putative hematopoietic transcription factor, SCL. NSCL-2 has been identified in both human and murine DNA. NSCL-2 complementary DNA clones were obtained from an 11.5-day murine embryo library. The coding region is 405 base pairs and encodes a predicted protein of 15.6 kilodaltons. There is 74% homology at the nucleotide level with the coding region of the murine SCL and 27% protein homology. Unlike the majority of previously described bHLH genes, the NSCL-2 coding region ends only six amino acids beyond the second amphipathic helix of the HLH domain. The NSCL-2 gene shows a markedly restricted pattern of expression predominantly confined to murine embryos at days 11-13 of development, although low level expression can be detected in murine embryos flanking this time point. Examination of 11- and 12-day mouse embryos by tissue in situ hybridization reveals expression of NSCL-2 in the developing nervous system, most likely in developing neurons. The NSCL-2 gene maps to murine chromosome 3. The temporally and tissue restricted pattern of expression of this gene and its identification as a member of a family of transcription factors relevant to growth and development in a wide variety of species suggest a role for NSCL-2 in the development of the eukaryotic nervous system.
3545	A new bind for Myc.	1579994	Recent studies centered on the c-Myc basic/helix-loop-helix/leucine zipper (B/HLH/LZ) motifs have led to the identification of a DNA recognition sequence for c-Myc and the isolation of a novel protein that forms a DNA-binding complex with c-Myc in vitro. These advances may make it possible to address directly the long-standing question of c-Myc function in vivo.
3546	HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors.	1312219	Proteins containing the basic-helix-loop-helix (B-HLH) domain have been shown to be important in regulating cellular differentiation. We have isolated a cDNA for a human B-HLH factor, denoted HEB, that shares nearly complete identity in the B-HLH domain with the immunoglobulin enhancer binding proteins encoded by the E2A and ITF2 genes (E proteins). Functional characterization of the protein expressed from this cDNA indicates that HEB is a third member of the E-protein class of B-HLH factors. HEB mRNA was found to be expressed in several tissues and cell types, including skeletal muscle, thymus, and a B-cell line. HEB, ITF2, and the E12 product of the E2A gene all bound to a similar spectrum of E-box sequences as homo-oligomers. All three factors also formed hetero-oligomers with myogenin, and the DNA-binding specificity and binding off-rates (dissociation rates) were modulated after hetero-oligomerization. Both homo- and hetero-oligomers of these proteins were able to distinguish between very closely related E-box sequences. In addition, HEB was shown to form hetero-oligomers with the E12 and ITF2 proteins. Finally, HEB was able to activate gene expression. These data demonstrate that HEB shares characteristics with other E proteins and show that HEB can interact with members of both the myogenic regulatory class and the E-protein class of B-HLH factors. HEB is therefore likely to play an important role in regulating lineage-specific gene expression.
3547	A human Id-like helix-loop-helix protein expressed during early development.	1741406	The interaction of helix-loop-helix (HLH) proteins is known to regulate the differentiation of several different tissues, including mammalian muscle and the insect peripheral nervous system. In myoblasts, the products of myogenic HLH genes such as MyoD and ubiquitous HLH proteins such as E12 are present at constant levels throughout development. An E12 monomer and a MyoD monomer form a DNA binding heterodimer that activates muscle-specific genes. These two proteins are unable to dimerize in proliferating myoblasts because a negative regulator HLH protein, Id, is present. We now report the sequence and structure of a human HLH gene related to Id, which has been designated Id-2. Two prominent Id-2 RNA molecules of 2.5 and 1.3 kilobases were found in a number of different human normal and neoplastic tissues. We believe the larger RNA is a precursor of the 1.3-kilobase mRNA that encodes an Id-2 protein of 134 amino acids. The HLH region of the Id-2 protein is 90% homologous to that of myogenic Id, but the homology is much less extensive outside the HLH region. The Id-2 gene is highly expressed during early fetal development in several tissues, including those of the central nervous system, but is not expressed in the corresponding mature tissues. Id-2 expression is modulated in association with retinoic acid-induced ganglionic differentiation of the neuroblastoma cell line SMS-KCNR. These findings suggest that Id-2 is an inhibitor of tissue-specific gene expression, although its distinctive pattern of expression during development suggests a role different from that of Id.
3548	mTFE3, an X-linked transcriptional activator containing basic helix-loop-helix and zipper domains, utilizes the zipper to stabilize both DNA binding and multimerization.	1732746	Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending.
3549	Search for peptide immunogens of the beta-subunit of human chorionic gonadotropin (hCG) capable of eliciting hormone specific and neutralizing antisera. Identification of an undecapeptide eliciting hCG-specific antisera.	1517013	The work reported herein describes our attempts to identify peptide immunogens of the beta-subunit of the pregnancy-specific placental hormone, human chorionic gonadotropin (hCG), capable of eliciting hormone specific and neutralizing antisera. Hydrophilicity profiles of the beta-subunits of hCG and the homologous pituitary hormone, human luteinizing hormone (hLH) were compared and two sequences which are hydrophilic but unique to hCG-beta were identified. They are 6-12 and 109-119 of hCG-beta. Results of the studies with the undecapeptide 109-119 of hCG-beta are reported in this paper. The undecapeptide amide was synthesized using the p-(acyloxy) benzhydrylamine resin and antisera to the peptide were elicited in rabbits using the peptide-diphtheria toxoid conjugate. The peptide is highly immunogenic as both the rabbits responded with high titers of antibodies to the peptide. The antipeptide antibodies bound to hCG but not to hLH showing thereby that the region 109-119 of hCG-beta is a unique determinant of hCG. However, the antibodies were found not to neutralize the biological activity of hCG.
3550	Human calcitonin gene regulation by helix-loop-helix recognition sequences.	1738589	Human calcitonin (CT) gene transcription is regulated by proximal 5' flanking sequences which mediate cAMP-induced expression, and by a distal basal enhancer region. Using transient expression of CT-CAT constructs, we showed that the basal enhancer is active in a CT-producing small cell lung cancer cell line (DMS53) and the thyroid C cell derived tumor line, TT, but is inactive in non-CT-producing cell lines. In deletional and direct mutational analyses of the distal enhancer region, disruption of two elements resembling recognition sequences for the helix-loop-helix (HLH) family of transcriptional regulatory proteins resulted in a significant loss of basal transcriptional enhancer action. These results suggest that HLH recognition motifs may mediate a significant portion of constitutive CT gene transcriptional activity in these cells. Nuclear protein extracts from DMS53 cells formed specific binding complexes with oligonucleotides containing two of these candidate enhancer sequences. However, proteins capable of binding to these CT gene HLH consensus recognition sites were not restricted to CT-producing cells. We conclude that members of the HLH protein family, some expressed ubiquitously and some expressed or activated in a tissue-restricted fashion, may combine to enhance CT gene transcription in tumor cells of neuroendocrine derivation.
3551	Progress and approaches in mapping the surfaces of human follicle-stimulating hormone: comparison with the other human pituitary glycoprotein hormones.	18407074	The pituitary glycoprotein hormones, including human follicle-stimulating hormone (hFSH), are involved in the physiologic functions of receptor binding, in subunit association during assembly and processing, and in untoward effects such as antibody formation during clinical intervention. These activities derive from specific macromolecular associations; a major research trend has been to map the surfaces of these glycoprotein hormones (hFSH, hLH, hCG, and hTSH) into discrete areas responsible for each activity by using a variety of biochemical approaches. Each surface domain or determinant of the hormone is likely to comprise discontinuous amino acid sequences, from one or both subunits, assembled into a precise, unique, macromolecular surface. The paradigm of antigen-antibody interaction may help to explain how the surfaces are assembled, how the common alpha-subunit combines with the unique beta-subunit of each, and how the receptor interacts with heterodimeric hormone.
3552	Helix-loop-helix proteins in the regulation of immunoglobulin gene transcription.	1739428	Transcriptional control of the Ig heavy chain and kappa light chain is dominated by single enhancers located within the body of each gene. These enhancers bind distinct, yet overlapping, sets of cell-type-specific and ubiquitous nuclear proteins. This review focuses on one particular subclass of enhancer-binding protein, termed helix-loop-helix (HLH) proteins, describing their role in the establishment of cell-type-specific transcription and suggesting how they may be regulated during B-cell maturation.
3553	Molecular characterization of NSCL, a gene encoding a helix-loop-helix protein expressed in the developing nervous system.	1729708	We report here the molecular cloning and chromosomal localization of an additional member of the helix-loop-helix (HLH) family of transcription factors, NSCL. The NSCL gene was identified based on its hybridization to the previously described hemopoietic HLH gene, SCL. Murine NSCL cDNA clones were obtained from a day 11.5 mouse embryo cDNA library. The coding region is 399 base pairs and encodes a predicted protein of 14.8 kDa. The nucleotide sequence shows 71% identity and the amino acid sequence shows 61% identity to murine SCL in the HLH domain. The NSCL protein-coding region terminates six amino acids beyond the second amphipathic helix of the HLH domain. Expression of NSCL was detected in RNA from mouse embryos between 9.5 and 14.5 days postcoitus, with maximum levels of expression at 10.5-12 days. Examination of 12- and 13-day mouse embryos by in situ hybridization revealed expression of NSCL in the developing nervous system. The NSCL gene was mapped to murine chromosome 1. The very restricted pattern of NSCL expression suggests an important role for this HLH protein in neurological development.
3554	[The human chorionic gonadotropin (hCG) receptor: a new class within the family of GTP protein coupled receptors. Epitope mapping of receptor-bound agonistic and antagonistic forms of hCG].	1636261	Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family. It is composed of an alpha and a beta subunit, the latter being closely homologous to that of human luteinizing hormone (hLH). HCG and hLH bind to the same receptor. The molecular mass of hCG is 38 kD, up to 30% of which is contributed by the carbohydrate moieties linked to each of the two subunits. Structure-function relationship studies have indicated that both subunits interact with the receptor and that altogether four different peptide domains (two on each subunit) are responsible for high-affinity receptor binding. The carbohydrate units are responsible for expression of agonist activity: deglycosylated hCG (degly-hCG) is unable to induce a biological response (cAMP increase), despite high-affinity binding. We have previously mapped the antigenic surface of hCG and described 14 different epitopes that can be recognized by specific monoclonal antibodies (MCA), i.e. five epitopes on the alpha subunit, five on the beta subunit and four epitopes which are formed through association of the two subunits (conformational or alpha beta epitopes). The number and topography of epitopes was found to be the same on degly-hCG. This framework of immunological coordinates was then applied to probe the orientation of hCG, as well as that of the competitive antagonist degly-hCG in their receptor-bound states. With a receptor-hormone-125I-MCA sandwich approach we found that while hCG still presented two of the 14 epitopes, i.e. beta 3 and beta 5, no epitope was accessible on receptor-bound degly-hCG. This differential pattern thus correlates with the signal transduction-competence of the respective ligand. Overall, these data indicate that most of the surface of hCG is masked by moieties of the receptor. Accordingly, that moiety must itself be folded in a way that it provides a large contact surface. No portion other than the 341-residue long extracellular domain would seem capable of doing that. Computer-assisted secondary structure predictions support this view. This domain is the novel feature that distinguishes glycoprotein-hormone receptors from the other members of the G protein-coupled receptor superfamily.
3555	Localization of murine HLH gene NSCL to chromosome 1 by use of recombinant inbred strains.	1617226	NaN
3556	A bacterial binding site which binds human chorionic gonadotropin but not human luteinizing hormone.	1576977	Exposed sites on Pseudomonas maltophilia (ATC #1637), which bind both human chorionic gonadotropin (hCG) and a native hCG-like ligand with equal high affinity, are described. This high-affinity binding site (Kd = 1.3 x 10(-10) binds hCG, but does not bind human luteinizing hormone (hLH), nor related human glycoprotein hormones, thyrotropin, and follicle stimulating hormone. A lower affinity, 2.3 x 10(-9), is also described which binds hLH and hCG equally well. This is the first description of a high-affinity binding site in nature, which distinguishes hCG from hLH.
3557	Stimulation of Candida albicans transition by human chorionic gonadotrophin and a bacterial protein.	1516564	Candida albicans, a dimorphic fungus, is involved commonly in human infections with the mycelium form more associated with pathogenicity. The influence of various hormones and a bacterial protein on the transition from blastospore to mycelium was assessed. Human luteinizing hormone (hLH), chorionic gonadotrophin (hCG), and an hCG-like material purified from a bacteria, Xanthomonas maltophilia (PCG), were able to increase the rate of transition when compared with the controls. The effect of the two hormones and the bacterial peptide were specific, as human follicle stimulating hormone (hFSH), thyroid stimulating hormone (hTSH), growth hormone (hGH), prolactin (hPrl) and rat and bovine LH (rLH, bLH), and bovine albumin and gamma globulin did not affect the transition. The binding of 125I-hCG or 125I-LH to spheroplasts of Candida albicans were competitively displaced by hCG, hLH, and PCG. Scatchard analysis of binding of all three ligands revealed two binding sites with a high-affinity nM Kd. Thus, hCG, hLH, and PCG induce transition of Candida albicans from a blastospore state to a mycelium form, suggesting that these hormones may modify the pathogenicity of Candida albicans.
3558	Members of the USF family of helix-loop-helix proteins bind DNA as homo- as well as heterodimers.	1450663	We have isolated human cDNA clones for USF2, a new member of the upstream stimulatory factor (USF) family of transcription factors. Analysis of these clones revealed the existence of highly conserved elements in the C terminal region of all USF proteins. These include the basic region, helix-loop-helix (HLH) motif, and, in the case of the human proteins, the C-terminal leucine repeat (LR). In addition, a highly conserved USF-specific domain is located immediately upstream of the basic region. Using in vitro translated proteins, we found that all members of the USF family bound DNA as dimers. The N-terminal portion of USF, including the USF-specific domain, was entirely dispensable for dimer formation and DNA-binding. However, deletion mutants of USF2 lacking the LR were deficient in DNA-binding activity. Interestingly, each of the USF proteins could form functional heterodimers with the other family members, including the sea urchin USF, which does not have a LR motif. This indicates that the conserved LR in human USF is not required for dimer formation, and influences only indirectly DNA-binding.
3559	Multimeric structures influence the binding activity of bHLH muscle regulatory factors.	1341052	Sucrose gradients and molecular sieve chromatography were used to determine the native molecular weight of the basic HLH proteins myogenin, MyoD and E12. The muscle bHLH proteins not only formed dimers but also associated in a variety of higher order complexes. Although homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer, homotetramers and larger forms do not recognize this DNA sequence. Little evidence for complexes larger than dimers was found for the ubiquitous bHLH protein E12. Most of the myogenin remains in large complexes when myogenin and E12 are mixed. The same result was obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin was found to be present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein that contains only the myogenin HLH region fused to glutathione-S-transferase also forms large homomeric complexes. A model to explain these results is that each helix of the HLH motif can associate with a different subunit to form chains or ring structures. The presence of myogenin in nuclear extracts as both dimers that recognize known DNA sequences as well as higher order complexes that do not raises significant issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.
3560	Activation of muscle-specific transcription by myogenic helix-loop-helix proteins.	1341046	Myogenin is a muscle-specific transcription factor that acts as a molecular switch to induce myogenesis. Myogenin shares homology with MyoD and other myogenic regulatory proteins within a basic region and helix-loop-helix (HLH) motif that mediate binding to a conserved DNA sequence (CANNTG) present in the regulatory regions of numerous muscle-specific genes. Binding of myogenin and other members of the MyoD family to DNA can be augmented upon heterodimerization with the widely expressed HLH protein E12. We have used the muscle creatine kinase (MCK) enhancer as a target to study the mechanism whereby myogenin activates muscle-specific transcription. Full activity of the MCK enhancer requires cooperative interactions between myogenin (or other myogenic HLH proteins that bind the same site) and a complex array of ubiquitous and cell type-specific nuclear factors. To define the domains of myogenin responsible for sequence-specific DNA binding, activation of muscle-specific transcription, and cooperativity with other transcription factors, we have generated an extensive series of mutants by site-directed mutagenesis and domain swapping. These mutants have revealed strong transcriptional activation domains in the N- and C-termini of myogenin that rely on a specific amino acid sequence within the DNA binding domain for activity. Myogenin's ability to induce muscle-specific transcription is subject to negative regulation by growth factor and oncogenic signals. Mechanisms through which growth signals may repress myogenin function are discussed.
3561	Tissue-specific expression of the skeletal alpha-actin gene involves sequences that can function independently of MyoD and Id.	1333317	The skeletal alpha-actin gene is a member of the sarcomeric contractile protein gene family and is specifically expressed in differentiated muscle. The skeletal alpha-actin gene is regulated efficiently by enhancer and regulatory sequences between nucleotide positions -1282 and -87. In the present study we have shown that the sequences 3' of nucleotide position -87 can functionally interact with the SV40 enhancer in a tissue-specific manner and can restrict the ubiquitous function of the SV40 enhancer to myogenic cells. Site-specific cassette mutagenesis was used to delimit the sequences upstream of the TATA motif (-32), between nucleotide positions -64 and -37, that mediate efficient expression in myogenic cells in the presence of the SV40 enhancer. The skeletal alpha-actin promoter was trans-activated by the helix-loop-helix (HLH) transcription factors MyoD, MRF-4, and Myogenin, in pluripotential 10T1/2 fibroblasts and trans-repressed by the HLH protein Id (inhibitor of differentiation) in myogenic C2C12 cells. This trans-regulation required sequences upstream of -87 and occurred independently of the two consensus E boxes (CANNTG) at positions +18 and +71. The -64/-37 region interacted with purified Sp1 and an unidentified protein(s), proximal regulatory factor(s) I (PRF-I). We conclude that the muscle-specific expression of the skeletal alpha-actin promoter is not simply determined by MyoD elements and enhancer and regulatory sequences, but that the minimal promoter contains important determinants of cell-specific transcription that can function independently of the helix-loop-helix transcription factors.
3562	Gonadotropin receptors in human ovarian follicles and corpora lutea throughout the menstrual cycle.	1330862	To study changes in the binding of gonadotropins to human follicles and corpora lutea, we have examined the binding of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the human ovary throughout the menstrual cycle, by both quantitative binding assays and surface binding autoradiography. The specific high-affinity low-capacity receptors for hLH were demonstrated in both human follicles and corpora lutea. Binding of 125I-hFSH was identified in the granulosa cells, but not in the thecal cells of the preantral and antral follicles at various stages of follicular development. The binding of 125I-hLH to the thecal cells increased during follicular development, and a dramatic increase was preferentially observed in the granulosa cells of the preovulatory follicles. In the corpora lutea, the binding of 125I-hLH increased from the early luteal phase to the midluteal phase and decreased towards the late luteal phase. The results of the present study suggest that changes in the binding of gonadotropins in the ovarian target cells during the menstrual cycle might play an important role in the regulation of follicular and luteal function.
3563	The mechanism of cyclosporine's action in the inhibition of testosterone biosynthesis by rat Leydig cells in vitro.	1310171	We have previously demonstrated that cyclosporine inhibits testosterone (T) biosynthesis in vivo. To better understand the mechanism by which CsA inhibits T synthesis, interstitial cells were isolated from rat testes and incubated in the standard medium 199 with or without CsA (0-10 micrograms/ml) in the presence or absence of human chorionic gonadotropin (hCG, 10(-7) M) and 8-bromo cyclic AMP (cAMP, 0.5 mM) for 3 hr at 32 degrees C. The levels of cAMP and T were determined by RIA. CsA did not inhibit the basal secretion of T, but inhibited hCG-stimulated T production in a dose-dependent manner (4 ng/10(6) cells vs. 10 ng/10(6) cells at a CsA dose of 5 micrograms/ml, P less than 0.05). Radioligand binding of 125I-hLH to testicular membranes was not affected by CsA, as CsA did not compete with hCG/LH for binding sites (25-28% binding with or without CsA). Similarly, the MIX-stimulated cAMP production was not affected by CsA (24.03 +/- 1.09 vs. 20.60 +/- 0.38 pmol/10(6) cells), suggesting that CsA does not inhibit the accumulation of the second messenger. However, when interstitial cells were incubated with CsA in the presence of cAMP, a significant dose-dependent decline in T secretion was observed (7 ng/10(6) cells vs. 20 ng/10(6) cells at a CsA dose of 5 micrograms/ml). To determine whether CsA inhibits the steps beyond cAMP stimulation of T secretion, the kinetic parameters (Km and Vmax) of steroidogenic enzymes, delta 4-3 keto-17 alpha hydroxylase (17 alpha-hydroxylase), and delta 4-3 keto-17 beta hydroxy steroid dehydrogenase (17B-HSD) were determined by using Michaelis Menten analysis. Results are shown in the presence of CsA vs. no CsA: Km and Vmax values for 17 alpha-hydroxylase were (2.32 vs. 7.98 microM) and (27.96 vs. 100.97 pmol/mg protein/min), respectively. For 17B-HSD the Km and Vmax were (2.14 vs. 1.52 microM) and (15 vs. 15 pmol/mg protein/min), respectively. These results indicate that CsA inhibits the activity of 17 alpha-hydroxylase uncompetitively and 17B-HSD activity competitively. In conclusion the primary site for CsA inhibition is the cAMP stimulation and, CsA inhibits T synthesis at multiple sites.
3564	The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.	1309591	Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.
3565	Dorsoventral development of the Drosophila embryo is controlled by a cascade of transcriptional regulators.	1299363	Maternal genes involved in dorsoventral (D/V) patterning of the Drosophila embryo interact to establish a stable nuclear concentration gradient of the Dorsal protein which acts as the morphogen along this axis. This protein belongs to the rel proto-oncogene and NF-KB transcriptional factor family and acts by controlling zygotic gene expression. In the ventral part of the embryo, dorsal specifically activates transcription of the gene twist and ventrally and laterally dorsal represses the expression of zerknüllt, a gene involved in the formation of dorsal derivatives. The extent of dorsal action is closely related to the affinity and the number of dorsal response elements present in these zygotic gene promoters. twist is one of the first zygotic genes necessary for mesoderm formation. It codes for a 'b-HLH' DNA-binding protein which can dimerize and bind to DNA in vitro and to polytene chromosomes in vivo. In addition, in cultured cells twist has been shown to be a transcriptional activator. Thus, the first events of embryonic development along the D/V axis are controlled at the transcriptional level.
3566	Repression of immunoglobulin enhancers by the helix-loop-helix protein Id: implications for B-lymphoid-cell development.	1944284	It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.
3567	The N-Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells.	1935896	Proteins encoded by the proto-oncogenes c-myc, L-myc, and N-myc contain at their carboxy-terminus a tripartite segment comprising a basic DNA binding region (BR), a helix-loop-helix (HLH) and a leucine zipper motif (Zip), that are believed to be involved in DNA binding and protein-protein interaction. The N-Myc oncoprotein is overexpressed in certain human tumors that share neuroectodermal features due to amplification of the N-myc gene. Using a monoclonal antibody directed against an N-terminal epitope of the N-Myc protein in immunoprecipitations performed with extracts of neuroblastoma cells, two nuclear phosphoprotein, p20/22, forming a hetero-oligomeric complex with N-Myc are identified. Both proteins are phosphorylated by casein kinase II in vitro. By partial proteolytic maps we show that p20 and p22 are structurally related to each other and that p20 is identical with Max, a recently described in vitro binding partner of myc proteins. Time course experiments show the presence of the complex in cellular extracts immunoprecipitated within a 5 min interval after the preparation of the cell extract. While the expression of N-myc is restricted, expression of both Max(p20/22) and the murine homolog Myn(p20/22) was observed in cells of diverse human and murine embryonal lineages as detected by heterologous complex formation. By introduction of expression vectors containing the wild type N-myc gene or N-myc genes with in frame deletions or point mutations into recipient cells and subsequent immunoprecipitation of the resulting N-Myc proteins we show that the HLH-Zip region is essential to the formation of the N-Myc-p20/22 complex.
3568	The helix-loop-helix protein rE12 and the C/EBP-related factor rNFIL-6 bind to neighboring sites within the c-fos serum response element.	1766666	We show that members of two major families of transcription factors, the helix-loop-helix and C/EBP families, interact with the c-fos serum response element (SRE). Two cDNA clones encoding SRE binding factors (clones 9 and 21) were isolated by the direct screening of a PC12 lambda gt11 cDNA library using SRE oligonucleotide sequences as probes. Clone 9 encodes the rat homolog of the human HLH transcription factor, E12 (called here rE12). Clone 21 encodes a b-zip domain polypeptide that is related to the liver transcription factor C/EBP, and is homologous to the human NFIL-6 transcription factor (called here rNFIL-6). Using in vitro-translated products we show that rNFIL-6 recognizes a 'CCAATT' motif which overlaps the c-fos dyad symmetry element (DSE), the binding site for serum regulatory factor (SRF). Factor rE12 binds to an E-box enhancer sequence, 'CATCTG', immediately adjacent to the rNFIL-6 site, within the SRE. Antibodies specific to rE12 and rNFIL-6 disrupt nucleoprotein complexes with these DNA-binding sites, confirming the interaction of native in vivo factors. We present evidence that rNFIL-6 and SRF binding are mutually exclusive, consistent with the overlap of their binding sites. The demonstration that rE12 and rNFIL-6 bind to the SRE at sites adjacent to the major c-fos regulatory element, the DSE, raises the possibility that helix-loop-helix and C/EBP families regulate the SRE and provide a new basis for the multifunctional properties of the SRE, including possible tissue specificity of expression.
3569	TFEB has DNA-binding and oligomerization properties of a unique helix-loop-helix/leucine-zipper family.	1748288	The DNA-binding factor TFEB contains adjacent helix-loop-helix (HLH) and leucine zipper (LZ) domains flanked by an upstream basic region. This arrangement of interactive motifs has recently been observed in several other transcription factors and in the Myc family of oncogenes. TFEB was isolated by virtue of its binding to the major late promoter of adenovirus. DNA binding by a soluble protein was achieved by deleting a hydrophobic amino-terminal domain and permitted the structural analysis of the oligomerization and DNA-binding properties of TFEB. TFEB specifically bound DNA as both a homodimer and a heterodimer with another b-HLH-LZ protein TFE3. The LZ domain was essential for homo- or hetero-oligomerization and high-affinity DNA binding. In the absence of DNA a tetramer-sized form of TFEB was observed that dissociates to bind added DNA as a dimer. Binding by TFEB and TFE3 to related, but different, naturally occurring DNA target sequences was observed with distinct binding preferences. Analysis of basic domain residues in this family of proteins revealed a pattern of sequence conservation predictive of an interacting alpha-helical face. Common oligomerization and DNA-binding features suggest the b-HLH-LZ domain structure to define a distinct family of DNA-binding factors.
3570	The Drosophila segmentation gene runt acts as a position-specific numerator element necessary for the uniform expression of the sex-determining gene Sex-lethal.	1748277	Female development in Drosophila is established through the activation of the X:A target gene Sex-lethal (Sxl) by an X:A ratio of 1. X-linked zygotic genes, termed numerator elements, comprise part of the X:A ratio and are primarily responsible for the activation of Sxl in females. We demonstrate that the X-linked segmentation gene runt is required for this process and has genetic and molecular properties of a numerator element. Genetically, runt has vital dose-dependent interactions with components of the X:A ratio and alterations in runt activity alter the sexual phenotype of triploid intersexes. Molecularly, loss of runt activity results in a failure to activate appropriately Sxl in the central region of female embryos. We also show that Sxl activation is influenced by the maternal anterior and terminal pattern-forming genes, bicoid (bcd) and torso (tor). These results indicate that the "uniform" activation of Sxl requires input from nonuniformly distributed products. We have demonstrated that runt is one such product and suggest that other genes with nonuniform input exist. runt is distinguished from previously identified regulators of Sxl by its nonuniform role and by the absence of an identifiable helix-loop-helix (HLH) motif, indicating that the activation of Sxl is not controlled solely by HLH proteins.
3571	Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.	1659530	Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.
3572	Expression of Id, a negative regulator of helix-loop-helix DNA binding proteins, is down-regulated at confluence and enhanced by dexamethasone in a mouse osteoblastic cell line, MC3T3E1.	1719977	The message of Id (for "inhibitor of DNA binding") was expressed in an osteoblastic cell line, MC3T3E1, when the cells were in early cultures, while undetectable after the cells became confluent. The abundance of Id message in MC3T3E1 cells in early cultures was increased when the cells were treated with dexamethasone. This effect was time and dose dependent in a range between 10(-11)M and 10(-7)M. Id message expression was not enhanced by TGF-beta, 1,25-dihydroxyvitamin D3, retinoic acid, interleukin 1-alpha, interleukin 6, tumor necrosis factor-alpha or parathyroid hormone. These observations indicate for the first time the presence of HLH protein family member, Id, in osteoblast-like cells and its regulation by dexamethasone.
3573	Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins.	1922066	The DNA binding activities of some basic region and putative helix-loop-helix (bHLH)-containing transcriptional factors can be inhibited by the Id protein. Because Id contains the HLH motif for dimerization but not the basic amino acid region for DNA binding, heterodimers of Id with bHLH transcriptional factors may not bind to DNA. We have isolated and characterized the gene and cDNA clones for a new Id protein, designated Id2. The Id2 protein contains a helix-loop-helix motif similar to that of the previously described Id protein (referred to here as Id1), but the two proteins are different elsewhere. Id1 and Id2 are encoded by two unlinked genes, as shown by chromosome mapping. The two Id proteins have similar inhibitory activities. They selectively bind to and inhibit the function of one set of bHLH proteins, typified by E2A.E47 and E2B.m3, but not that of the other set, including TFE3, USF, and AP4. The Id proteins also homodimerize poorly. Expression of both Id genes is down-regulated during differentiation in a variety of cell types.
3574	Two genetically and molecularly distinct functions involved in early neurogenesis reside within the Enhancer of split locus of Drosophila melanogaster.	1752423	Molecular correlation of the genetic aspects of the function of the neurogenic gene Enhancer of split [E(spl)] has previously been hampered by the densely transcribed nature of the chromosomal region within which it resides. We present data indicating that two distinct molecular species contribute to E(spl) function. Analysis of new E(spl) alleles has allowed us to define two complementing functions within the locus. Subsequent phenotypic analysis of different E(spl) deficiencies combined with P element-transformed constructs has demonstrated that these two functions correspond to: (1) a family of helix-loop-helix (HLH) protein-encoding genes and (2) the single copy gene E(spl) m9/10, whose product shares homology with G-protein beta subunits. The zygotically active E(spl) HLH genes can, at least partially, substitute for one another's functions and their total copy number determines the activity of the locus. E(spl) m9/10 acts synergistically with the E(spl) HLH genes and other neurogenic genes in the process of neurogenesis. The maternal component of E(spl) m9/10 has the most pronounced effect in neurogenesis, while its zygotic component is predominantly required during postembryonic development. The lethality of trans-heterozygotes of null E(spl) deficiency alleles with a strong Delta point mutation is a result of the concomitant reduction in activity of both E(spl) HLH and m9/10 functions. Immunocytochemical localization of the E(spl) m9/10 protein has revealed that it is a ubiquitously distributed nuclear component in embryonic, larval and imaginal tissues.
3575	The four human muscle regulatory helix-loop-helix proteins Myf3-Myf6 exhibit similar hetero-dimerization and DNA binding properties.	1945842	The muscle regulatory proteins Myf3, Myf4, Myf5, and Myf6 share a highly conserved DNA binding and dimerization domain consisting of a cluster of basic amino acids and a potential helix-loop-helix structure. Here we demonstrate that the four human muscle-specific HLH proteins have similar DNA binding and dimerization properties. The members of this family form protein complexes of comparable stability with the ubiquitously expressed HLH proteins E12, E2-2, and E2-5 and bind to the conserved DNA sequence CANNTG designated as E-box with similar efficiency in vitro. The binding affinities of the various complexes are greatly influenced by the variable internal and flanking nucleotides of the consensus motif. Combinations of Myf proteins with one another and with lyl-1, and HLH protein from human T cells, do not bind to DNA in vitro. Our results suggest that combinatorial associations of the various tissue-specific and more widely expressed HLH factors do not result in differential recognition of DNA sequences by Myf proteins.
3576	The helix-loop-helix domain: a common motif for bristles, muscles and sex.	1755826	Three apparently unrelated developmental processes--mammalian myogenesis, the choice of neural fate and sex determination in Drosophila--are controlled by a common mechanism. Most of the genes governing these processes encode transcriptional factors that contain the helix-loop-helix (HLH) motif. This domain mediates the formation of homo- or heterodimers that specifically bind to DNA through a conserved basic region adjacent to the HLH motif. Dimers differ in their affinity for DNA and in their ability to activate transcription from HLH binding-site containing promoters. In addition, the activity of HLH proteins is inhibited by dimerization with another class of HLH proteins that lack a basic domain entirely or have an altered one. These structural properties provide a molecular mechanism to explain the synergistic and antagonistic functional relations among the HLH encoding genes that control several developmental pathways.
3577	Immunohistochemistry of the pituitary pars distalis of the musk shrew, Suncus murinus.	1663882	Immunocharacteristics of the pituitary pars distalis cell types of the musk shrew, Suncus murinus, were studied by the unlabeled antibody enzyme technique, using peroxidase-antiperoxidase or avidin-biotin-peroxidase complex. The thyrotropin (TSH)-, gonadotropin (GTH)-, corticotropin (ACTH)-, prolactin (PRL)-, and growth hormone (GH)-secreting cells of the PD were identified on the basis of their immunoreactivity with different heterologous antisera. The TSH cells showed specific immunoreactivity with antisera against human (h) TSH beta and rat (r) TSH beta. Cells showing immunoreactivity with the antisera against hLH beta and ovine (o) LH beta were designated as GTH cells as no immunoreactivity was observed with antisera against hFSH beta and oFSH beta. The ACTH cells as well as the cells of the pars intermedia were revealed by anti-ACTH1-24 and anti-ACTH1-10 sera. Whereas the PRL cells were recognized by their immunoreactivity with antisera against hPRL and oPRL, the GH cells were identified with anti-hGH, anti-oGH, and anti-bovine (b) GH sera. TSH and GTH, TSH and ACTH, GTH and ACTH, ACTH and GH, ACTH and PRL, and GH and PRL cells were visualized in the same section using the dual immunoperoxidase technique. Comparison of the immunohistochemically identified cells with those described histochemically reveals several discrepancies, which expose the limitations of the latter techniques identifying adenohypophysial cells.
3578	Helix-loop-helix genes translocated in lymphoid leukemia.	1949153	A new class of DNA-binding proteins with a helix-loop-helix (HLH) structure has recently been described. Many of these transcriptional regulators are known to play a central role in cell-specification and differentiation processes. Four members of the HLH family are now implicated in the development of human lymphoid malignancies as a result of aberrant expression following chromosomal translocation events. This review focuses on two of these family members: SCL and LYL-1.
3579	The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes.	1764999	In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three 'E box' consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.
3580	The purification and development of a radioimmunoassay for beta-core fragment of human chorionic gonadotrophin in urine: application as a marker of gynaecological cancer in premenopausal and postmenopausal women.	1719119	The beta-core fragment of human chorionic gonadotrophin (hCG) is a major part of the immunoreactive hCG-like material found in the urine of normal pregnant women. Patients with non-trophoblastic gynaecological malignancies have been found to have raised levels of urinary beta-core. We describe the purification of beta-core, the preparation of a polyclonal sheep antiserum and the development of radioimmunoassay. The minimum detection limit of this assay was 0.025 micrograms beta-core/l. There was no significant cross-reaction with the free alpha-subunit, hLH, hFSH and hTSH (less than 0.7%), and only partial cross-reaction with intact hCG and free beta-subunit of hCG (6.9 and 18%). Within-assay variability ranged from 2.03 to 12.5% and between-assay variability from 2.25 to 13.4%. The assay was applied to urine samples from 92 normal non-pregnant premenopausal women, 54 normal postmenopausal women and 65 women with active gynaecological disease (47 postmenopausal and 18 premenopausal). In normal premenopausal women the values ranged from less than 0.025 to 0.62 micrograms beta-core/l (median 0.043 micrograms beta-core/l). The values for normal postmenopausal women ranged from less than 0.025 to 0.64 micrograms beta-core/l (median 0.26 micrograms beta-core/l). Postmenopausal women with gynaecological malignancy had values which ranged from less than 0.025 to 4.0 micrograms/beta-core/l (median 0.31 micrograms beta-core/l); premenopausal women in this group had values which ranged from less than 0.025 to 1.15 micrograms beta-core/l (median 0.12 micrograms beta-core/l). On molecular sieve chromatography, the material found in the urine of normal postmenopausal women showed the physicochemical characteristics of authentic beta-core.(ABSTRACT TRUNCATED AT 250 WORDS)
3581	Binding sites for LH in Candida albicans: comparison with the mammalian corpus luteum LH receptor.	1919390	We have described recently the presence of binding sites for human LH (hLH) and human chorionic gonadotrophin (hCG) in microsomal and cytosol fractions prepared from the dimorphic, pathogenic fungus, Candida albicans. We have now compared the properties of Candida LH/hCG-binding sites with those of the ovine luteal LH receptor. Sheep luteal LH-binding sites were associated with luteal membranes, and little or no binding activity was present in cytosol fractions. In contrast, significant LH/hCG-binding activity was present in Candida cytosol. Moreover, there were marked differences in sensitivity to inhibition by metal ions, association and dissociation rates, and affinity constants between sheep and Candida LH-binding sites. Scatchard plots of 125I-labelled hLH binding to sheep luteal receptors demonstrated a single high-affinity component (association constant (Ka) 0.3 litres/pmol) which was displaceable by hCG. In contrast, Scatchard plots of binding to Candida microsomes and cytosol fractions demonstrated two components, one with high affinity (Ka 0.18 litres/pmol) and low capacity and a second site with lower affinity (Ka 4 litres/nmol) and high capacity, both of which were displaceable by unlabelled hCG. Gel permeation chromatography of cytosol demonstrated two distinct peaks of LH-binding activity with approximate molecular weights of greater than 1,000,000 and 30,000-50,000. Scatchard plots of 125I-labelled hLH binding to the higher molecular weight peak demonstrated a single, high-affinity LH-binding site (Ka 0.18 litres/pmol), whereas the lower molecular weight fraction contained both high- (Ka 0.17 litres/pmol) and low-affinity (Ka 4 litres/nmol) LH-binding sites. Both partially purified and highly purified hCG and hLH preparations displaced binding of 125I-labelled hLH and hCG to sheep luteal LH receptors at similar concentrations. 125I-labelled hLH/hCG binding to Candida membranes was also displaceable by low levels (ng) of partially purified hCG preparations, but much higher levels (micrograms) of highly purified hLH and hCG were required. This paradoxical observation suggested the presence of radiolabelled contaminants, or damaged forms induced during radioiodination of hormone tracers, which can bind more strongly to Candida membranes than unlabelled hCG and hLH but which do not bind to sheep LH receptors. However, no evidence for hLH tracer contaminants with differential binding to Candida and sheep luteal receptors was obtained following gel exclusion chromatography or fractionation on Concanavalin A-Sepharose. (Although three distinct 125I-labelled hLH fractions were resolved on Concanavalin A-Sepharose, presumably reflecting differences in their carbohydrate compositions, all three tracer peaks bound equivalently to both Candida membranes and ovine luteal LH receptors).(ABSTRACT TRUNCATED AT 400 WORDS)
3582	Two site-specific deletions and t(1;14) translocation restricted to human T-cell acute leukemias disrupt the 5' part of the tal-1 gene.	1886719	Analysis of several cases of t(1:14)(p32;q11) translocation present in 3% of T-cell acute leukemias (T-ALL) has revealed the tal-1 gene. This gene encodes a helix-loop-helix protein. It has been found to be expressed in normal bone marrow and in leukemic T-cell and erythroleukemia cell lines, but not in normal T cells. Recently, a site-specific deletion, tald, renamed tald1 in this paper, has been detected in a high proportion of pediatric T-ALL, which arose by a site-specific DNA recombination between tal-1 and a new locus termed SIL. In this study we searched for structural rearrangements within tal-1 in a panel of 134 non-selected leukemic patients (including 66 with T-ALL). Only 6% of patients with T-ALL harbored the tald1 deletion. A second specific deletion termed tald2 was observed in another 6% of T-ALL patients; it involves another site within tal-1 plus the same site as tald1 in the SIL locus. Similarly to tald1 deletion, tald2 junctions harbor structural characteristics that are reminiscent of aberrant recombinase activity. Moreover, we report a detailed analysis of the tal-1 gene structure. Transcription analysis and in vitro translation data are consistent with the differential expression of several TAL-1 protein species containing the HLH motif but differing in their amino terminus. Taken together, our data indicate that t(1;14) translocations and both tald deletions disrupt the 5' part of the tal-1 gene, placing its entire coding sequences under the control of the regulatory elements of the TCR-delta gene or the SIL gene, both of which are normally expressed in T-cell lineage.
3583	Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.	1786292	The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)
3584	Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.	1649701	In this report we provide four lines of evidence indicating that E12/E47-like proteins interact in vivo with the myogenic HLH proteins MyoD and myogenin. First, cotransfection of MyoD and E47 in COS cells indicates that these factors synergistically enhance transcription of a reporter gene containing an oligomerized MyoD-binding site. Second, mobility-shift assays of muscle cell nuclear extracts, "double shifted" with specific antisera, have identified complexes binding to the MEF1 site that contain either MyoD or myogenin in association with E12/E47-like proteins. Third, association with E47 alters the phosphorylation state of MyoD. Fourth, C3H10T1/2 cells expressing antisense E2A transcripts contain low levels of E2A gene products and display less terminal muscle differentiation when infected with retroviral MyoD or when challenged to differentiate with 5-azacytidine treatment. In addition we demonstrate that MyoD, in conjunction with E12/E47-like proteins, is functioning as a regulatory nodal point for activation of several other downstream muscle regulators.
3585	Distribution and characterization of helix-loop-helix enhancer-binding proteins from pancreatic beta cells and lymphocytes.	1861981	Transcription of a number of mammalian genes is controlled in part by closely-related DNA elements sharing a CAxxTG consensus sequence (E boxes). In this report, we survey cell extracts from a variety of mammalian cell lineages for ability to bind to the E box denoted IEB1/kappa E1, which plays an important role in expression of both insulin and immunoglobulin kappa genes. Insulin enhancer factor 1 (IEF1), a binding activity previously identified in beta cells, was also present in pituitary endocrine cells but absent in 7 other mammalian cell lines tested. A distinct binding activity, lymphoid enhancer factor 1 (LEF1), was observed in several lymphoid cell lines, but was absent from all nonlymphoid cells tested. IEF1 and LEF1 were distinct according to electrophoretic mobility, and DNA binding specificity. As previously reported, both beta cell and lymphoid cell factors are recognized by antibodies to helix-loop-helix (HLH) proteins, indicating that they may contain functional helix-loop-helix dimerization domains. To directly demonstrate this, we showed that the binding factors are able to interact in vitro with the HLH domain of a characterized HLH protein. These results support the notion that HLH proteins play a key role in cell-specific transcriptional regulation in cells from endocrine and lymphocyte lineages.
3586	CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins.	1893793	CENP-B is a centromere associated protein originally identified in human cells as an 80 kDa autoantigen recognized by sera from patients with anti-centromere antibodies (ACA). Recent evidence indicates that CENP-B interacts with centromeric heterochromatin in human chromosomes and may bind to a specific subset of human alphoid satellite DNA. CENP-B has not been unambiguously identified in non-primates and could, in principal, be a primate-specific alphoid DNA binding protein. In this work, a human genomic DNA segment containing the CENP-B gene was isolated and subjected to DNA sequence analysis. In vitro expression identified the site for translation initiation of CENP-B, demonstrating that it is encoded by an intronless open reading frame (ORF) in human DNA. A homologous mouse gene was also isolated and characterized. It was found to possess a high degree of homology with the human gene, containing an intronless ORF coding for a 599 residue polypeptide with 96% sequence similarity to human CENP-B. 5' and 3' flanking and untranslated sequences were conserved at a level of 94.6% and 82.7%, respectively, suggesting that the regulatory properties of CENP-B may be conserved as well. CENP-B mRNA was detected in mouse cells and tissues and an immunoreactive nuclear protein identical in size to human CENP-B was detected in mouse 3T3 cells using human ACA. Analysis of the sequence of CENP-B revealed a segment of significant similarity to a DNA binding motif identified for the helix-loop-helix (HLH) family of DNA binding proteins. These data demonstrate that CENP-B is a highly conserved mammalian protein that may be a member of the HLH protein family and suggest that it plays a role in a conserved aspect of centromere structure or function.
3587	C-myc and the yeast transcription factor PHO4 share a common CACGTG-binding motif.	1861859	The basic-helix-loop-helix (b-HLH) motif is common to a number of proteins involved in transcriptional regulation and cell-type determination. The b-HLH motif is also present in the S. cerevisiae transcription factor PHO4 which positively regulates the acid phosphatase gene PHO5. In this report we show that the b-HLH region of PHO4 is sufficient to confer specific DNA-binding to the sequence CACGTG and, by comparison of the basic regions of PHO4 with those of other recently isolated CACGTG-binding proteins, we identify a specific subset of conserved amino acids in the basic region likely to confer DNA-binding specificity. On the basis of these observations we predict successfully the effect of substituting the PHO4 basic region with that from c-myc and show that the chimaeric protein activates transcription from the CACGTG elements present in the PHO5 UAS. From these data it is clear that the myc basic region confers specific binding to the sequence CACGTG.
3588	Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.	1646392	Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.
3589	Structure, chromosome mapping, and expression of the mouse Lyl-1 gene.	2067848	The mouse Lyl-1 gene was cloned and shown to consist of four exons with extensive nucleotide and structural homology to the human LYL1 gene. The Lyl-1 gene was localized to the central region of mouse chromosome 8 which defines a new region of synteny with human chromosome 19p. The predicted mouse Lyl-1 protein is 78% identical to human LYL1. The region of highest similarity occurs in the basic DNA binding and helix-loop-helix dimerization motifs which are nearly identical in mouse and man differing by only one conservative amino acid substitution. Expression of the Lyl-1 gene was found to be low in murine spleen and undetectable in other tissues by Northern blot analysis. In lymphoid cell lines, Lyl-1 was expressed in most B lineage cells but downregulated during terminal differentiation and was not expressed in most T lineage cells. In a human T ALL cell line carrying a translocation that juxtaposed LYL1 with the beta TCR gene, the translocated LYL1 gene was transcriptionally active whereas the nontranslocated gene was transcriptionally silent. We conclude that LYL1 has the properties of a lineage- and differentiation-specific HLH protein that contributes to T-cell neoplasia through its deregulated expression following chromosomal translocation.
3590	Regulation of achaete-scute gene expression and sensory organ pattern formation in the Drosophila wing.	2044964	Adult Drosophila possess a large number of sensory organs, including large and small bristles and other types of sensilla, each arising from a single mother cell at particular positions in a reproducible pattern. Genetic studies have shown that sensory organ pattern formation is partly coordinated by a number of structurally similar, potential heterodimer-forming, helix-loop-helix (HLH) regulatory proteins. Here, by localizing regulatory gene expression during the development of normal and mutant imaginal discs, we show that two positive regulators of sensory neurogenesis, the proneural achaete and scute proteins, initially trans-activate each other and are transiently expressed in identical patterns, including clusters of wing ectodermal cells and the individual sensory mother cells that arise from them. Two negative regulators, hairy and extramacrochaete, suppress sensory neurogenesis by selectively repressing achaete and scute gene expression, respectively, but in different spatial domains and at different developmental stages. Surprisingly, we also find that the level of achaete-scute activity influences the level of hairy expression, thereby providing feedback control upon achaete-scute activity and sensory organ formation. Some or all of these interactions may involve specific dimerization reactions between different combinations of HLH proteins.
3591	The leucine zipper of TFE3 dictates helix-loop-helix dimerization specificity.	2044953	TFE3 is a DNA-binding protein that activates transcription through the muE3 site of the immunoglobulin heavy-chain enhancer. Its amino acid sequence reveals two putative protein dimerization motifs: a helix-loop-helix (HLH) and an adjacent leucine zipper. We show here that both of these motifs are necessary for TFE3 to homodimerize and to bind DNA in vitro. Using a dominant negative TFE3 mutant, we also demonstrate that both the HLH and the leucine zipper motifs are necessary and sufficient for protein-protein interactions in vivo. TFE3 is unable to form stable heterodimers with a variety of other HLH proteins, including USF, a protein that is structurally similar to TFE3 and binds a common DNA sequence. The analysis of "zipper swap" proteins in which the TFE3 HLH was fused to the leucine zipper region of USF indicates that dimerization specificity is mediated entirely by the identity of the leucine zipper and its position relative to the HLH. Hence, in this "b-HLH-zip" class of proteins, the leucine zipper functions in concert with the HLH both to stabilize protein-protein interactions and to establish dimerization specificity.
3592	Enhancer-binding activity of the tal-1 oncoprotein in association with the E47/E12 helix-loop-helix proteins.	2038315	Almost 30% of patients with T-cell acute lymphoblastic leukemia (T-ALL) bear structural alterations of tal-1, a presumptive proto-oncogene that encodes sequences homologous to the helix-loop-helix (HLH) DNA-binding and dimerization domain. Analysis of the tal-1 gene product reveals that its HLH domain mediates protein-protein interactions with either of the ubiquitously expressed HLH proteins E47 and E12. The resultant tal-1/E47 and tal-1/E12 heterodimers specifically recognize the E-box DNA sequence motif found in eucaryotic transcriptional enhancers. Hence, the tal-1 protein shares biochemical properties with other tissue-specific HLH proteins that control cell type determination during myogenesis (e.g., MyoD1) and neurogenesis (e.g., achaete-scute). The data suggest that HLH heterodimers involving tal-1 may function in vivo as transcriptional regulatory factors that influence cell type determination during hematopoietic development.
3593	Evaluation of 29 monoclonal and polyclonal antibodies used in the diagnosis of pituitary adenomas. A collaborative study from pathologists of the Club Français de l'Hypophyse.	1923943	Fifteen polyclonal antibodies (pAbs) and 14 monoclonal antibodies (mAbs) directed against hGH, hPRL, beta hFSH, beta hLH, beta hTSH and alpha-subunit were assessed by five different laboratories on normal and adenomatous pituitary tissues. This study aims at providing pathologists with a selected panel of antisera suitable for diagnosis, and appreciating the interest of the recently introduced mAbs. All the anti-hGH Abs proved to be specific (3 pAbs and 4 mAbs); three mAb out of four gave a few false-negative reactions. Three out of six polyclonal anti-hPRL showed cross-reactivity with hGH; anti-hPRL mAbs gave a strong staining with no false-negativity detected so far. MAbs proved to be more efficient for detecting glycoprotein hormones and alpha subunit than pAbs, which, in several cases, gave widespread cross-reactivity. This lack of specificity could explain the noticeable discrepancies reported so far in the appraisal of gonadotropic and somatoprolactinic adenomas.
3594	Transcriptional regulation of HLA-DRA gene.	1754710	Transcriptional regulation of class II genes is complex. DRA, for example, is expressed in both tissue-specific and lymphokine-inducible fashion. High levels of expression in B and activated T cells depend upon a lymphoid-specific transcriptional enhancer located in the first intron and on the upstream and downstream promoter elements. Conserved upstream promoter Z and X sequences (CUS) are the main determinants of this complex regulation. At least four distinct trans-acting factors that might be differentially expressed in various cell types bind to these Z and X boxes. These are members of JUN, helix-loop-helix (HLH), C/EBP and ETS families of proteins.
3595	Baculovirus-expressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer.	1645648	Two recombinant baculoviruses BcV-myf4 and BcV-myf5 have been constructed to synthesize the human myogenic determination factors myogenin (myf4) and myf5 in eucaryotic cells. Both recombinant proteins are localized to the nucleus of virus-infected Spodoroptera frugiperda (sf) insect cells and can be recovered as soluble factors. The virus-produced proteins exhibit high-affinity binding to a muscle-specific DNA sequence in the presence of the ubiquitous helix-loop-helix (HLH) protein E12, but only marginal binding in unsupplemented sf nuclear extracts. Both baculovirus-encoded myogenic factors are able to heterooligomerize with E12 in the absence of DNA-binding sites. We conclude from our results that these muscle-specific HLH proteins produced in eucaryotic cells largely depend on dimerization with E12 or similar HLH proteins to recognize the myosin-light-chain-enhancer-MEF-1-binding site. We have no evidence for intracellular protein modifications exerting major effects on the interaction between these factors and DNA.
3596	Expression from the murine p53 promoter is mediated by factor binding to a downstream helix-loop-helix recognition motif.	1851994	Expression of the p53 gene plays an important role in the regulation of cellular proliferation and malignant transformation. Overexpression of mutant forms of p53 is in fact a common feature of many transformed cells. Studies dealing with the transcriptional regulatory regions of the p53 gene indicate that, unlike most promoters transcribed by RNA polymerase II, the p53 promoter contains no TATA-like sequence upstream of the transcription start site. Here we demonstrate that the murine p53 promoter contains a cis-acting element that maps downstream to the transcription initiation site. The integrity of this element is required for high-level expression from the promoter in transformed cells. By DNase I protection and mobility-shift analysis, we show that a nuclear factor binds to this downstream element through the consensus recognition sequence for the helix-loop-helix (HLH)-containing proteins of the myc/MyoD family of transcriptional regulators. We propose that the activity of one or more members of this family of transcription factors is an important determinant in the expression of p53 and that at least one level of p53 overexpression in transformed cells may thus be due to aberrant expression of the relevant factor(s). Furthermore, the possibility that the regulation of expression of p53 occurs, in part, by means of a potential HLH-containing factor provides a possible mechanism for the suppression of proliferation by the MyoD family of transcriptional regulators.
3597	Role of luteal cell nucleus in the expression of gonadotropin action.	1651965	Gonadotropin receptors are not only present in cell membranes, but also in nuclei of bovine and human luteal cells. hCG/hLH can directly regulate several nuclear functions. To further investigate the role of luteal cell nucleus in the expression of gonadotropin action, the effect of enucleation of luteal cells on gonadotropin receptors and gonadotropin response was studied. Luteal cytoplasts were prepared by colchicine treatment of purified whole luteal cells followed by centrifugation at 37 C in a Percoll gradient. The cytoplasts were 85 to 90% pure with a recovery of about 57%. Cytoplasts were viable as determined by trypan blue exclusion (87%) and metabolically competent as determined by 3H-leucine incorporation into proteins. On the day of preparation, the viability and metabolic competency of cytoplasts were similar to control cells, i.e. untreated and colchicine treated whole luteal cells. In addition, cytoplasts and control cells showed a similar decline in number and viability during storage at 4 C. While control cells continue to be metabolically competent, cytoplasts showed a dramatic decline by 48 h of storage at 4 C. Neither the cytoplasts nor control cells degraded 125I-hCG. The kinetics of 125I-hCG association and dissociation, specificity and affinity of binding to cytoplasts were similar to control cells. However, the number of available gonadotropin receptors in cytoplasts was significantly lower than in control cells. Cytoplasts contained lower progesterone levels and more importantly, they could not be stimulated by 10 nM hCG or 10 mM dibutyryl cyclic AMP to produce more progesterone. Controls cells, on the other hand, contained higher progesterone levels and responded to hCG and dibutyryl cyclic AMP stimulation. In summary, removal of nuclei from luteal cells results in a partial loss of gonadotropin receptors and complete loss of steroidogenic response to hCG and dibutyryl cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)
3598	Association of Myn, the murine homolog of max, with c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation.	1840505	Myn, a novel murine approximately 18 kd basic/helix-loop-helix/"leucine zipper" (B/HLH/LZ) protein, forms a specific DNA-binding complex with the c-Myc oncoprotein through the HLH/LZ motif in both proteins. c-Myc/Myn recognizes a c-Myc-binding site (GACCACGTGGTC) with higher affinity than either protein by itself. CpG methylation of the recognition site greatly inhibits DNA binding, suggesting that DNA methylation may regulate the c-Myc/Myn complex in vivo. In 3T3 fibroblasts, Myn mRNA levels are induced several-fold by serum with delayed early kinetics, suggesting regulation by immediate-early gene products. Coexpression of Myn in a myc/ras rat embryo fibroblast focus formation assay specifically augmented c-myc transforming activity. We suggest that interaction of Myn with c-Myc stabilizes sequence-specific DNA binding in vivo.
3599	Transforming growth factor beta represses the actions of myogenin through a mechanism independent of DNA binding.	1850837	Myogenin belongs to a family of regulatory factors that can activate myogenesis when transfected into nonmyogenic cells. A conserved DNA sequence, known as an E box, serves as the target for binding and trans-activation by myogenin. Using 10T1/2 fibroblasts that constitutively express a transfected myogenin cDNA, we show that myogenin accumulates in the nucleus but is unable to initiate myogenesis when cells are maintained with transforming growth factor beta (TGF-beta) or high serum. Although the final effect of TGF-beta and high serum--inhibition of myogenesis--was the same, their effects on the DNA-binding properties of myogenin in vitro differed. TGF-beta did not affect the ability of myogenin to bind DNA, whereas serum diminished the in vitro DNA-binding activity of myogenin. The helix-loop-helix (HLH) protein Id, postulated to inhibit DNA binding of other HLH proteins, was induced by high serum but not by TGF-beta. The presence of Id correlated with the failure of myogenin to bind the muscle creatine kinase enhancer in vitro. These findings suggest that serum can inhibit myogenesis by attenuating the DNA-binding activity of myogenin, possibly as a consequence of Id protein expression, whereas TGF-beta acts through a mechanism distal to DNA sequence recognition by myogenin and independent of Id.
3600	Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter.	1850096	Recent studies have shown that two genes regulating myogenesis (MyoD and myogenin) are coexpressed with cardiac alpha-actin during early stages of skeletal muscle development. Myogenin and MyoD are members of a family of regulatory proteins which share a helix-loop-helix (HLH) motif required for dimerization and DNA binding. Myogenin and MyoD form heterodimers with the ubiquitous HLH protein E12 which bind cis-acting DNA elements that have an E box (CANNTG) at their core. E boxes are present in the control regions of numerous muscle-specific genes, although their functional importance in regulating many of these genes has not yet been evaluated. In this report we examine the possibility that myogenin (or MyoD) directly transactivates the cardiac alpha-actin promoter. Heterodimers of myogenin and E12 (or MyoD and E12) specifically bound a restriction fragment extending from -200 to -103 relative to the start of cardiac alpha-actin transcription. Methylation interference footprints pinpointed the site of interaction to an E box immediately adjacent to a previously identified CArG box (CArG3). Site-directed mutations to the DNA-binding site revealed that either an intact E box or an intact CArG3 is required for induction of the cardiac alpha-actin promoter in myoblasts and for transactivation by myogenin in cotransfected fibroblasts. However, deletion and substitution experiments indicate that the complex E box/CArG3 element alone does not confer muscle-specific expression to a minimal promoter. These results suggest that direct and indirect pathways involving multiple cis-acting elements mediate the induction of the cardiac alpha-actin promoter by myogenin and MyoD.
3601	A chorionic gonadotropin-secreting human pituitary cell.	2005199	Recent data from our laboratory show that hCG is secreted in a pulsatile manner in parallel with human LH (hLH) in nonpregnant normal humans. Furthermore, GnRH stimulates hCG and hLH release. Using a monoclonal antibody specific for the beta-chain of hCG and not reacting with hLH, we have identified a heretofore unknown cell type in human pituitaries which stains only for hCG. The light microscopic, immunocytochemical, and ultrastructural features are described. These data coupled to those from multiple earlier studies indicate that hCG is secreted by these cells in normal nonpregnant humans.
3602	Hypoplastic left heart syndrome with rhabdomyoma of the left ventricle.	1866332	A case of primary cardiac tumor with hypoplastic left heart syndrome (HLH) is presented. Sonographic examination at 32 weeks' gestation revealed a large tumor in the left ventricle of the fetal heart. The newborn died of congestive heart failure at 11 days of age. Autopsy demonstrated a large tumor in the left ventricle obliterating most of the left ventricular cavity, aortic atresia, and hypoplastic ascending aorta. Microscopically, the cardiac tumor showed "spider-cells" characteristics of rhabdomyoma.
3603	Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo.	1849072	We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin.
3604	Comparison of steroidogenic potencies of homologous and heterologous gonadotropins in rat and mouse Leydig cells.	1820967	The in vitro steroidogenic potencies have been determined in rat and mouse Leydig cells for two homologous human gonadotropins, lutropin (hLH) and choriogonadotropin (hCG), and two heterologous gonadotropins, hCG beta wild-type and the product of an hCG beta clone containing a premature termination codon at position 122, each associated with a co-expressed bovine alpha subunit. hCG was found to be more potent than hLH in rat, but not mouse Leydig cells, and the heterologous gonadotropin containing the truncated hCG beta subunit was equipotent to that with hCG beta wild-type in both rat and mouse Leydig cells. Persistent steroidogenesis was determined by measuring testosterone production following pre-incubation with each of the above four gonadotropins and with ovine LH, subsequent washing of the cells, and re-incubation in the absence and presence of additional hormone. Interesting differences were found with the five gonadotropins in rat and mouse Leydig cells. The testosterone response to all gonadotropins in rat Leydig cells was essentially the same whether or not additional hormone was added after the initial cell incubation and washing. In contrast, only hLH and hCG yielded identical responses in mouse Leydig cells in the presence and absence of additional hormone, testosterone production being invariably lower with ovine LH and the two expressed heterologous gonadotropins unless they were present in the second incubation. In summary, the hCG beta C-terminal sequence from residues 122-145 makes no discernible contribution to the in vitro potency in rat or mouse Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)
3605	Molecular biology of myogenic regulatory factors.	1806761	A family of proteins has recently been identified, each member of which has the capacity to initiate muscle differentiation in many non-muscle cell types. These factors, which include MyoD1, myogenin, myf-5 and MRF4, share homologies with each other and belong to a superfamily of Myc-related proteins. Expression of these regulatory proteins results in auto-activation and cross-activation of other members of the family and in the transcriptional activation of the markers of terminal differentiation. Sequence analysis has shown a conserved basic domain in each protein that is required for binding to specific DNA sequences of the E-box type and for myogenic activation. A conserved helix-loop-helix (HLH) domain allows homo- and heterodimerization of these muscle-specific proteins with each other and with ubiquitously expressed proteins such as the E2A gene products (E12/E47). This review describes the discovery and characterization of these muscle regulatory proteins and their actions in the context of proposed models for the determination and differentiation of muscle tissue.
3606	Experience of human chorionic gonadotropin assays in gestational trophoblastic tumors.	1680961	From May 1979 through December 1988, 146 patients with gestational trophoblastic tumors (71 hydatidiform mole, 3 partial mole, 15 choriocarcinoma and 57 persistent trophoblastic tumors) were studied. A total of 1178 daily urine samples were collected before and/or after treatment, and in the course of follow-up. H93 RIA (an HCG specific assay), H80 RIA (an assay detecting hCG and hLH) and a hCG alpha assay measured levels in the urine specimens. Three hCG declining patterns (pattern D, P and R) based on the H93 RIA assay were noted. Patients showing pattern D had the most favorable outcome (no mortality at all). However, pattern P and R had a 10% and 14.3% mortality rate, respectively. The ratios of H80/H93, hCG alpha/H93, hCG alpha/H80 in the urine specimens were similar in both pattern D and R (excluding samples from a patient who expired later). However, the ratios of H80/H93, hCG alpha/H93, hCG alpha/H80 of samples from the patient (CK) who expired later were significantly different from those of the pattern D and R. This was suggestive of a marked unbalanced secretion of hCG and its subunit in the urine specimens of patient CK. The molecular forms in pattern D were similar to the standard hCG. However, the molecular form in pattern R of 3 fatal choriocarcinomas showed a great variation, from smaller to larger than the standard hCG. The isoelectric points of hCG in pattern D and R were all acidic. In clinical practice, we can measure the ratios of H80/H93, hCG alpha/H93 and hCG alpha/H80, molecular forms, and isoelectric points of hCG.(ABSTRACT TRUNCATED AT 250 WORDS)
3607	Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc.	2006410	The myc protooncogene family has been implicated in cell proliferation, differentiation, and neoplasia, but its mechanism of function at the molecular level is unknown. The carboxyl terminus of Myc family proteins contains a basic region helix-loop-helix leucine zipper motif (bHLH-Zip), which has DNA-binding activity and has been predicted to mediate protein-protein interactions. The bHLH-Zip region of c-Myc was used to screen a complementary DNA (cDNA) expression library, and a bHLH-Zip protein, termed Max, was identified. Max specifically associated with c-Myc, N-Myc, and L-Myc proteins, but not with a number of other bHLH, bZip, or bHLH-Zip proteins. The interaction between Max and c-Myc was dependent on the integrity of the c-Myc HLH-Zip domain, but not on the basic region or other sequences outside the domain. Furthermore, the Myc-Max complex bound to DNA in a sequence-specific manner under conditions where neither Max nor Myc exhibited appreciable binding. The DNA-binding activity of the complex was dependent on both the dimerization domain and the basic region of c-Myc. These results suggest that Myc family proteins undergo a restricted set of interactions in the cell and may belong to the more general class of eukaryotic DNA-binding transcription factors.
3608	Pancreatic beta-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNA-binding proteins.	1996119	The pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by the interaction of unique trans-acting beta-cell factors with a cis-acting DNA element found within the insulin enhancer (5'-GC CATCTG-3'; referred to as the insulin control element [ICE]) present in the rat insulin II gene between positions -100 and -91. This sequence element contains the consensus binding site for a group of DNA-binding transcription factors called basic helix-loop-helix proteins (B-HLH). As a consequence of the similarity of the ICE with the DNA sequence motif associated with the cis-acting elements of the B-HLH class of binding proteins (CANNTG), the ability of this class of proteins to regulate cell-type-specific expression of the insulin gene was addressed. Cotransfection experiments indicated that overexpression of Id, a negative regulator of B-HLH protein function, inhibits ICE-mediated activity. Antibody to the E12/E47 B-HLH proteins attenuated the formation, in vitro, of a previously described (J. Whelan, S. R. Cordle, E. Henderson, P. A. Weil, and R. Stein, Mol. Cell. Biol. 10:1564-1572, 1990) beta-cell-specific activator factor(s)-ICE DNA complex. Both of these B-HLH proteins (E12 and E47) bound efficiently and specifically to the ICE sequences. The role of B-HLH proteins in mediating pancreatic beta-cell-specific transcription of the insulin gene is discussed.
3609	Differential trans-activation of a muscle-specific enhancer by myogenic helix-loop-helix proteins is separable from DNA binding.	1847137	The muscle creatine kinase (MCK) enhancer was used as a target to study the specificity of DNA binding and trans-activation by members of the helix-loop-helix (HLH) family of myogenic regulatory factors, MyoD1, myogenin, myf-5, and MRF4. Whereas all four myogenic factors bound with similar affinities to the MCK enhancer in the presence of the widely expressed HLH protein E12, only MyoD1, myogenin, and myf-5 efficiently trans-activated the enhancer in transiently transfected 10T1/2 and 3T3 cells. That MRF4 binds the MCK enhancer without activating transcription suggests that domains in addition to those required for DNA binding are important for transcriptional activation and supports the notion that the different members of the HLH family of myogenic regulatory factors may selectively regulate unique sets of muscle-specific genes.
3610	Doping control of testosterone and human chorionic gonadotrophin: a case study.	2030059	Doping control for testosterone and human Chorionic Gonadotrophin (hCG) requires special attention as a difference must be made between the endogenous and exogenous origin of both substances. The detection of exogenous testosterone is based on the ratio of testosterone- to epitestosterone-glucuronide (T/E) in urine. The problems with this ratio are discussed. For hCG analysis in urine the utilization of sandwich-type hCG specific assays instead of hCG/hCG beta competitive assays is recommended. A case study in which an athlete self-administered testosterone and hCG before a competition is described. The T/E ratio and hCG concentration in urine were followed during this period of self-administration. The results demonstrate the relevance of the T/E ratio and of the selected hCG assay. The ratio of testosterone to human Luteinizing Hormone (T/hLH) in serum also indicated the use of hormones. Although the athlete's urine was negative for exogenous testosterone directly after competition, he would have been found positive for hCG.
3611	Studies of human pituitary LH containing internally cleaved beta subunit.	2015051	We have investigated the origin of the internal peptide bond cleavage found in the beta subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between alpha and beta subunits and on binding of hLH to its receptor. The content of cleaved beta subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked beta subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked beta subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37 degrees C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS).(ABSTRACT TRUNCATED AT 250 WORDS)
3612	B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits.	1990271	Recent studies have identified a family of DNA-binding proteins that share a common DNA-binding and dimerization domain with the potential to form a helix-loop-helix (HLH) structure. Various HLH proteins can form heterodimers that bind to a common DNA sequence, termed the E2-box. We demonstrate here that E2-box-binding B-cell- and myocyte-specific nuclear factors contain subunits which are identical or closely related to ubiquitously expressed (E12/E47) HLH proteins. These biochemical function for E12/E47-like molecules in mammalian differentiation, similar to the genetically defined function of daughterless in Drosophila development.
3613	Conversion of human choriogonadotropin into a follitropin by protein engineering.	1899483	Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.
3614	Activation of the cAMP cascade by steroidogenic hormones and glucagon in the pathogenic fungus Candida albicans.	1851467	Incubation of Candida albicans yeast cells with human luteinizing hormone (hLH), human chorionic gonadotrophin (hCG) or glucagon produced a significant rise in cAMP total levels. The effect of these hormones in permeabilized cells of the fungus produced a 2-3 fold increase in the Mg2+, GTP-dependent adenylyl cyclase activity as well as full activation of the cAMP-dependent protein kinase (PKA) activity. These results indicate that the interaction of the mammalian hormones with the fungus triggered the cAMP activation cascade in a similar way to that found in higher eukaryotic organisms.
3615	Molecular cloning and chromosomal localization of the murine homolog of the human helix-loop-helix gene SCL.	1704135	The human SCL gene is a member of the family of genes that encode the helix-loop-helix (HLH) class of DNA-binding proteins. A murine SCL cDNA was isolated from a normal macrophage cDNA library by using HLH-specific oligonucleotides as hybridization probes. The coding region is 987 base pairs and encodes a predicted protein of 34 kDa. The nucleotide sequence of the coding region shows 88% identity to the human SCL gene, and the amino acid sequence is 94% identical. The HLH motif and upstream hydrophilic region are entirely conserved in the murine and human proteins. The identity between the mouse and human sequences was less marked in the 5' and 3' untranslated regions. Two murine SCL transcripts that differ in the 3' noncoding region have been detected in fetal liver and various cell lines. Variation was also observed in the 5' untranslated region. Interestingly, immediately downstream of the protein-termination codon, both the human SCL sequence and the murine homolog share an E-box element--the suggested target site for DNA binding of HLH proteins. The murine SCL homolog was mapped to the central part of chromosome 4.
3616	Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region.	1987636	The function of the c-Myc oncoprotein and its role in cell growth control is unclear. A basic region of c-Myc is structurally related to the basic motifs of helix-loop-helix (HLH) and leucine zipper proteins, which provide sequence-specific DNA binding function. The c-Myc basic region was tested for its ability to bind DNA by attaching it to the HLH dimerization interface of the E12 enhancer binding factor. Dimers of the chimeric protein, termed E6, specifically bound an E box element (GGCCACGTGACC) recognized by other HLH proteins in a manner dependent on the integrity of the c-Myc basic motif. Methylation of the core CpG in the E box recognition site specifically inhibited binding by E6, but not by two other HLH proteins. Expression of E6 (but not an E6 DNA binding mutant) suppressed the ability of c-myc to cooperate with H-ras in a rat embryo fibroblast transformation assay, suggesting that the DNA recognition specificity of E6 is related to that of c-Myc in vivo.
3617	Characterization of a factor(s) from partially purified human gonadotrophin preparations which inhibit(s) the binding of radiolabelled human LH and human chorionic gonadotrophin to Candida albicans.	1999672	We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of 125I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16,000-21,000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for beta-core protein, a cleavage product of the beta subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified beta-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites. Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to beta-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.
3618	NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin.	1991473	Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]
3619	The c-ets-1 proto-oncogene has oncogenic activity and is positively autoregulated.	2284095	The proto-oncogene ets-1 is a member of the ets family of genes that share homology with the viral oncogene, v-ets, of the avian leukemia virus E26. By using expression vectors, we demonstrate that the ets-1 gene transforms NIH3T3 cells and the ets-1 transfected cells form colonies in soft agar and induce tumors in nude mice. We have also determined that the ets-1 protein contains homology with the helix-loop-helix motif of the HLH family proteins, but lacks the basic domain upstream of helix I. Transfection of the NIH3T3 cells with ets-1 vectors results in the activation of the endogenous ets-1 gene. Using hybridization probes that can distinguish between transcripts from endogenous and exogenous templates, we show that the endogenous ets-1 gene is activated by the expression of the transfected exogenous ets-1. In contrast, the expression of transfected ets-2 has no effect on the endogenous ets-1 gene expression. The results indicate that the ets-1 proto-oncogene is positively autoregulated by its product.
3620	[The follow-up of trophoblastic disease by using an hCG-CTP enzyme immunoassay].	2176247	In the management of a chorionic disease, human chorionic gonadotropin (hCG) is the most reliable tumor marker. However, hCG has a low titer, so that it cannot be strictly distinguished from the human luteinizing hormone (hLH) by the traditional method that uses a hemagglutinin reaction (HAR) or by a radioimmunoassay (RIA). Recently, however, the detection of the beta-COOH-terminal peptide of hCG (hCG-CTP) by an enzyme immunoassay has made it possible to clearly distinguish hCG from hLH, and from April, 1987 to December, 1989, 13 trophoblastic diseases have been managed using this new technique. Results have shown that human chorionic gonadotropin-CTP, when compared to other methods of measurement, is the most sensitive tumor marker and as it is the most accurate, it should be used in careful follow-up observations of a chorionic disease.
3621	CeMyoD accumulation defines the body wall muscle cell fate during C. elegans embryogenesis.	2175254	We have cloned a gene from the nematode C. elegans that is closely related to the vertebrate MyoD gene family. The nematode gene product, CeMyoD, is a nuclear protein that is expressed specifically in body wall muscle cells. Antibody staining of early embryos shows that CeMyoD accumulates in early blastomeres that will subsequently produce only body wall muscle cells. CeMyoD is not detected in pharyngeal muscle cells or in nonmyogenic lineages. A CeMyoD-beta-galactosidase fusion gene is accurately expressed in myogenic cells that also express CeMyoD. In addition, the beta-galactosidase reporter is expressed as early as the 28 cell stage of embryogenesis in specific blastomeres prior to their clonal commitment to body wall muscle. This early fusion gene activity reveals that part of the specificity for CeMyoD transcription can arise very early in development and that subsequently, negative events may restrict CeMyoD expression in progeny cells not destined to become muscle.
3622	Purification and characterization of the human ovarian LH/hCG receptor and comparison of the properties of mammalian LH/hCG receptors.	2242036	Methods previously published by us [Wimalasena et al., J Biol Chem 260: 10689-10697, 1985; Wimalasena et al., J Biol Chem 261: 9416-9420, 1986] were utilized to solubilize the human corpus luteal leuteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor with 3-[(3-cholamide-propyl)dimethylammonio]-1-propanesulfonate (CHAPS) and to purify the receptor by two steps of hCG-Sepharose affinity chromatography. The specific binding capacity (SBC) of the purified human receptor was 7510 pmol/mg protein, and KA = 2.2 x 10(9) M-1 when iodo hCG was competed by hCG; the yield was 4-7% of starting activity. When hLH was used in competition with hCG, specific binding capacity was 7900 pmol/mg protein and KA 1.0 x 10(9) M-1. Silver staining and autoradiography demonstrated a single protein of Mr 78,000 under reducing and Mr 58-62 x 10(3) under nonreducing conditions. Rat ovarian LH/hCG receptor was purified by similar methods and the KA of 3.5 x 10(10) M-1 for hCG was substantially different from the KA for hLH which was 2.1 x 10(9) M-1. Mr of the rat protein was 78-82 x 10(3) (reduced) and 58-62 x 10(3) (nonreduced) when analyzed by silver staining and autoradiography. For the first time, human LH/hCG receptor has been purified to apparent homogeneity, and its Mr of 78,000 was essentially identical to the Mr values of purified rat and porcine receptors.
3623	Receptor-mediated elevation of adenylate cyclase by luteinizing hormone in Candida albicans.	2079618	Human luteinizing hormone (hLH) and the GTP analogue guanosine 5'-O-(3-thio)triphosphate stimulated morphogenesis in the dimorphic fungal pathogen Candida albicans. hLH bound specifically to subcellular fractions from C. albicans and stimulated adenylate cyclase activity in C. albicans microsomes. We provide the first demonstration of guanine-nucleotide-binding proteins (G-proteins) in C. albicans, and propose that the stimulation of C. albicans morphogenesis by hLH is mediated by a receptor-coupled adenylate cyclase system involving G-proteins.
3624	Specific binding sites for 125I-labelled human luteinizing hormone (hLH) in microsomal fractions from Drosophila.	2125284	Microsomal fractions prepared from Drosophila flies and larvae bound 125I-labelled human luteinizing hormone (hLH) in a displaceable manner. Binding increased linearly with increasing microsome concentration, and was dependent on the pH and metal ion concentration of the medium, and on the temperature and duration of incubation. Scatchard plots of 125I-hLH binding demonstrated the presence of two distinct binding sites, one with high affinity and low capacity, the other with low affinity and high capacity. 125I-hLH binding was inhibited in a dose-dependent fashion by partially purified human gonadotropin preparations, but not by other proteins, hormones and peptides. Highly purified human chorionic gonadotropin (hCG) or hLH preparations were much less effective at inhibiting 125I-hLH binding to Drosophila microsomes than partially purified gonadotropins. This was due to the presence of a contaminant present in partially purified commercial hCG preparations. Chromatography of crude hCG preparations clearly resolved the LH-binding inhibitor from hCG. Moreover, whereas hCG (and epidermal growth factor-like factors present in crude hCG preparations) were not bound to CM-Sepharose, the Drosophila LH-binding inhibitor was adsorbed strongly. Fractionation of 125I-hLH tracer on concanavalin A-Sepharose suggested that Drosophila binding sites did not preferentially bind a subfraction of the hormone tracer. These data suggest that high affinity binding sites for hLH/hCG-like molecules are present in Drosophila microsomes.
3625	Site-specific recombination of the tal-1 gene is a common occurrence in human T cell leukemia.	2209547	The tal-1 gene is altered as a consequence of the t(1;14) (p32;q11) chromosome translocation observed in 3% of patients with T cell acute lymphoblastic leukemia (T-ALL). tal-1 encodes a helix-loop-helix (HLH) domain, a DNA binding and dimerization motif found in a number of proteins involved in cell growth and differentiation. We now report that an additional 25% of T-ALL patients bear tal-1 gene rearrangements that are not detected by karyotype analysis. These rearrangements result from a precise 90 kb deletion (designated tald) that arises independently in different patients by site-specific DNA recombination. Since the deletion junctions resemble the coding joints of assembled immunoglobulin genes, tald rearrangements are likely to be mediated by aberrant activity of the immunoglobulin recombinase. Moreover, t(1;14)(p32;q11) translocations and tald rearrangements disrupt the coding potential of tal-1 in an equivalent manner, and thereby generate a common genetic lesion shared by a significant proportion of T-ALL patients.
3626	Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity.	2123466	Enhancer binding protein AP-4 is a transcription factor that activates both viral and cellular genes by binding to the symmetrical DNA sequence, CAGCTG. Here, we report the molecular cloning and characterization of human AP-4 cDNAs. The deduced amino acid sequence reveals that AP-4 is a helix-loop-helix (HLH) protein. Like other members of this family, the AP-4 HLH motif and the adjacent basic domain are necessary and sufficient to confer site-specific DNA binding. However, unlike other HLH proteins, AP-4 also contains two additional protein dimerization motifs consisting of leucine repeat elements LR1 and LR2. The analysis of various deletion and point mutants for their ability to dimerize in the presence or absence of DNA reveals several unusual features. Although the HLH basic region is sufficient for DNA recognition and binding, dimer formation between different truncated versions of AP-4 in solution requires an intact LR1 or LR2 domain. AP-4 is unable to form heterodimers with other helix-loop-helix family members such as the immunoglobulin enhancer binding factor, E12. In contrast, an AP-4 derivative, delta C222, which lacks LR1 and LR2 but retains an intact HLH, can form heterodimers with E12. Moreover, AP-4 molecules containing LR2 or LR1 are unable to form mixed dimers with carboxy-terminally truncated AP-4 molecules such as delta C222, but retain the ability to form complexes with longer versions of AP-4 that contain LR1 and/or LR2. Our findings strongly suggest that AP-4 contains multiple protein-protein interfaces that function to promote homodimer formation and restrict heterocomplexes. These findings provide a mechanism by which different members of the helix-loop-helix family of transcription factors can form functional dimers in a specific fashion with their appropriate partners to control transcriptional networks during cellular differentiation.
3627	Development and validation of a radioligand receptor assay for measurement of luteinizing hormone in human serum.	2394770	Studies of circulating LH physiology and pathophysiology are dependent upon measurements of immuno- and bioactivity, both of which have methodologic limitations. We have developed and validated a RRA which allows direct measurement of receptor-bindable LH in human serum. Using a cultured Leydig tumor cell line (MA-10) known to express the CG/LH receptor as the receptor source and polyacrylamide-gel electrophoresis purified hCG as the radioligand, we have established an assay system with the requisite sensitivity (0.04 ng/tube) to measure circulating LH, without significant alteration in total specific binding upon addition of up to 150 microL gonadotropin-free serum when compared to no serum. Standard curves of hLH diluted in gonadotropin-free serum were not statistically different in slope or ED50 from buffer curves. Dilutions of human serum from postmenopausal women and men with Klinefelter's syndrome containing LH measured in the assay were parallel to the standard curve. Further validation of the RRA included measurement of LH by RRA and RIA in daily serum samples from normal women across the menstrual cycle (n = 6) where there was excellent correlation (P less than 0.001) between RRA and RIA measurements with the exception of the mid-cycle surge where the RRA/RIA ratio fell to 0.5. This LH RRA will be useful in further studies of the physiology and biochemistry of LH in human serum.
3628	Ectoderm nuclei from sea urchin embryos contain a Spec-DNA binding protein similar to the vertebrate transcription factor USF.	2081463	The Spec gene family of Stronglyocentrotus purpuratus is expressed exclusively in aboral ectoderm cells during embryogenesis. To investigate the regulation of Spec gene activity, the region around the Spec1 transcriptional initiation site was analyzed for sites of protein-DNA interaction. One high-affinity site bound a factor termed SpF1 within the Spec1 5' untranslated leader region at position +39 to +60. The core sequence recognized by SpF1, CACGTG, is the same as that of the upstream stimulatory factor (USF), a widely occurring vertebrate transcription factor containing a myc-HLH motif. A comparison of USF- and SpF1-binding activities suggested that SpF1 was a sea urchin version of USF. SpF1 activity was detectable only in ectoderm cells of the embryo, implying that it has a role as a cell type-specific transcription factor. SpF1-binding sites were also found upstream of the Spec2a and Spec2c genes in the same conserved sequence block as Spec1. Extracts from Lytechinus pictus embryos showed an SpF1-like activity, suggesting that SpF1 is conserved in sea urchins. Surprisingly, changes in the Spec1, Spec2a, or Spec2c genes that removed or modified the SpF1-binding site had no effect on expression when reporter gene fusions containing these mutations were injected into sea urchin eggs and analyzed for expression during embryogenesis. We propose that, while SpF1 may not be essential for expression of the exogenously introduced reporter genes, it may be required for proper regulation of the endogenous Spec genes.
3629	Molecular analysis of the t(1;19) breakpoint cluster region in pre-B cell acute lymphoblastic leukemias.	2078515	The t(1;19) chromosomal translocation in acute lymphoblastic pre-B cell leukemias involves the gene E2A for helix-loop-helix (HLH) proteins E12 and E47, ubiquitous transcriptional proteins implicated in the regulation of various lymphoid and nonlymphoid genes. To characterize the molecular features of the t(1;19)(q23;p13) translocation, we molecularly cloned breakpoint DNA from t(1;19)-carrying pre-B cell leukemias. In all cases, breakpoints on chromosome 19 occurred within 2 kb of each other in a single intron of the E2A gene. This clustered arrangement resulted in specific truncation of the E2A gene and transcript, with loss of sequences encoding the basic DNA-binding and HLH dimerization motifs from the derivative 19 chromosome. In contrast, breakpoints on chromosome 1 were distributed over a large region and could not be linked to exonic sequences of the PBX1 gene, although identical chromosome 1 sequences are joined to E2A sequences in 1;19 fusion transcripts. These data show that the 1;19 translocation consistently results in exchange of 3' exons encoding the HLH motifs of E2A with DNA from chromosome 1 to form a fusion gene on the derivative 19 chromosome.
3630	Two rat homologues of Drosophila achaete-scute specifically expressed in neuronal precursors.	2392153	In vertebrates, the peripheral nervous system is embryologically derived from the neural crest. Although the earliest neural crest cells seem to be multipotent, the molecular mechanisms responsible for the restriction of these cells to different sublineages are not understood. We therefore searched for developmental control genes expressed in crest cells or their derivatives. One important class of regulatory molecules comprises proteins with common DNA-binding and dimerization domains, the basic helix-loop-helix (B-HLH) region. Members of this family include MyoD, a mammalian myogenic determination molecule, and proteins encoded by genes of the achaete-scute complex of Drosophila, which have an important role in neuronal determination. From a sympathetic neuronal precursor cell line derived from the neural crest we have now isolated two different mammalian genes that are homologous to genes of the achaete-scute complex. The sequence of the B-HLH-encoding region of these genes is more similar to that of the genes of the achaete-scute complex than it is to that of other, mammalian members of the B-HLH family. At least one of these genes is transiently expressed in the embryonic rat nervous system, is not detected in non-neuronal tissues or cell lines, and is induced by nerve growth factor in PC12 cells.
3631	Negative control of the helix-loop-helix family of myogenic regulators in the NFB mutant.	1696180	We have characterized a nondifferentiating mouse muscle cell line, NFB, that represses the activity of the helix-loop-helix (HLH) family of myogenic regulators, yet expresses sarcomeric actins. The NFB MyoD gene is silent, but can be activated upon transfection of a long terminal region-controlled chicken MyoD cDNA, resulting in myogenesis. When NFB cells are fused with H9c2 rat muscle cells in heterokaryons, the level of rat MyoD transcripts declines. Thus, the stoichiometry of MyoD and the putative repressor controls myogenesis. Although NFB cells express myogenin and Myf-5 transcripts, the activity of these regulators is also repressed:myogenesis is not induced in 10T1/2 fibroblasts and is repressed in L6 muscle cells upon fusion with NFB cells. We conclude that the myogenic HLH regulators are not required for sarcomeric actin gene activation and that myogenesis is subject to dominant-negative control.
3632	Isolation of TSH and LH/CG receptor cDNAs from human thyroid: regulation by tissue specific splicing.	2293030	A TSH receptor (TSH-R) cDNA has been isolated from a human thyroid lambda GT11 library. Unexpectedly, several cDNAs encoding the human LH/CG receptor (LH/CG-R), previously thought to be expressed solely in gonadal cells, were also isolated from the thyroid library. The receptors are structurally related, consisting of a signal sequence, a large extracellular amino terminal domain, seven membrane spanning domains, and a short carboxyl-terminal portion. The TSH-R is encoded by a single 4.2 kilobase mRNA specific to the thyroid. Introns were not present in any hTSH-R cDNAs examined, however, sequencing of several LH/CG-R cDNAs and RNase protection experiments demonstrated that the majority of hLH/CG-R mRNA in the thyroid is incompletely spliced. Consequently, tissue-specific splicing may be an important step in the regulation of the glycoprotein hormone receptor family.
3633	Ethanol has direct inhibitory effects on steroidogenesis in human granulosa cells: specific inhibition of LH action.	2121059	We have evaluated the direct effects of ethanol (EtOH) on the production of progesterone (P) and estradiol-17 beta (E2) by cultured human granulosa cells obtained during in vitro fertilization procedures. On day 3 of culture, cells were divided into control and ethanol (20 mM) groups and stimulated by hFSH (50 ng/ml), hLH (0-50 ng/ml), FSH+LH, 8 Br-cAMP (0.25 mM) and androstenedione (10(-7) M). Experiments were terminated on days 7 and 9 and DNA, P, and E2 were measured. Ethanol inhibited P and E2 secretion stimulated by LH; however, there was no significant effect of ethanol on P and E2 production in the control group or when the cells were stimulated by FSH or cAMP. EtOH also had no effect on androstenedione stimulated E2 production. There was no significant difference in the DNA contents of the human granulosa cells in the ethanol group as compared with the control group. These results are the first demonstration of a direct effect of ethanol on cultured granulosa-lutein cells and suggest that ethanol may inhibit action of LH on the corpus luteum. A direct selective toxic effect of EtOH on the ovary may be responsible for some of the reproductive abnormalities observed in alcoholic women.
3634	Conformation of the alpha-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies.	1703093	The conformation of the common alpha-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the alpha-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the alpha-subunit in free and combined form. The alpha-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various beta-subunits as indicated by shifts in the displacement curves of binding of [125I]hCG alpha to the polyclonal antiserum. The extent of the change was dependent on the beta-subunit present with minimum change being observed with hLH beta, intermediate with hCG beta and maximum change with hFSH and TSH beta-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the alpha-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the alpha-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated alpha-subunit. Displacement analysis using [125I]hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the beta-subunit present. Based on these results, it is concluded that the alpha-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist.
3635	Birth control vaccines and immunological approaches to the therapy of noninfectious diseases.	2345290	Vaccination usually means the immunization of persons or animals against foreign infectious organisms for disease prevention. However, it has now been demonstrated that immunization against certain self substances to which tolerance normally exists can elicit beneficial effects to humans and other animals without inducing autoimmune disease. Clinical trials in women have been conducted with vaccines against reproductive antigens for the prevention of pregnancy and research is under way to develop more advanced formulations. Other self antigens have been described that might be used for developing methods of immunological therapy for such diseases as cancer, ulcers, and complications of diabetes. Emphasis is placed on the need for careful studies in appropriate animal models before any clinical application of such procedures is suggested.By deliberately upsetting the body's recognition of self, and inducing an immune response against specific self antigens, birth control vaccines and disease therapies may be possible. The vaccine must be highly specific, the antigen must be present in small amounts or at limited times, and the benefit-to-risk ratio must be thoroughly evaluated. Target antigens being considered for birth control vaccines include sperm antigens or enzymes, zona pellucida of the ova and the pituitary hormone hCG. Anti-sperm immunity has been observed naturally occurring, and induced experimentally. Anti-ovum vaccines have the drawback of possibly reacting with ovarian tissue. The gonadotropin hormone receiving the most attention is hCG, or specifically its carboxy-terminal subunit. The rest of the peptide is too similar to hLH. Anti-hCG could act by preventing the luteotrophic effect of the hormone, or by attacking the hCG-producing cells of the blastocyst. Vaccines based on hCG with a tetanus toxoid carrier and a synthetic hCG peptide vaccine have been tested successfully in Phase 1 clinical trials. Other therapies based on immunization against self antigens include anticancer therapy, excess growth hormone and the Zollinger- Ellison syndrome of gastric ulcer disease. All of these approaches need to be monitored carefully to ensure that no damage is done to the host from circulating immune complexes.
3636	Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains.	2112746	A common DNA binding and dimerization domain containing an apparent "helix-loop-helix" (HLH) structure was recognized recently in a number of regulatory proteins, including the E47 and E12 proteins that bind to the kappa E2 motif in immunoglobulin kappa gene enhancer. The effect of site-directed mutagenesis on E47 protein multimerization and DNA binding was examined. Mutations in either putative helix domain disrupted protein dimerization and DNA binding. No DNA binding was observed when mutations were introduced in the basic region, but these mutants were able to dimerize. These basic region mutants were not able to bind to DNA as heterodimers with the wild-type E47 proteins, demonstrating that two functional basic regions are required for binding to DNA. Therefore the basic region mutants are "transdominant."
3637	Urinary surge of human luteinizing hormone in infertile women.	1972101	An improved semiquantitative immunoassay method allows human luteinizing hormone (hLH) in urine to be detected from 2.5 to 640 mIU/ml has developed. With this method, the hLH surges in every 3 h urine samples while awake were measured in 8 normal and 12 infertile women. The result was that the urinary hLH surge in infertility was insufficient regardless of the high urinary E2 value. This result may play, at least in part, an etiologic role in infertility.
3638	Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.	1692832	The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common alpha subunit but differ in their hormone-specific beta subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) beta subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH beta-CG beta chimeras appeared to fold similar to hCG beta, since they combined with hCG alpha and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg8-Arg10,Gly47-Ala51, and Gln89-Leu92 form a major epitope region and appear to be adjacent to each other on the surface of hCG beta. Gly47-Ala51 and Gln89-Leu92 are recognized by dimer-specific mAbs while Arg8-Arg10 is recognized by mAbs which have highest affinity for the free beta subunit. These observations suggest that the conformation of this region of the beta subunit changes when the alpha and beta subunits combine. Residues which are C-terminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCG beta and hLH beta by the presence of Asn77 in hCG beta and can be detected after hCG binds to receptors. These findings were used to develop a model of hCG beta which predicts the locations of these residues and their positions relative to the alpha subunit and receptor interfaces.
3639	Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy.	2185892	The centromere and its binding proteins constitute the kinetochore structure of metaphase chromosomes, which is crucial for the high accuracy of the chromosome segregation process. Isolation and analysis of the gene encoding a centromere binding protein from the yeast S. cerevisiae, CBF1, are described in this paper. DNA sequence analysis of the CBF1 gene reveals homology with the transforming protein myc and a family of regulatory proteins known as the helix-loop-helix (HLH) proteins. Disruption of the CBF1 gene caused a decrease in the growth rate, an increase in the rate of chromosome loss/nondisjunction, and hypersensitivity to the antimitotic drug thiabendazole. Unexpectedly, the cbf1 null mutation concomitantly resulted in a methionine auxotrophic phenotype, which suggests that CBF1, like other HLH proteins in higher eukaryotic cells, participates in the regulation of gene expression.
3640	Purification and characterization of lutropin receptor from membranes of pig follicular fluid.	2340270	Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).
3641	The protein Id: a negative regulator of helix-loop-helix DNA binding proteins.	2156629	We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes.
3642	The Drosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein.	1690605	The Drosophila extramacrochaetae (emc) locus participates in sensory organ patterning by antagonizing, in a mechanistically unknown way, the neurogenic activity of the achaete-scute complex (AS-C). Our cloning of emc DNA and molecular mapping of emc mutations have identified a transcription unit as the most likely candidate for the emc function. It encodes a protein that has a dimerizing helix-loop-helix (HLH) motif but lacks a basic region presumably important for DNA binding. AS-C and other proneural proteins have both domains. We propose that the emc product antagonizes neurogenesis by sequestering proneural proteins in complexes inefficient for DNA interaction. These and other findings suggest the existence of a network of synergistic and antagonistic interactions, mediated by HLH proteins, that participates in the establishment of the neural fate.
3643	extramacrochaetae, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins.	1690604	The function of the extramacrochaetae (emc) gene is required to establish the normal spatial pattern of adult sensory organs in Drosophila. emc acts to suppress sensory organ development in certain regions of the body surface, apparently by antagonizing the function of the achaete and scute genes of the achaetescute complex (AS.C). We have found that emc encodes a novel member of the helix-loop-helix (HLH) family of proteins. The emc protein shares the dimerization domain of other HLH proteins but lacks their DNA binding motif. We propose a model in which the emc protein negatively regulates sensory organ determination by forming heterodimers with the HLH proteins encoded by the AS-C and/or daughterless, thereby altering or interfering with their activity.
3644	Maturity at collection and the developmental potential of rhesus monkey oocytes.	2189504	The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
3645	Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches.	1694262	Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.
3646	Specific, high-affinity binding sites for human luteinizing hormone (hLH) and human chorionic gonadotrophin (hCG) in Candida species.	2108673	The presence of specific binding sites for [125I]-labelled hLH and hCG is described in Candida species. Binding was present in three strains of Candida albicans, and in Candida tropicalis, and was greatest in microsomes, though binding was also present in cytosol fractions. hLH and hCG mutually competed for these binding sites. Other hormones did not bind and did not compete for hLH binding sites. Scatchard plots showed two classes of binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomes and cytosol. This is the first report of specific binding sites for mammalian peptide hormones in a yeast.
3647	The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation.	2155707	A 60 amino acid domain of the myogenic determination gene MyoD is necessary and sufficient for sequence-specific DNA binding in vitro and myogenic conversion of transfected C3H10T1/2 cells. We show that a highly basic region, immediately upstream of the helix-loop-helix (HLH) oligomerization motif, is required for MyoD DNA binding in vitro. Replacing helix1, helix2, or the loop of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (required for peripheral neurogenesis) has no substantial effect on DNA binding in vitro or muscle-specific gene activation in transfected C3H10T1/2 cells. However, replacing the basic region of MyoD with the analogous sequence of other HLH proteins (the immunoglobulin enhancer binding E12 protein or T4 achaete scute protein) allows DNA binding in vitro, yet abolishes muscle-specific gene activation. These findings suggest that a recognition code that determines muscle-specific gene activation lies within the MyoD basic region and that the capacity for specific DNA binding is insufficient to activate the muscle program.
3648	Analysis of menstrual records of women immunized with anti-hCG vaccines inducing antibodies partially cross-reactive with hLH.	2182289	Menstrual data of 13 control subjects and 88 subjects immunized with three beta-hCG-based vaccine formulations were analysed. Immunization did not change the menstrual regularity; bleeding days were normal (3-7 days) and 89% of the menstrual cycles were within the normal range of 22-35 days. Irregular (short or long) cycles were observed in both immunized and control groups. These were, however, unrelated to prevailing anti-hCG antibody titres or to cross-reactivity of antibodies with hLH.
3649	A highly sensitive and specific sandwich enzyme immunoassay for detection of human chorionic gonadotropin in trophoblastic disease.	2160806	A highly sensitive and specific two-site enzyme immunoassay (EIA) kit for human chorionic gonadotropin (hCG) has been examined and applied to the detection of hormones in the serum of normal individuals and patients with trophoblastic disease. This EIA uses a solid phase coupled antibody to the unique COOH terminal peptide region of the hCG beta-subunit (beta-CTP) and an enzyme conjugated monoclonal antibody to the hCG beta-subunit. The overnight 2 step assay measured as little as 0.2 mIU/ml serum without hLH interference and a rapid one step assay was newly developed in our laboratory. Assays of serum from normal men and nonpregnant women of reproductive age indicated that most individuals did not have detectable serum levels of hCG immunoreactivity, although a minority had minute amounts. In contrast, women with non-functional ovaries had detectable levels of hCG immunoreactivity with a mean value of 0.86 mIU/ml serum. The extremely high sensitivity and specificity of the EIA in detecting hCG provides substantial advantages for the detection of early pregnancy and for monitoring trophoblastic tumors.
3650	Synthesis of cystine peptides 21-25/70-73 and 35-39/56-59 of the beta-subunit of human choriogonadotropin.	1691156	Syntheses of two asymmetrical cystine peptides with the amino acid residues 21-25/70-73 and 35-39/56-59, based on the linear amino acid sequence and the disulfide bond assignment in the beta-subunit of human choriogonadotropin (hCG-beta), are described. S-trityl and S-acetamidomethyl peptide fragments of each cystine peptide were prepared in solution phase and were subjected to oxidation with I2/MeOH to form the disulfide bridge. The cystine peptides were characterized by their amino acid analyses and fast atom bombardment mass spectrometry. Immunological characterization by several homologous radioimmunoassay systems showed that peptide 21-25/70-73 had significant hCG, hCG-beta, and hLH activities while peptide 35-39/56-59 failed to reveal any immunoreactivity.
3651	[A computerized method for optimization of radioimmunoassays].	2368936	The authors have developed a one step quantitative method for radioimmunoassay optimization. The method is rapid and necessitates only to perform a series of saturation curves with different titres of the antiserum. After calculating the saturation point at several antiserum titres using the Scatchard plot, the authors have produced a table that predicts the main characteristics of the standard curve (Bo/T, Bo and T) that will prevail for any combination of antiserum titre and percentage of sites saturation. The authors have developed a microcomputer program able to interpolate all the data needed to produce such a table from the results of the saturation curves. This computer program permits also to predict the sensitivity of the assay at any experimental conditions if the antibody does not discriminate between the labeled and the non labeled antigen. The authors have tested the accuracy of this optimization table with two in house RIA systems: 17-beta-estradiol, and hLH. The results obtained experimentally, including sensitivity determinations, were concordant with those predicted from the optimization table. This method accelerates and improves greatly the process of optimization of radioimmunoassays.
3652	The role of LH in the autofeedback inhibition of LH and FSH secretion in ovariectomized rats.	2127254	This study examined the role of exogenous LH in the autofeedback regulation of LH and FSH release in ovariectomized rats. The rats were implanted with third ventricular cannulae three weeks after ovariectomy and fitted with silastic jugular cannulae one week later. Baseline hormone levels were established on the day of experimentation in conscious, unrestrained animals. Thereafter, experimental animals received intraventricularly (IVT) either a 9 ug or 20 ug dose of a purified preparation of human (h)LH that did not crossreact in our rLH RIA. In response to 20 ug, but not 9 ug of LH, plasma levels of both LH and FSH were significantly reduced during the following one hour period compared to values in controls receiving buffer IVT. Administration of ovine (o)LH (6 ug, IVT), a preparation which crossreacts in the rLH RIA, significantly elevated plasma levels of detectable LH during the experimental period indicating that LH reaches the blood stream from the third ventricle and, thus, may effect endogenous hormone release at either the pituitary or hypothalamic levels. However, in animals preinjected with 9 or 20 ug of hLH IVT one hour earlier the surge of both LH and FSH in response to LHRH (25 ng iv) was not different from that in the buffer-injected controls receiving LHRH which indicates that pituitary responsiveness was not suppressed by the effective dose of hLH. The results of this study suggest that the inhibitory shortloop feedback of LH on endogenous LH and FSH secretion in ovariectomized rats occurs at the level of the hypothalamus.
3653	Hormonal modulation of invitro biosynthesis of inhibin like peptide by human prostate.	2126421	The hormonal regulation of inhibin biosynthesis by human benign hyperplastic prostate tissue was studied by determining the incorporation of 3H-leucine. 3H-inhibin, synthesized de novo, was immunoprecipitated from the culture media using specific antibodies to human prostatic inhibin. Testosterone and estradiol had no effect on inhibin biosynthesis whereas hFSH and PMSG had stimulatory effect. A decrease in inhibin biosynthesis was noticed on the addition of LHRH, TRH, hCG, hLH and human prolactin.
3654	Quantitative immunoenzymatic assay of human lutropin, with use of a bi-specific monoclonal antibody.	2105180	In this immunoenzymatic assay for human lutropin (hLH) we used bi-specific antibodies (BiAbs) obtained from the fusion of two hybridomas producing antibodies to beta-D-galactosidase and hLh. The BiAb complexed with the enzyme beta-D-galactosidase was used as tracer in a double-determinant assay. We compared the assay involving the BiAb (Bi-EIA) with an immunoenzymatic assay (EIA) in which the same capture antibody was used but the tracer was an enzyme-conjugated hLH-specific monoclonal antibody produced by the same parental cell line used to produce the BiAb. The coefficient of correlation (r) between the two assays was 0.979 but the Bi-EIA was more sensitive (detection limits: 0.8 int. units/L for the Bi-EIA, 2.0 int. units/L for the EIA) and more specific (less than 0.04% vs less than 1.2% cross-reactions with human choriogonadotropin). Mean intra- and interassay CVs for the Bi-EIA were 2.9% and 5.9%, respectively. Correlation (r) with an immunoradiometric assay (IRMA, Serono kit) was 0.960, with radioimmunoassay (RIA, Biodata kit) 0.909, and with an enzyme-linked immunosorbent assay (ELISA kit, Specialty Medical Industries Inc.) 0.888, (n = 25). Evidently, bi-specific antibodies can be used successfully in immunoenzymatic assays, and with potentially greater sensitivity and specificity than assay with a traditional antibody-enzyme conjugate.
3655	Binding of bovine and porcine pituitary glycoprotein hormone alpha-subunit to TSH antibody in serum of patients with Graves' disease.	1717006	The characteristics of autoantibodies reactive with bovine (b) TSH were examined in the sera of six patients with Graves' disease selected on the basis of highly negative values in the TSH receptor assay. Test sera were incubated with other 125I-labeled pituitary glycoprotein hormones and their isolated subunits (alpha and beta) [human (h) TSH, bTSH, porcine (p) TSH, pFSH, bFSH, bLH and equine (e) chorionic gonadotropin (CG)] (purity was confirmed by gel-filtration on Sephadex G-100 and SDS-PAGE), and the antibody bound fraction was precipitated by the addition of anti-human gamma-globulin (goat). Almost all sera showed detectable binding to bTSH, pTSH, pFSH, pTSH-alpha, bFSH-alpha, bLH-alpha, but not to hTSH, hTSH-alpha, hTSH-beta, hFSH, hLH, hCG, pTSH-beta, bLH-beta, eCG-alpha. Exceptions were very low binding to bLH-beta by one serum and to pTSH-beta, by two sera. The level of binding (B/T%) of the patients' sera to pTSH-alpha, bFSH-alpha and bLH-alpha was 3.0-27.7%, 2.6-45.3% and 2.2-39.0%, respectively; that of sera from normal healthy adults was 1.9 +/- 0.3%, 0.8 +/- 0.2% and 0.9 +/- 0.2% (mean +/- SD), respectively. These results indicate that the TSH antibodies recognize mainly an epitope in the alpha subunit of bovine and porcine pituitary glycoprotein hormones (TSH, FSH, LH).
3656	[Preparation and evaluation of anti-human growth hormone immunoserum].	2576553	Using small dosage of human growth hormone to immunize rabbit or guinea pig, it is able to induce anti-hGH antibody formation with high titre and high affinity that could be applied to hGH RIA. In the present study five rabbits and three guinea pigs were immunized with 125-200 micrograms and 250-285 micrograms per animal of hGH respectively, followed by boosters of 10-20 or 160-250 micrograms of hGH at 2-4 week intervals for 6 or 3 months. Blood was drown 1-2 weeks before each booster for determination of antibody formation. Antibody titre and affinity were successively observed and specificity of antibody was determined for the final bleeding. It was shown that titres of immune sera from guinea pigs were much higher than those of rabbit immune sera, but vice versa for antibody affinity. This might be due to larger immunogen dose used for guinea pigs than for rabbits. Fourteen different peptide hormones were tested in reference to cross-immunoreactivity to anti-hGH antibody. It could be demonstrated that the major cross-reactive hormones are hFSH and hLH, and hTSH also reacts to rabbit anti-hGH immune sera at a lesser degree. These cross reactivities are obviously owing to the molecular homogeneities between hGH and these hormones especially of their alpha-subunits.
3657	Transcription control and differentiation: the HLH family, c-myc and C/EBP.	2561453	NaN
3658	Endocrine control of human spermatogenesis.	2513453	We performed a series of studies in normal men to determine the relative roles of FSH and LH in the regulation of human spermatogenesis. Experimental gonadotropin deficiency was induced in normal men by administration of high-dosage testosterone (T) enanthate, resulting in suppression of spermatogenesis to severely oligospermic or azoospermic levels. In these gonadotropin deficient normal men, we found that selective replacement of either LH activity, with human chorionic gonadotropin (hCG) or hLH, or FSH activity with hFSH increased sperm counts into the 20-50 million/ml range, but quantitatively normal spermatogenesis was not achieved. To determine whether both LH and FSH together are required for quantitatively normal spermatogenesis, selective FSH deficiency was induced in normal men by administration of hCG. In the setting of high LH-like activity and markedly suppressed FSH levels induced by hCG, sperm production was partially suppressed again into the 20-50 million/ml range. Replacement of FSH activity, with either hFSH or human menopausal gonadotropin in hCG-treated normal men stimulated sperm production into the control range in all subjects. These studies demonstrate that neither normal serum levels of FSH or LH are required for qualitatively normal spermatogenesis in man, since sperm counts in the range of 20-50 million/ml are found in states of selective FSH or LH deficiency. However, normal levels of both FSH and LH are required to achieve quantitatively normal sperm production in man.
3659	Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta.	2481812	Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.
3660	Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.	2503252	A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.
3661	In vitro fertilization and embryo transfer in the rhesus monkey.	2508776	Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.
3662	Evaluation of the human gonadotroph free alpha-subunit secretory pools by administration of gonadotropin hormone-releasing hormone into normal subjects at different phases of the ovarian cycle.	2504805	The specificity of a monoclonal antibody RIA for the measurement of free alpha-subunit in plasma is presently documented. This RIA was used to explore the pituitary gonadotroph free alpha-subunit reserve in normal ovulating women stimulated with gonadotropin hormone-releasing hormone (GnRH). RIA specificity was established by means of competitive inhibition curves with various glycoprotein hormone preparations, and Sephadex G-100 exclusion chromatography of purified hFSH (1-2), hLH (LER 960) and pituitary extract. The 3.8% and 5.5% crossreactivities of hFSH 1-2 and hLH LER 960 were shown by exclusion chromatography to result in part from free alpha-subunit contamination in these purified preparations. Pituitary extract chromatography indicated hFSH and hLH cross-reactivity below 2.5% and 1.5%, respectively. Normal females were stimulated with GnRH throughout the cycle: 3 tests were performed on Day 7, 1 test on Day 13, 16, 17 and 22, respectively, and 2 tests on Day 24. GnRH stimulation consisted of an initial 100 micrograms bolus (time 0) followed at 2 h by a 12.5 micrograms/h constant infusion, and a second 100 micrograms bolus at 5 h. In all subjects, baseline free alpha-subunit values were below 2 ng/ml. Total free alpha-subunit secretion was markedly enhanced in subjects Day 13 and 16, in concert with total hLH and hFSH secretion. In the three subjects Day 7, free alpha-subunit was released only after the second GnRH bolus. In periovulatory subjects, free alpha-subunit secretion became apparent after the initial bolus and with constant GnRH infusion. In the three subjects Day 22 and 24, peak levels of free alpha-subunit were obtained after the second GnRH bolus.(ABSTRACT TRUNCATED AT 250 WORDS)
3663	Comparison of different regimens of human gonadotropins for superovulation of rhesus monkeys: ovulatory response and subsequent luteal function.	2498448	This study was designed to identify suitable treatment regimens of human gonadotropin for superovulation of rhesus monkeys. At menses, female monkeys were given one of three regimens: Plan A [days 1 to 6, 60 IU human follicle-stimulating hormone (hFSH); days 7 to 9, 60 IU hFSH/60 IU human luteinizing hormone (hLH)], Plan B [days 1 to 3, 75 IU FSH/20 IU LH; days 4 to 6, 60 IU FSH/20 IU LH; days 7 to 9, 45 IU FSH/45 IU LH], or Plan C [days 1 to 9, 60 IU FSH/60 IU LH]. On day 10, human chorionic gonadotropin (hCG; 1000 IU) was administered. Serum estrogen levels peaked on the day of hCG treatment (day 10) in Plans A and C but earlier (day 8) in Plan B. An oviduct lavage recovered 1 to 3 oocytes in Plan B but 3 to 13 oocytes in the other treatment groups. Peak progesterone levels in the luteal phase were greater (P less than 0.05) in animals receiving Plan A or C than Plan B. Regardless of treatment group, progesterone levels declined abruptly 7 days after ovulation induction; the length of the luteal phase in all groups was significantly less than that of normal menstrual cycles. We conclude that regimens of hFSH and hLH (i.e., Plans A and C), followed by hCG, reliably superovulate rhesus monkeys. However, the premature decline in luteal function around the typical time of implantation may compromise pregnancy initiation and maintenance.
3664	Characterisation of antibodies to urinary gonadotrophin peptide.	2926152	There is now general recognition that human chorionic gonadotropin (hCG), its beta subunit and its fragments are valuable diagnostic markers of trophoblastic and some non-trophoblastic malignancies. Urinary gonadotropin peptide (UGP) contains at least one epitope which cross-reacts with the beta-subunit of hCG. In order to assess the potential of UGP as a tumour marker in its own right, it was paramount that any measurements made could be regarded as specific for UGP and not cross-reactive with either hCG or human luteinising hormone (hLH). Four antibodies were tested, two polyclonal (AK12 and DR-Pool) and two monoclonal (2C2 and 6D3). Initial screening using radioiodinated (125I) UGP, hCG, hCG beta-subunit and hLH showed that the polyclonal antibodies bound to all four gonadotrophins, whilst the monoclonal antibodies bound only to the radioiodinated UGP. The antibodies were tested in both radioimmunoassay (RIA) and immunoradiometric assay (IRMA-sandwich assay) systems. The parameters measured were sensitivity and specificity for UGP. The polyclonal antibodies used in the RIA system produced a sensitive assay (0.2 ng/ml UGP) which was relatively specific; cross-reactions for the AK12 antibody (at 50% inhibition) were 5% for hCG, 11% for its beta-subunit, 0.4% for the alpha-subunit and 2.6% for hLH. The monoclonal antibodies performed optimally in the IRMA system. Immobilised 2C2 and radiolabelled (125I) AK12 produced a system that had a sensitivity of 0.4 ng/ml UGP and cross-reactivity (50% maximum binding) of 2% for the hCG beta-subunit and less than 1% for hCG, hLH and the hCG alpha-subunit.
3665	An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys.	2926311	A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1.1 micrograms/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1.5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.
3666	Non-familial hemophagocytic lymphohistiocytosis.	2748450	A case of non-familial hemophagocytic lymphohistiocytosis (HLH) is described. The patient had a chronic course being alive and doing relatively well 3 years since onset. The specific pathological features of this disorder are discussed with emphasis on the fact that a remarkable loss of lymph node structure is sustained by a mature lymphohistiocytic infiltrate. Erythrophagocytosis is assessed as an important but not specific finding. Lymphocyte depletion is an important feature of the lesion. The presence of overlapping features in HLH, infection-associated hemophagocytic syndrome (IAHS) and X-linked recessive lymphoproliferative syndrome (XLS) is emphasized.
3667	Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin.	2747659	The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.
3668	Amplification or rearrangement of the beta-human chorionic gonadotrophin (beta-hCG)--human LH gene cluster is not responsible for the ectopic production of beta-hCG by bladder tumour cells.	2476154	The beta-subunit of human chorionic gonadotrophin (hCG) is coded on chromosome 19 by the beta-hCG-hLH gene cluster. Genomic DNA has been isolated from bladder tumour cell lines which ectopically express beta-hCG. The beta-hCG-hLH gene cluster was probed for possible rearrangement or amplification and cells karyotyped for chromosome 19 abnormalities. No rearrangement or amplification of the gene cluster and no consistent abnormalities of chromosome 19 were found. The expression of beta-hCG by bladder tumours is therefore likely to be the result of altered gene regulation and not a rearrangement or amplification of this gene cluster.
3669	Characterization of a monoclonal antibody inhibiting the biological activity of human chorionic gonadotrophin.	2645317	A mouse monoclonal antibody (MCA), raised against human chorionic gonadotrophin (HCG) and coded 130A, was characterized with several types of immunoassay and with in-vitro and in-vivo bioassays. MCA 130A binds strongly to the intact HCG molecule but less so to either alpha- or beta-subunit; The dissociation constant of the MCA-HCG complex was found to be in the order of 70 pmol/l. MCA 130A inhibits the biological activity of HCG very effectively, both in vitro and in vivo. In a competitive immunoassay MCA 130A binds 30 x better to HCG than to human luteinizing hormone (HLH). The selectivity in the bioassays was much higher, e.g. in vitro, 50% inhibition of HLH-stimulated testosterone production requires 2600 x as much MCA as is needed for inhibiting HCG-stimulated production. Possible reasons for this difference in selectivity are discussed.
3670	Characterization of a monoclonal antibody which inhibits the biological activity of human chorionic gonadotrophin in human corpora lutea.	2537341	In the present study a murine monoclonal antibody (MCA), directed against human chorionic gonadotrophin (HCG), was investigated in vitro utilizing human corpora lutea (CL). The CL were excised from women of fertile age undergoing various gynaecological operations. The CL specimens were cut into pieces and incubated in standard medium for 2 or 4 h in the presence of HCG, luteinizing hormone (HLH) or prostaglandin (PG) E2 alone or in combination with the MCA directed against HCG. After incubation, the tissue levels of cyclic adenosine 3',5' monophosphate (cAMP) and the media contents of progesterone (P) were determined. HCG, HLH and PGE2 stimulated both cAMP and P formation in CL of all ages, while the MCA alone had no effect on these parameters. The MCA, however, effectively counteracted the stimulatory effect of HCG on both cAMP and P formation on a 1:1 molar basis. This antagonistic effect was clearly reversible and could be overcome by increasing the HCG concentrations. The stimulatory effect of HLH and PGE2, on the other hand, was not influenced by the antibody, even when administered at high concentrations. These in-vitro data show that the MCA studied effectively counteracts the stimulatory effect of HCG on human luteal tissue with high specificity.
3671	MyoD and achaete-scute: 4-5 amino acids distinguishes myogenesis from neurogenesis.	2562185	The myogenic determination gene MyoD contains a 60 amino acid domain that is necessary for both sequence-specific DNA binding and myogenic conversion of transfected C3H10T1/2 mouse embryo fibroblasts. We have generated deletion, insertion, and substitution mutants to probe the structure and function of this region, both in vitro and in vivo. Our results are consistent with a previous proposal that a helix-loop-helix (HLH) motif mediates protein dimerization. A highly basic region located immediately upstream of the HLH motif is required for DNA binding, which occurs if and only if the HLH motif is capable of dimerization. All mutants of MyoD that do not bind DNA in vitro fail to activate myogenesis or expression of a co-transfected muscle-specific reporter gene in transfected C3H10T1/2 cells. Replacing either helix 1, helix 2, or the loop sequence of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (which is required for neurogenesis) has no substantial effect on DNA binding or biological activity. However, replacing the basic region of MyoD with the analogous sequence of the kappa immunoglobulin enhancer binding protein E12, or the T4 achaete-scute protein, produces proteins still capable of specific DNA binding in vitro, yet without significant biological activity. These findings suggest that within the 13 amino acid sequence of the MyoD basic region lies a recognition code that determines muscle-specific gene expression, although specific DNA binding per se is not sufficient to activate the muscle program.
3672	Biopotency of highly purified porcine FSH and human LH on gonadal function.	2493064	The gonadotrophin preparations that have been used previously to study different aspects of testis function are of limited purity. We have, therefore, purified existing gonadotrophin preparations further. The demonstration of their purity and biological activity are reported as well as their suitability for in-vivo use. After separation of the subunits of the intact hormones by high-performance liquid chromatography followed by analytical sodium dodecylsulphate-polyacrylamide gel electrophoresis, no contaminating proteins could be detected in either the FSH or LH preparation. After immunoadsorption, contamination by other pituitary hormones (TSH, prolactin, FSH, LH and GH) was found to be less than 0.002% by weight for all the hormones tested. The biological activity of porcine FSH (pFSH) measured in a Steelman-Pohley assay was 150-170 times more potent than the NIH-FSH-P1 reference preparation. The biopotency of human LH (hLH) in the ovarian ascorbic acid depletion test was measured and appeared to be 8100-8300 IU/mg against the 68/40 International Standard. The dose-dependent effects of pFSH and hLH on testis weight and the number of FSH and LH receptors were measured in immature (22-day-old) hypophysectomized rats treated for 7 consecutive days. Treatment with FSH induced a dose-dependent increase in testis weight (threefold) when compared with the control. The concentration and total number of LH receptors were increased (two- to sixfold) in a dose-dependent manner. The number of FSH receptors per testis increased while the number of FSH receptors per mg of protein remained unchanged. Administration of human LH to immature hypophysectomized rats had no effect on either LH or FSH receptors, regardless of the dose administered.(ABSTRACT TRUNCATED AT 250 WORDS)
3673	Primary and secondary structural determinants in the receptor binding sequence beta-(38-57) from human luteinizing hormone.	3233214	The intercysteine "loop" sequence 38-57 in the beta subunit has been shown to be a determinant for expression of biological activity in human lutropin (hLH) and choriogonadotropin (hCG) [Keutmann, H. T., Charlesworth, M. C., Mason, K. A., Ostrea, T., Johnson, L., & Ryan, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2038]. Together with other sequences, the 38-57 region may contribute to a multicomponent receptor binding domain in hLH/hCG. Because the structural features influencing activity in this important region are not easy to evaluate in the full-length subunit, we have used analogues of hLH beta-(38-57) prepared by solid-phase synthesis. The peptides were tested for inhibition of 125I-labeled hCG binding to rat ovarian membrane receptors. Secondary structure was analyzed by circular dichroism (CD) and by reactivity with antibodies to the native 38-57 peptide. An analogue lacking the 38-57 disulfide linkage retained 20% receptor binding and full immunoreactivity. "Far"-ultraviolet CD profiles were essentially identical with those of the disulfide-intact peptide; a transition from 10% to 30% alpha-helix in 90% trifluoroethanol was characteristic of both. The peptide thus appears not to require the disulfide bridge to retain a looped conformation with amphipathic secondary structure. An essential positive charge at position 43 was shown by complete loss of activity upon substitution of Asp or Ala for the Arg found in all known species of LH. Other analogues showed a requirement for a neutral residue at position 47, also highly conserved.(ABSTRACT TRUNCATED AT 250 WORDS)
3674	Immunometric assay for lutropin (hLH) based on the use of universal reagents for enzymatic labelling and magnetic separation and monitored by enhanced chemiluminescence.	3263445	A solid-phase immunometric assay of human lutropin (hLH) is described. Two different anti-hLH antibodies were utilized as capture antibodies, and anti-IgG antibodies covalently coupled to magnetic particles and horseradish peroxidase, respectively, served as 'universal' detection reagents. An anti-hLH antibody raised in rabbits was incubated with a goat anti-rabbit IgG covalently bound to magnetic particles. The resulting complex was added to a separately incubated mixture of hLH and monoclonal anti-hLH antibody. Following incubation, the immunocomplex was sedimented in a magnetic field and the supernatant discarded. Finally a sheep anti-mouse antibody (F(ab')2 fragment) conjugated to horseradish peroxidase as label was added. Following a further incubation, the particles were sedimented in the magnetic field and washed. The hLH content of the sample was quantitated by measuring 'enhanced chemiluminescence'. The sensitivity of the assay was 2.5 +/- 0.9 IU/l (mean +/- SD), the within-run variation ranged from 7.9 to 11%, the between-run variation from 12.9 to 19.8%. Cross-reaction with hFSH or hTSH could not be detected, but was approximately 0.1% with hCG. The results correlated well with those obtained by radioimmunoassay (r = 0.84).
3675	Stokes radius determination of radioiodinated polypeptide hormones by gel filtration.	3239767	A simple technique for determination of the molecular (Stokes) radius of radioiodinated proteins was developed using the same column and chromatographic conditions employed in routine radioimmunoassay tracer purification. The calibration curve for five radioiodinated standard proteins presented a highly significant correlation (r = -0.996; P less than 0.001) and allowed precise molecular radius determination for labeled human growth hormone (hGH), luteotropin (hLH), follicle-stimulating hormone (hFSH), thyrotropin (hTSH), prolactin (hPRL), and corticotropin (hACTH), enabling detection of differences of the order of +/- 3%. The validity of the method was verified by determining the molecular radius of hGH in both "cold" (unlabeled standards and unknowns) and "hot" (radioiodinated standards and unknowns) systems. The technique can be applied in a very simple manner, requiring just one simple additional calibration run before Sephadex G-100 tracer purification. Furthermore, it can be applied to any protein, even when only extremely limited amounts are available. Since the standards and unknowns are labeled and chromatographed under identical conditions, potential common alterations of the molecule due to oxidation, iodine incorporation, tracer-carrier interactions, etc., are automatically corrected for.
3676	Antigenic features of human follicle stimulating hormone delineated by monoclonal antibodies and construction of an immunoradiomometric assay.	2458911	The characterization of human (h) FSH with 181 monoclonal antibodies (MCA) allowed the elucidation of its antigenic topography. One- and two-site, limited as well as excess reagent type radioimmuno- and enzymoimmunoassays revealed three main categories of MCA molecular binding specificities; two thirds of all antibodies were directed against the alpha-subunit and one fourth toward the beta-chain, and less than one tenth recognized the conformationally (c) intact holohormone. With high frequency immunization schedules these specificities were shifted toward a higher proportion of beta-MCA. On the basis of intra- and interspecies cross-reaction studies as well as epitope contiguity analyses by sandwich assays, the three main categories could be further subdivided into nine epitopes: 1) five epitopes associated with the alpha-subunit, two of which were suprisingly shared by other species, and two being iodination sensitive, 2) two evolutionary conserved structures on the beta-subunit, adjacent to each other, and 3) two c-determinants, one of these present also on hTSH. The epitopes were arranged in three major antigenic domains, which seems to be a common homologous construction principle of the four human glycoprotein hormones: a central domain, consisting of three identically arranged alpha- and similarly located c-epitopes, is flanked by a single spatially distinct domain on each subunit. The establishment of an epitope map was followed by the construction of an immunoradiometric assay with a sensitivity of 0.25 ng hFSH/ml and an apparent cross-reactivity vs. hLH, hTSH, and hCG of less than 1%.
3677	Epitope mapping of human luteinizing hormone using monoclonal antibodies.	2458249	To establish structure-function relationships for human (h) LH, 14 murine monoclonal antibodies (MABs) to hLH were characterized in terms of their affinity of binding (Ka), their specificity for intact glycoproteins and their subunits, their paratopic relationships, and their ability to interfere with the biological activity of hLH. The Ka values obtained ranged between 2.4 x 10(6) and 1.0 x 10(10) for intact hLH and between 2.3 x 10(6) and 7.5 x 10(8) liters/M for the free alpha- and beta-subunits, indicating that, in general, the antibodies showed higher avidity for the intact hormone. Six MAB recognized both the intact and free alpha-subunit of hLH, cross-reacted with intact hCG, hFSH, and hTSH, and thus appeared to be alpha-directed. Four MAB were beta-directed, recognizing both intact hLH and its free beta-subunit. One of these beta-directed MABs also cross-reacted with intact hCG, hFSH, and hTSH, while two others recognized both intact and free beta-subunits of hLH and hCG. The fourth beta-directed MAB was quite specific for intact hLH and its beta-subunit. The remaining four MABs recognized epitopes only on the intact hormone; three recognized intact hLH and hCG, and the fourth was specific for intact hLH. Their paratopic relationships tested in competitive binding studies resulted in either mutual competition or complementarity, sometimes with cooperativity. Biointerference, defined as the ability to inhibit hLH-induced testosterone biosynthesis in dispersed rat Leydig cells, indicated that three of the alpha- and one of the beta-directed antibodies neutralized the biological response of hLH in this bioassay in a dose-responsive manner. Their ability to inhibit hLH bioactivity largely paralleled their affinity constants. Our data have allowed us to establish a tentative topographic relationship of epitopes to the biological region of the molecule of hLH, foreshadowing (in additive binding studies) some of the possible combinations of antibodies that might allow us to design two- or multiple-site immunometric assays in which measurement of immunoactive LH reflects biological activity. In addition, these studies suggest that both the alpha- and beta-subunits participate in LH receptor binding and/or biological activity.
3678	Relative roles of follicle-stimulating hormone and luteinizing hormone in the control of inhibin secretion in normal men.	3138288	The glycoprotein hormone inhibin is produced by the Sertoli cells of the testis under the influence of follicle-stimulating hormone (FSH) and is postulated in turn to inhibit FSH secretion. Luteinizing hormone (LH) is not recognized to have an important role in the control of inhibin secretion in any species. To determine the relative roles of FSH and LH in the control of inhibin secretion in man, we examined the effects of selective FSH and LH replacement on serum inhibin levels in normal men whose endogenous gonadotropins were suppressed by testosterone (T). After a 3-mo control period, nine men received 200 mg T enanthate i.m. weekly for 3-9 mo. During T treatment, serum LH and FSH levels were markedly suppressed and serum inhibin levels fell to 40% of control values. While continuing T, 3-5 mo of treatment with purified hFSH (n = 4) or hLH (n = 4) increased the respective serum gonadotropin level into the upper normal range and significantly increased inhibin levels back to 64 and 55% of control values, respectively. Supraphysiological LH replacement with high doses of human chorionic gonadotropin (n = 3) returned serum inhibin levels to 63% of control values. In no case did inhibin levels return fully to control levels. In conclusion, serum inhibin levels fell during gonadotropin suppression and were partially and approximately equally restored by either FSH or LH treatment. FSH presumably acts directly on the Sertoli cell to increase inhibin secretion whereas LH may act via increases in intratesticular T levels and/or other factor(s).
3679	Masked gonadotropin-binding sites in human corpora lutea during menstrual cycle and pregnancy.	3396694	To evaluate the possible existence of masked receptors for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) in the human corpus luteum, we examined the effect of neuraminidase pretreatment on the specific binding of (125I)hLH to luteal particulates obtained from 11 patients during the menstrual cycle and 4 patients during early pregnancy. The pretreatment with neuraminidase significantly enhanced the specific binding of hLH to corpora lutea at the different stages of the luteal phase. Scatchard analyses revealed that neuraminidase increased the number of LH binding sites without altering the affinity for LH. Furthermore, the specific binding of hLH to human corpora lutea during pregnancy significantly increased after the pretreatment with neuraminidase. The data suggest that distinct populations of receptors for LH/hCG are masked within the human luteal cells.
3680	Preparation and characterization of antibodies to the urinary fragment of the human chorionic gonadotropin beta-subunit.	2454812	hCG is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and is normally synthesized by trophoblastic tissue. Its measurement by immunoassay is widely employed as a test for pregnancy, but can be complicated by cross-reactivity with human (h) LH. Immunoassays based on the beta-subunit of hCG have been employed to decrease this cross-reactivity with hLH, but when these assays are used with urine specimens, the antibodies employed also detect a fragment of hCG beta, which can lead to significant differences in measurement. To overcome these problems, we have developed a series of monoclonal antibodies to the beta fragment of hCG recovered from pregnancy urine. Some of the antibodies that bind to this beta fragment are directed to a region of hCG beta that is different from the epitopes recognized by antibodies raised against the intact beta-subunit. The new epitopes available in the hCG beta fragment form the basis for novel immunoassays. These beta fragment antibodies are used in conjunction with other antibodies, directed to different epitopes of the hormone, to produce a series of immunoradiometric assays that can discriminate among intact hormone, free hCG beta, and hCG beta fragment. The hCG beta fragment antibodies described herein have affinities between 10(9) and 10(11) M-1 for the beta fragment and exhibit varying degrees of discrimination between the hCG beta fragment, the beta-subunits of hCG and hLH, and intact hCG and hLH.
3681	Immunological cross-reactivity of antibodies with species chorionic gonadotropin is a critical requirement for efficacy testing of human gonadotropin vaccines in sub-human primates.	3418617	A key question in the evaluation of a contraceptive vaccine is its efficacy in the prevention of pregnancy. Primates have been employed for evaluation of the efficacy of candidate vaccines against human chorionic gonadotropin (hCG). Discrepancies have been noted between the immuno-reactive antibody titres (against hCG) determined by RIA and their ability to prevent pregnancy in test animals. To gain further information on factors involved in fertility control, other characteristics of antibodies, such as affinity for hCG, bioneutralization capacities for hCG, hLH and monkey chorionic gonadotropin, have been determined in antisera from ten bonnet monkeys (Macaca radiata) at times when they were infertile and in bleeds preceding conception. The data reported show that the most important correlate for efficacy is the bioneutralization capacity of the antibodies for the species CG; animals became pregnant when the neutralization capacity diminished to low levels (less than 45 IU/l = 5 ng/ml of hCG equivalent). The bioneutralization of heterospecies CG decreased with the increase in affinity for hCG. These findings have implications for the choice and suitability of primate species for the various candidate vaccines being developed. Vaccines inducing highly specific antibodies against hCG, and restricted in their reactivity with primate CG, may not be testable in heterospecies primate models.
3682	Characterization of a factor from human ovarian follicular fluid which stimulates Leydig cell testosterone production.	3389053	A factor from human ovarian follicular fluid (hFF) has been characterized which stimulates testosterone production of human, rat, mouse and hamster Leydig cells. hFF was obtained from women participating in an in vitro fertilization programme. Basal and hCG stimulated testosterone production of rat interstitial cells, and Percoll purified Leydig cells were significantly stimulated by hFF. The steroidogenic response of the cells was 3-5 fold higher than that obtained after stimulation with maximal doses of hCG. A serum pool from the same patients was found to be about 30 times less potent than hFF in stimulating steroidogenesis. The stimulatory activity was retained after precipitation with ammonium sulphate and dialysis. Precipitation with ethanol, ether and acetone resulted in a partial loss of activity, whereas extraction with charcoal or heating at 100 degrees C for 10 min resulted in significant loss of activity. When hFF was fractionated by gel chromatography, the stimulatory activity was eluted in a molecular weight region between 30 and 50 kD. The stimulatory factor was further purified by chromatofocusing and was eluted as a homogeneous peak with an isoelectric point between 8.8 and 9.5. The SDS-PAGE analysis of these fractions, however, revealed that the active substance was not homogeneous. The purified factor was immunologically distinct from hCG and hLH. These studies demonstrate for the first time the presence of a factor in the hFF which may potentiate the action of LH in the ovary.
3683	Luteal function following ovarian stimulation in rhesus monkeys for in vitro fertilization: atypical response to human chorionic gonadotropin treatment simulating early pregnancy.	3131158	This study determined if corpora lutea of hyperstimulated cycles in rhesus monkeys could be "rescued" by the pregnancy signal, chorionic gonadotropin (CG), given at the typical time of implantation. At menses, female monkeys received human follicle-stimulating hormone (hFSH, 60 IU, days 1 to 6) followed by human menopausal gonadotropin (hMG, 60 IU hFSH/60 IU luteinizing hormone [hLH], days 7 to 9). On day 10, human chorionic gonadotropin (hCG) was given to mimic the LH surge. Nine days later, a regimen of daily increasing doses of hCG (15 to 360 IU twice a day) was initiated to simulate rescue of the corpus luteum in early pregnancy. Serum levels of progesterone (P) increased through day 5 of the luteal phase but then declined. Circulating levels of bioactive LH were significantly less on days 7 to 9 of the luteal phase than at this stage in the natural cycle. The hCG regimen extended (P less than 0.05) the luteal phase in five of six animals. The hCG treatment elicited a persistent increase (P less than 0.05) in circulating P levels, rather than a transient rise typical of normal or simulated pregnancy in natural cycles. The authors conclude that (1) corpora lutea of hyperstimulated cycles can respond to CG, but (2) there are differences in luteal function during both the luteal phase and simulated early pregnancy that may be due to inadequate luteal development or the abnormal gonadotropin milieu existing after ovulation or both.
3684	Characterization of antisera distinguishing carbohydrate structures in the beta-carboxyl-terminal region of human chorionic gonadotropin.	2452075	The beta-COOH-terminal peptide region (beta CTP) of hCG is an important immunochemical domain that is specific to hCG but absent from homologous hLH. Three types of polyclonal antibodies can be elicited to the beta CTP portion of hCG. The first and most common recognizes the beta CTP amino acid sequence independent of its carbohydrate content (carbohydrate-oblivious). It can be elicited against synthetic beta CTP as well as against native or asialo beta CTP. The second type binds best to desialylated beta CTP, (galactose-requiring). The third type binds well only to sialylated beta CTP (sialic acid-requiring). All three types of antisera recognize the sialylated beta CTP as equivalent to the whole hormone on a molar basis, indicating that this peptide region of the whole hormone is neither sequestered nor conformationally altered. We have characterized the epitopes for the sialic acid-requiring and galactose-requiring beta CTP antisera. Both require elements of the primary amino acid sequence of beta hCG as well as specific elements of the carbohydrate side-chains and, therefore, are not directed solely to either peptide or carbohydrate determinants. The galactose-requiring beta CTP antiserum was generated to an ovalbumin conjugate of a desialylated form of beta CTP, beta 123-145. It binds desialylated forms 1000 times better than it binds native hCG, and binds neither synthetic beta CTP nor asialo-agalacto beta 123-141. The antiserum requires residues 123-141 and a portion of an O-serine-linked oligosaccharide chain. Its binding requirements are, thus, very different from those of the carbohydrate-oblivious beta CTP antiserum, which requires the last two residues of the hCG beta polypeptide chain and is not sensitive to the presence or absence of carbohydrate. The sialic acid-requiring beta CTP antiserum, which was generated to sialylated beta 123-145-thyroglobulin conjugate, binds well to sialylated beta CTP, but poorly to asialo beta CTP and not at all to the carbohydrate-free synthetic peptide. This antiserum requires the entire beta 123-145 sequence as well as sialic acid-terminated oligosaccharide side-chains. Since the sialylated peptide beta 115-141 binds poorly to it, this antiserum resembles the carbohydrate-oblivious anti-beta CTP; both require the COOH-terminal amino acids of hCG beta. However, the former requires intact O-linked carbohydrate moieties for binding. These antisera can be used to assess the primary protein and carbohydrate structures of beta CTP at low concentrations in urine.(ABSTRACT TRUNCATED AT 400 WORDS)
3685	Measurement of serum human lutropin (hLH): hCG-interference evaluated for two hLH-specific IRMA kits.	3359619	NaN
3686	Failure of gonadotropin therapy secondary to chorionic gonadotropin-induced antibodies.	3279065	Seventeen years after first receiving treatment with hCG (at age 8 yr), a man with hypogonadotropic hypogonadism no longer responded to gonadotropin therapy. He had received hCG for 6 months when he was 8 yr old, from age 18-21 yr and from age 21-25 yr, when the resistance developed. Anti-hCG antibodies were found in his serum. Three sequential treatment regimens were tried to obviate the effect of these antibodies. 1) hCG treatment (2000 IU, three times per week) concomitant with weekly plasmapheresis (since the patient's response to an hCG challenge test was improved after a reduction of antibody titer by plasmapheresis) resulted in only a temporary increase in testosterone production. 2) Treatment with human (h) LH (400 IU/week) and hFSH (25 IU/week) was used because of the low cross-reaction of the antibodies with hLH and a response to a hLH-challenge test. This treatment maintained serum testosterone levels within the normal range for long periods, but had to be discontinued when the supply of hLH was exhausted. 3) Pulsatile LHRH administration (25 ng/kg, sc, every 2 h) for 2 months did not induce the release of pituitary gonadotropins. These results indicated that 1) conventional hCG treatment was impaired by antibody-induced changes in the kinetics of hCG after its im administration; 2) hLH was an effective substitute for hCG, and the combined hLH-hFSH administration initiated a moderate amount of spermatogenesis; and 3) the patient differs from most individuals with hypogonadotropin hypogonadism in that he did not have normal responses to repetitive LHRH administration.
3687	Immunobiology of human chorionic gonadotropin.	3049323	A comprehensive review of the immunobiology of human chorionic gonadotropin (hCG), including the structure of both alpha and beta chains, immunogenicity of various segments and epitopes of each, secretion and function of the hormone, determinants of receptor recognition, and finally, clinical studies of possible contraceptive beta-hCG-based vaccines, is presented. hCG is composed of 2 glycosylated peptides. The alpha subunit is identical to that found in hLH, hFSH and hTSH. The beta subunit, which is limiting in the sense that it is secreted in smaller amounts, defines the biological activity of hCG. hCG is secreted throughout pregnancy from 170 hours after fertilization to a peak at 8-10 weeks of and is essential for maintenance of early pregnancy by progesterone secreted by the corpus luteum. Although native hCG evokes antibodies, they cross react with LH, so such a vaccine would not be useful for contraception. Beta-hCG has been purified and also produced by monoclonal antibodies, and shown to produce antibodies and infertility in baboons. Phase I clinical trials of immunologically purified beta-hCG complexed to tetanus toxoid were conducted on 63 women in an international study in the mid-1970s, but results were mixed in terms of antibody titer and duration. New vaccines have been designed based on more sophisticated adjuvants, beta- hCG-terminal peptides, and polyvalent vaccines and are being tested in 4 Phase I trials currently, sponsored by the Population Council, the Indian government-sponsored program, and the WHO.
3688	Fibrocystic breast disease: the significance of beta-human chorionic gonadotropin and other polypeptides in breast cyst fluid.	2450789	Breast cyst fluids from 118 women, aged 29 to 69 years, were analyzed by radioimmunoassays for beta-human chorionic gonadotropin (beta-hCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and thyroid-stimulating hormone (TSH). Blood was drawn at the same time in many cases to compare hormonal levels in serum with those in the breast cyst fluids (BCF). The levels of beta-hCG in BCF were relatively high, with a mean (+/- standard error of the mean [SEM]) of 58.9 +/- 16.8 mIU/ml; serum levels of beta-hCG were negligible. LH and TSH also were elevated in BCF compared with serum levels, exhibiting mean values (+/- SEM) of 26.7 +/- 4.3 mIU/ml and 6.4 +/- 0.44 muIU/ml, respectively. The levels of FSH and PRL in BCF were equivalent to the levels in the serum. The presence of biologically active hCG was suggested in several BCF samples using the rat ovarian hyperemia test. Samples of BCF were assessed for the capacity to stimulate Leydig cell testosterone production in vitro in the presence or absence of an anti-hLH antiserum. Testosterone production was significantly (P less than 0.05) enhanced, even in the presence of the antiserum. These data suggest that BCF contains biologically active hCG.
3689	Assessment of serum luteinizing hormone during ovarian stimulation with gonadotrophins.	3128573	An immunoradiometric assay (IRMA), using monoclonal antibodies with high affinity for human luteinizing hormone (HLH), was evaluated for quantitative measurement of serum LH after human chorionic gonadotrophin (HCG) administration in patients undergoing stimulation of multiple follicular development. Compared to a radioimmunoassay (RIA) commonly used to monitor serum LH, LH IRMA was more effective by several orders of magnitude in discriminating between HLH and HCG and showed no cross-reactivity at HCG concentrations normally found in serum after hormone treatment. Assays of serum samples obtained from 10 patients receiving HCG as part of an HMG/HCG protocol to induce ovulation for IVF/GIFT also demonstrated that RIA values were greatly affected by exogenous HCG. It was estimated that 17-32% of serum HCG was measured as serum LH in RIA. In contrast, determinations of serum LH by IRMA was not biased by exogenous HCG. Data from IRMA indicated that eight of the 10 patients showed a significant rise in LH secretion, relative to mean baselines, at either 12 or 36 h after administration. In one patient the rise had already occurred before HCG administration. When an LH rise occurred, either before or after HCG injection, mean values were 2- to 9-fold higher than those of baseline levels. Assuming that LH rises greater than 12 mIU/ml may relate to an endogenous surge of LH, none of the patients showed a surge prior to HCG administration. On the contrary, the occurrence of an 'LH surge' after HCG was apparent in four patients.(ABSTRACT TRUNCATED AT 250 WORDS)
3690	125I-luteinizing hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: effects of ethanol exposure.	3398712	In vitro exposure to alcohols unmasks additional binding sites for gonadotropin in cell/membrane preparations of the corpus luteum of rhesus monkeys. In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125I-human luteinizing hormone (hLH) than were available in particulate preparations (p less than 0.05). However, the soluble receptors displayed a 3-fold lower affinity for 125I-hLH (p less than 0.05). Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific 125I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.
3691	Quantitation of human chorionic gonadotrophin by the planimetry of latex agglutination-inhibition results on microtiter plates.	3360919	A new type of heavy latex particle (LP) coated with the conjugates of human chorionic gonadotrophin (hCG) and bovine serum albumin (BSA) was used as indicator for the microtiter agglutination-inhibition assay of hCG using a monoclonal antibody (McAb) to hCG. The precipitation patterns of LP were measured by an automatic planimetry system. The detection limit of this method was 4 to 8 IU/l. Human luteinizing hormone (hLH) and follicle-stimulating hormone (FSH) caused hardly any cross-reaction in the presence of 3,350 IU/l and 1,500 IU/l, respectively. This planimetric immunoassay (PMIA) method provides a potentially useful method for hormone assay.
3692	Absence of immunoreactive luteinizing hormone following gonadotropin-releasing hormone agonist therapy in women with endometriosis.	2967234	Pituitary desensitization following infusion of gonadotropin-releasing hormone (GnRH) is measurable if bioactivity instead of immunoreactivity is considered. We hypothesized that GnRH agonist therapy induces the same kind of desensitization, but that radioimmunoassays (RIA) for gonadotropins based on polyclonal antibodies cannot show this effect because they recognize inactive fragments of gondadotropins. To test this hypothesis we measured luteinizing hormone (LH) with two different assays: one RIA was based on a polyclonal rabbit anti-hLH, while the other one was an immunoradiometric assay (IRMA) based on 2 different mouse monoclonal anti-hLH. LH measurements were performed on plasma samples obtained from 13 women with laparoscopically proven endometriosis and treated with microcapsules of the GnRH agonist D-Trp6-GnRH (Ferring) once a month. The correlation between LH measurements with both assays in 36 control plasma samples and in another 13 samples obtained before treatment in women with endometriosis was excellent (r = 0.959). In contrast, in women treated with GnRH agonist, the RIA yielded values ranging from undetectable to 12 mIU/ml, whereas 60 out of 66 values were undetectable with the IRMA. We conclude that the monoclonal anti-hLH antibodies in the IRMA either recognize an epitope close to the active site and/or do not recognize the biologically inactive LH fragments which are known to be produced during GnRH agonist therapy. Thus, monoclonal-antibody-based IRMA provide a new and interesting clinical tool to follow the effects of therapies which desensitize the gonadotropic function of the pituitary.
3693	Probing the receptor-interaction of glycoprotein hormones with monoclonal antibodies.	2455048	Having recently analyzed with monoclonal antibodies (MCA) the immunologic surface of human chorionic gonadotropin (hCG) as consisting of 9 distinct epitopes exposed on the molecule in a characteristic topographical manner (Schwarz, S., Berger, P., and Wick, G., Endocrinology 118, 189-197, 1986) we now attempted to confirm this result on a more general basis, e.g. by incorporating MCA that were just recently obtained and previously not included. What we were, however, interested most was the question whether the tentative model of the epitope map of hCG could represent a "target" with which hCG-related hormones such as LH, FSH, and TSH (the family of glycoprotein hormones, GPH) would (partially) match. Indeed, repeating various immunizations with GPH of human as well as animal origin revealed a remarkable reproducibility in terms of several anticipated epitope specificities of MCA. This indicates that MCA can be regarded as reliable probes for mapping epitopes and, as we have presumed, of receptor interaction domains of GPH as well. Extending the originally used 2-site MCA binding exclusion approach by an interspecies crossreactivity (Xr) analysis we now are able to refine our epitope model of hCG such that 2 additional epitopes were found which were not previously resolvable. Most surprisingly, two of 5 epitopes on the alpha subunit were now also detected on various non-human GPH, which is in striking contrast to a seemingly well established dogma. Yet all five alpha epitopes of hCG are present on hLH, hFSH, and hTSH as well and arranged in the same spatial relationship to each other as on hCG. Even the 2 conformational epitopes and their close topographical relationship to the alpha-epitopes appear to be remarkably conserved on all human GPH. Among the beta-epitopes we have found one that is not shared by hLH and that - surprisingly - is not the C-terminal peptide (CTP) by which hLH differs from hCG. On the basis of this refined epitope map a way was paved along which it should be feasible to elucidate the sterical relationship of the epitopes to the receptor interaction domain(s) of hCG. To this end the MCA were tested in principally two ways: first, as to which of the 11 MCA with different epitope specificities would be able (or not) to inhibit by preincubation the binding of radiolabeled hCG (or hLH, respectively) to rat testis LH/hCG receptors? Secondly (and inversely) which of the 11 epitopes of hCG would still be accessible to binding by radiolabeled MCA when the (unlabeled) hormone is bound to the receptor?(ABSTRACT TRUNCATED AT 400 WORDS)
3694	Pulsatile secretion of human chorionic gonadotropin in normal adults.	3696177	We used very sensitive and specific monoclonal-antibody sandwich assays for human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) to measure both hormones in serum samples from normal men and women. When single serum samples from 92 men were studied, 73 percent had detectable hCG. In normal men, the amount of detectable hCG averaged 8.9 pg per milliliter, with a range of less than 3.0 to 160 pg per milliliter (biologic potency = 13,450 IU per milligram). In postmenopausal women the hCG level averaged 111 pg per milliliter and ranged from 32 to 510. In women of reproductive age the hCG level varied with the menstrual cycle. Gonadotropin-releasing hormone administered to 10 normal men increased both hCG and hLH. When daily serum samples were studied throughout a normal menstrual cycle, hCG concentrations paralleled those of hLH; follicular-phase concentrations were higher than those of the luteal phase, and there was a midcycle ovulatory peak of hCG coincident with the hLH peak. When hCG was measured every 10 minutes for six hours in eight postmenopausal women, distinct pulses were detected in parallel with those of hLH: hLH pulsed at a mean (+/- SEM) frequency of 0.56 +/- 0.08 pulses per hour; hCG pulsed at 0.54 +/- 0.07 pulses per hour. The mean pulse durations were 89 +/- 22 and 56 +/- 20 minutes for hLH and hCG, respectively. We conclude that hCG is produced in a pulsatile fashion, probably by the pituitary, in all normal adults.
3695	Characterization of human LH isohormones from fresh pituitary tissue.	3691956	The pattern of human LH (hLH) microheterogeneity was determined using freshly obtained pituitary tissue. Chromatofocusing across a pH 9-6 gradient produced several, distinct peaks of hLH immunoreactivity between pH 8.5 and 6.8, as well as a 'salt peak'. Chromatofocusing of the 'salt peak' across a pH 7-4 gradient yielded distinct peaks of hLH at pH 6.1, 5.6, and 5.4. Pituitary tissue obtained at surgery produced a virtually identical pattern. By gel filtration the elution volume of each major chromatofocusing peak was similar to that of intact hLH, rather than those of the hLH subunits. The bioactivity/immunoreactivity ratios of the major chromatofocusing peaks fell dramatically with decreasing pH, from approximately 8 at pH 8.5 to approximately 1 at pH less than 6. These results indicate that human pituitary LH exists in multiple, isomeric forms that differ markedly in charge and bioactivity. These differences appear to be inherent in the native, intact molecules and not the result of autolysis or of dissociation into subunits.
3696	Antigenic regions on the beta chain of human chorionic gonadotropin and development of hormone specific antibodies.	2450842	Five peptides corresponding to four regions of the beta chain of human chorionic gonadotropin (hCG), were synthesized, purified and characterized. The four regions studied were selected on the basis of sequence differences between the beta chain of hCG (beta hCG) and the beta chains of related hormones. The peptides were found to bind rabbit and mouse anti-hCG antibodies as well as rabbit anti-beta chain antibodies, but did not bind antibodies against the alpha chain or against other hormones. All the peptides, even in their free form, were able to elicit high titer antisera in both rabbits and mice. In all cases, anti-peptide antisera bound to the immunizing peptide as well as to the native hCG and the isolated beta chain. These anti-peptide antisera did not bind to unrelated peptides, the alpha chain of hCG or to other hormones with very similar beta chains such as human luteotropic hormone (hLH), ovine luteotropic hormone (oLH) and equine chorionic gonadotropin (eCG). Since the areas represented by these peptides elicit antibodies that are specific for human beta hCG, they can formulate the basis for the development of discriminatory reagents for the beta chain of hCG.
3697	Development of an assay for a biomarker of pregnancy and early fetal loss.	3319556	Human chorionic gonadotropin (hCG) is a glycoprotein hormone, secreted by the syncytiotrophoblast cells of the fertilized ovum, that enters the maternal circulation at the time of endometrial implantation. It is composed of two nonidentical subunits; alpha and beta, with molecular weights of 14 kD and 23 kD, respectively. Its alpha subunit is identical in primary structure to its glycoprotein homologs, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). Human chorionic gonadotropin binds to the same receptor as hLH and displays the same biological response, namely, to stimulate the declining function of the corpus luteum to produce progestins and estrogen late in the menstrual cycle. The differences in the structures of hCG and hLH have been exploited to develop antibodies that can measure hCG specifically in the presence of hLH. Two-site antibody binding assays have been developed, based on a surface immunological concept of hCG epitopes, that involve four distinct regions to which antibodies against hCG can bind simultaneously. Antibody cooperative effects, in conjunction with kinetic advantages derived from the concentration factors by use of the sandwich assay technique (immunoradiometric assay, IRMA), have enabled development of extremely sensitive and specific measurement protocols for urinary hCG. The assay described herein permits the detection of pregnancy on an average 25.4 days after the first day of the preceding menses, as opposed to 29.5 days for conventional radioimmunoassay techniques. In addition, the greater sensitivity and specificity of this assay method has permitted the detection of episodes of fetal loss not detected by radioimmunoassay of urine specimens. A large scale epidemiological study is in progress using this assay technique as a way to identify pregnancies that are lost before becoming clinically apparent. This methodology provides a valuable tool for the determination of the rate of early fetal loss.
3698	Significant steroidogenic activity of luteinizing hormone is maintained after enzymatic removal of oligosaccharides.	3155265	Several reports have described the destruction of the N-linked oligosaccharides on glycoprotein hormones by hydrogen fluoride treatment and have noted the accompanying marked reduction, or complete loss, in biological activity. This has led to the concept that the oligosaccharides have an obligatory role in glycoprotein hormone steroidogenic function. Using a less radical and more complete method for removing sugar units, endoglycosidase treatment and ovine LH (oLH) and human LH (hLH) as examples, we examined the role of oligosaccharides in hormone function. Ovine LH and hLH were digested with endoglycosidase F. After treatment cleavage of oligosaccharides was demonstrated by compositional studies, greater than 87% cleavage was demonstrated and only N-acetylglucosamine or N-acetylglucosamine-Fucose shown to remain attached to the peptide, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate size change) and by chromatofocusing (appearance of a single basic peak). Biological activities and relative potencies of preparations were then assessed in an in vitro assay, in which the ability of samples to promote testosterone production by testicular interstitial cells was measured. Although endoglycosidase F treatment reduced relative potencies 2- to 3-fold in the bioassay, (possibly in part due to subunit dissociation) it did not lessen abilities to induce maximal testosterone response (that of native hLH and oLH). These findings contrast with those obtained from hydrogen fluoride studies and indicate that the oligosaccharides, per se, do not play an obligatory role in the steroidogenic activity of LH.
3699	An improved in vitro bioassay for follicle-stimulating hormone (FSH): suitable for measurement of FSH in unextracted human serum.	3113917	FSH bioactivity was measured by means of FSH-dependent aromatase activity (conversion of androgen substrate to estradiol). Assay sensitivity was optimized by the use of immature (7-10 days old) rats as Sertoli cell donors, serum-free medium for incubation, phosphodiesterase inhibitor (methylisobutylxanthinine), serial dilution of FSH in medium containing 1% BSA, delayed addition of FSH for 72 h after cell plating, and 19-hydroxyandrostenedione (2.5 X 10(-6) M) as the aromatizable androgen substrate. The method consisted of subjecting the decapsulated immature rat testes to a 2-step collagenase dispersion, plating the cells in medium [Dulbecco's Modified Eagle's Medium-Ham's F-10 (1:1)] containing growth factors and methylisobutylxanthinine for 72 h, adding increasing doses of FSH to the standard curve or small volumes of serum to the test vials as well as 19-hydroxyandrostenedione for 24 h, and measuring estradiol by RIA in dilutions of the medium. Using NIAMDD human (h) FSH-2 as the bioassay standard, the useful range of the assay was 0.01-5.0 ng/ml. Specificity was determined by the addition of graded doses of hLH, hTSH, ACTH, hGH, hPRL, and hCG. The minor degree of FSH bioactivity observed in a few hormone preparations was accounted for by the degree of FSH contamination in them. Mean intra- and interassay coefficients of variation were 9% and 11%, and the index of precision was 0.049. This bioassay was used to determine the bioactive FSH content of pituitary extracts, tissue culture media, elutions from columns, and isoelectrically focused samples. More importantly, small quantities of human sera gave responses parallel to the standard curve in a minimum of two dilutions. The bio- to immunoreactive ratios, expressed as the mean +/- SEM (NIAMDD-hFSH-2), were 0.66 +/- 0.2 in boys (n = 6), 0.78 +/- 0.2 in pubertal girls (n = 6), 1.18 +/- 0.2 in men (n = 13), 1.24 +/- 0.1 in postmenopausal women (n = 30), 1.94 +/- 0.3 in the follicular phase (n = 19), 6.2 +/- 1.4 in the ovulatory phase (n = 19), and 1.6 +/- 0.4 in the luteal phase (n = 19) of the normal menstrual cycle. These results indicate that the bio- to immunoreactive hFSH ratio in the circulation, is dependent upon the hormonal milieu of the subject.
3700	Two-monoclonal-antibody "sandwich"-type assay of human lutropin, with no cross reaction with choriogonadotropin.	3113769	We have developed a sensitive, specific, noncompetitive sandwich-type assay for human lutropin (hLH). Two monoclonal antibodies are used, and there is no cross reaction with human choriogonadotropin (hCG) or human follitropin (hFSH), and little or none with human thyrotropin (hTSH). There also is no reaction with the free beta chains of hLH and hCG. The detection limit is less than 0.5 int. units of hLH per liter of serum, and the dose-response curve is linear between 0 and 10 int. units/L. The intra-assay CV averaged 5.4% at low doses of hLH; the interassay CV averaged 12.5%.
3701	Monoclonal antibody mapping of the antigenic surface of human thyrotropin and its subunits.	2441979	Although the amino acid sequence of the alpha- and beta-subunits of glycoprotein hormones in various species has been deciphered, data on their tertiary structure are not abundant. This impedes correlation between structure and function. The availability of monoclonal antibodies to human TSH (hTSH) offers the opportunity to enumerate the antigenic determinants present on the surface of hTSH and its subunits and to examine their spatial relationships. Twenty-eight monoclonal antibodies to hTSH were obtained from several fusions, and screens carried out separately in the laboratories involved in this study. Affinities for hTSH ranged from 10(8)-10(11) M-1. Cross-reactivity with bovine TSH (bTSH), human gonadotropins (hLH, hFSH, and hCG), and the alpha- and beta-subunits of hTSH distinguished 10 groups of monoclonal antibodies (mAb) according to their main cross-reactions: 1) hTSH alpha, hLH, hFSH, and hCG; 2) hTSH alpha, bTSH, hLH, hFSH, and hCG; 3) hFSH; 4) bTSH and hFSH; 5) bTSH, hLH, and hFSH; 6) bTSH, hLH, hFSH, and hCG; 7) hTSH beta; 8) hTSH beta and bTSH; 9) hTSH beta and hFSH; and 10) hTSH beta, hLH, hFSH, and hCG. mAb were incorporated into 2-site binding assays to probe hTSH by a 28 X 28 matrix, the free alpha-subunit by a 4 X 4 matrix, and the free beta-subunit by a 18 X 18 matrix. Regarding intact hTSH, 12 different clusters of mAb were distinguished and interpreted as reflecting 12 distinct antigenic regions on the surface of the hTSH molecule. Two of them were localized on the alpha-subunit, and 6 on the beta-subunit; 4 were only expressed by the holo-hormone and, thus were designated conformational antigenic regions (alpha beta). Surface mapping of the free alpha- and beta-subunits was virtually identical to that observed with the holo-hormone. Modification of the operative conditions of mAb reacting only with holo-hTSH shows that they recognize the alpha-subunit, but not the beta-subunit of hTSH. These results indicate that 1) hTSH beta presents epitopes that are evolutionary conserved; 2) hTSH alpha presents several epitopes that are species specific and 2 that are not hormone specific; 3) dissociation of hTSH does not modify the antigenic surface expressed by both subunits when they are associated; and 4) some of the conformational determinants expressed only by holo-hTSH are more likely derived from the alpha-subunit than from the beta-subunit.
3702	Elution and rebinding of gonadotropins to receptors in corpora lutea: comparisons among treatments and species.	3651550	Several treatment regimens have been used to dissociate bound gonadotropins from their target tissues to quantify numbers of occupied receptors. To compare the efficacy of these methods, we evaluated the ability of a number of treatments to elute bound gonadotropins from corpora lutea of various species. In addition, we examined the capacity of luteal tissue to rebind gonadotropins after efficacious elution (greater than 90% dissociation of bound gonadotropin). Particulate (20,000 g) preparations of luteal tissue from pseudopregnant rats and from nonpregnant pigs and rhesus monkeys were incubated with 125I-labeled human luteinizing hormone (hLH, NIH-LH-11) or 125I-labelled chorionic gonadotropin (hCG, CR119) for 20 h at 25 degrees C to occupy gonadotropin receptors. Heat treatment (60 degrees C) eluted greater than 80% of bound 125I-hLH from rat tissue within 30 min, but similar treatment dissociated only 35% of 125I-hLH bound to porcine tissue (p less than 0.01). Likewise, heat treatment eluted only 57% of 125I-hLH bound to macaque tissue. At 4 degrees C, the following treatments dissociated greater than 90% of specifically bound 125I-hLH from porcine and rat luteal particulates within 5 min: acetic acid (pH 2.3), formic acid (pH 2.3), propionic acid (pH 2.3), acetic acid: HCl (pH 2.3 and 3.3), and MgCl2 (4 M). After 1-2 h at 4 degrees C, exposure to urea (4 M) or acetic acid:HCl (pH 4.3) also eluted greater than 90% of bound 125I-hLH.(ABSTRACT TRUNCATED AT 250 WORDS)
3703	Biochemical evidence that human placental lactogen and human chorionic gonadotropin are not stored in cytoplasmic secretion granules.	3651549	The intracellular storage sites for the human placental hormones placental lactogen (hPL) and chorionic gonadotropin (hCG) are unknown. To determine whether hPL and hCG are stored in cytoplasmic secretion granules, we have compared the localization of hPL and hCG in placental homogenates following differential and density-gradient centrifugations to those of prolactin (PRL) and luteinizing hormone (LH) in human and rat pituitary homogenates. In the differential centrifugation studies, 93.1 +/- 4.1% (mean +/- SE) of the hPL and 79.4 +/- 6.0% of the hCG were detected in the postmicrosomal supernatant of placental homogenates. In contrast, 95-98% of the hPRL and hLH in the pituitary homogenates were detected in particulate fractions. Following centrifugation on sucrose-density gradients, particulate hPL and hCG were distributed diffusely throughout the gradients, while greater than 90% of the pituitary hormones sedimented as single peaks with densities of 1.22 g/cm3. When human placental and rat pituitary tissues were homogenized together prior to differential and density-gradient centrifugations, similar marked differences were observed between the distribution of the placental and pituitary hormones. These results strongly suggest that the placental hormones hPL and hCG, unlike pituitary PRL and LH, are not stored in large secretory granules. Differences in the intracellular storage sites of the hormones may explain, in part, differences in the regulation of peptide hormone secretion by placental and pituitary tissues.
3704	[Neuroimmunomodulation of the immune response by the endocrine system: effect of luteinizing hormone (LH) on the proliferative response of lymphocytes].	3452673	Proliferative response of splenic lymphocytes from adult (2 months old) male mice to human luteotrophic hormone (hLH) was studied in vitro. hLH induced lymphoblastic transformation at a concentration of 0.1 micrograms/ml or higher. This hormone potentiated the mitogen activities of concanavalin A (ConA) and phytohemagglutinin (PHA) for T cells and of pokweed mitogen for B and T cells. The data suggest that peripherally circulating LH may regulate a certain function of lymphoid cells.
3705	Direct stimulation of nucleoside triphosphatase activity in human ovarian nuclear membranes by human chorionic gonadotropin.	3036904	We previously reported that nuclei isolated from ovaries of premenopausal women contain binding sites for hCG/human LH (hLH). This study was undertaken to determine the possible functional significance of these nuclear binding sites. Upon addition to isolated ovarian (mostly luteal cells) nuclear membranes, hCG and hLH stimulated nucleoside triphosphatase (NTPase), an enzyme involved in nucleocytoplasmic transfer of mRNA, but not Mg2+-ATPase or NADH cytochrome c reductase activities, in a concentration-dependent manner. Heat-denatured hCG, isolated alpha- and beta-subunits of hCG, human FSH, PRL, and porcine relaxin had no effect on the enzyme. Addition of hCG antiserum blocked hCG's ability to stimulate NTPase activity. cAMP, which is a second messenger in hCG- and hLH-stimulated steroidogenesis, had no effect on NTPase activity. These results, which demonstrate that hCG acts on human ovarian nuclei directly, raise the possibility that internalized hCG might influence nuclear function(s) before it is eventually degraded in the lysosomes of ovarian cells.
3706	Specific binding sites for LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. I: Validation of methods.	3108440	The specific binding of 125I-labelled human chorionic gonadotrophin (hCG), human low-density lipoprotein (hLDL), human FSH (hFSH) and human prolactin (hPRL) to homogenates of human corpus luteum tissue was measured. Specific binding of 125I-labelled hCG was dependent on the temperature and duration of incubation, was inhibited by divalent metal ions or chelating agents, and increased linearly with homogenate concentration. Recovery of bound hormone was more effective using Millipore filtration or polyethylene glycol precipitation compared with centrifugation alone. Binding of 125I-labelled hCG was inhibited specifically by low levels of hCG and human LH (hLH) but not by ovine LH or bovine LH. Incubation of human luteal tissue with ice-cold citrate buffer (pH 3) released more than 90% of specifically bound 125I-labelled hCG within 5 min. This treatment inactivated LH receptors, but did not affect the immunoactivity of hLH released, enabling the measurement of released hormone by radioimmunoassay. Scatchard plots of binding of 125I-labelled LDL to human corpus luteum demonstrated a single class of binding sites. Binding was saturable, increased linearly with increasing concentration of homogenate, and was displaceable by low concentrations of unlabelled LDL. Binding of 125I-labelled hPRL to human luteal homogenates was increased by Mg2+ and was specific for lactogenic hormones (human prolactin, human growth hormone and ovine prolactin). Binding of 125I-labelled hFSH was not dependent on divalent metal ion concentration (in marked contrast to hFSH binding to immature pig granulosa cell receptors) and was displaced by hFSH preparations but not by hPRL, ovine LH or hCG at 1 microgram/ml. These results establish optimal conditions and hormone specificities for the measurement of human luteal gonadotrophin and LDL receptors, and methods for the estimation of hLH/hCG endogenously bound to human corpus luteum tissue.
3707	Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle.	3106871	Gonadotropin binding sites were localized by autoradiography after incubation of human ovarian sections with 125I-labeled gonadotropins. The binding sites for 125I-labeled human follicle-stimulating hormone (125I-hFSH) were identified in the granulosa cells and in the newly formed corpora lutea. The 125I-labeled human luteinizing hormone (125I-hLH) binding to the thecal cells increased during follicular maturation, and a dramatic increase was preferentially observed in the granulosa cells of the large preovulatory follicle. In the corpora lutea, the binding of 125I-hLH increased from the early luteal phase and decreased toward the late luteal phase. The changes in 3 beta-hydroxysteroid dehydrogenase activity in the corpora lutea corresponded to the 125I-hLH binding. Thus, the changes in gonadotropin binding sites in the follicles and corpora lutea during the menstrual cycle may help in some important way to regulate human ovarian function.
3708	Characterization of monoclonal antibodies against human thyrotropin and use in an immunoradiometric assay and immunohistochemistry.	3571984	Monoclonal antibodies were prepared against human thyrotropin. 13 different antibodies were characterized. Ten antibodies were of the IgG1 subclass. The affinities of the antibodies were in the range 10(9)-10(11) mol-1 X l. Four of them were specific for hTSH and did not react with hLH, hFSH, hCG or alpha hCG. Four reacted with these hormones and recognized the alpha subunit of hCG. One cross-reacted only with hFSH. The remaining four antibodies recognized the holo-hTSH only, and thus were designated as anti-conformational determinants. Monoclonal antibodies reacting with different antigenic determinants on the hTSH molecule defined seven clusters. Two of them were used to develop a simplified two-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized on tubes (anti-beta TSH) and another (anti-alpha) labelled with 125I. This assay was highly specific and demonstrated a sensitivity level of 0.1 microIU/ml. Two monoclonal antibodies were used in immunohistochemistry and their quality and specificity was assessed in the detection of hTSH immunoreactivity in human pituitary biological sections.
3709	Tryptophan fluorescence studies of subunit interaction and rotational dynamics of human luteinizing hormone.	3109472	Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].
3710	A receptor-binding region in human choriogonadotropin/lutropin beta subunit.	3470775	Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. We prepared a series of peptides representing the intercysteine "loop" sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) beta subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG beta-(38-57) and hLH beta-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 X 10(-4) and 2.03 X 10(-5) M, respectively, while other peptide hormones and fragments from elsewhere in the beta subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 X 10(-5) M (hCG) and 2.18 X 10(-5) M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG beta-(38-57) peptide, native hCG and beta subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. Helical-wheel projection predicted an amphipathic region in the N-terminal portion of the 38-57 sequence, and circular dichroic measurements showed an increase in ordered structure, especially alpha-helix, when the 38-57 peptides were transferred from an aqueous to a more lipophilic (90% trifluoroethanol) environment. These results indicate that the 38-57 region of beta subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.
3711	Radioimmunoassays of human luteinizing and follicle-stimulating hormones using the same radioactive tracer.	3114867	The National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) provides the hFSH-I-3 preparation, to be employed as a tracer in the radioimmunoassay (RIA) of human follicle-stimulating hormone (hFSH). The contaminating LH contained in that preparation led us to study whether the iodination of such a material could render it a suitable tracer for RIA of both LH and FSH. hFSH-I-3 was labelled with 125I by the chloramine-T method and was further purified on Sephadex G-75 column. The LH-RIA was performed using this preparation and anti-LH at a final dilution of 1:37,500, with a sensitivity of 3 mIU LH/ml (2nd international reference pattern). The method was validated by comparing the LH values obtained in different serum samples with those obtained using the standard RIA (125I-LH/anti-LH); the correlation coefficient (r) was equal to 0.9988. No LH overestimation due to the putative cross-reaction with FSH was found. This was demonstrated by testing serum samples containing high (greater than 100 mIU/ml) and low (less than 10 mIU/ml) concentrations of FSH before and after the treatment with anti-LH. Under these conditions, serum samples from postmenopausal women, pregnant women, normal men and women in basal conditions and after the LH-RH administration, and from a patient with Klinefelter's syndrome, were evaluated. In conclusion, NIDDK 125I-hFSH-I-3 can be used as a tracer for the radioimmunological quantitation of both hLH and hFSH, which results not only inexpensive, but also allows to reduce the amount of the stored radioactive materials.
3712	Production of inhibin bioactivity by human granulosa-lutein cells: stimulation by LH and testosterone in vitro.	3559445	Granulosa-lutein cells from human preovulatory ovarian follicles were cultured for up to 12 days to determine their capacity for production of inhibin in vitro. Using a highly sensitive sheep pituitary cell bioassay we observed time-related changes in basal inhibin production, maximal during the first 4 days of culture (48 +/- 15 units/million cells every 2 days, means +/- S.E.M.; n = 5 patients) falling to values five times lower by day 12. After 4-6 days of culture in the presence of human LH (hLH) inhibin production was enhanced in proportion to the hLH dose (maximum five fold at 10 ng/ml); hFSH over the same dose-range had no effect. Progesterone production in response to hLH followed a similar pattern to that of inhibin and was also unresponsive to hFSH. In the absence of exogenous aromatase substrate, basal and gonadotrophin-stimulated oestradiol production was negligible after the first 4 days. Addition of testosterone (1 mumol/l) to the culture medium increased oestrogen formation several hundred-fold with no effect on progesterone production. Inhibin production was also increased by 50-100% in the presence of testosterone. These results demonstrate that LH and testosterone stimulate the production of inhibin by granulosa-lutein cells in vitro. It is suggested that inhibin production occurs under hormonal control in the corpus luteum as well as in the preovulatory follicle in the human ovary.
3713	Association of Turner syndrome with hypoplastic left-heart syndrome.	3812392	Three cases of hypoplastic left-heart syndrome (HLH) associated with Turner syndrome (45,X) were seen during an 11-year interval. Several isolated case reports are present in the literature. This association is probably not fortuitous but, rather, may represent the most extreme form of a spectrum of left-sided heart anomalies seen in patients with Turner syndrome. The association of Turner syndrome with HLH, as well as other known genetic etiologies of HLH, underscores the need for a detailed genetic evaluation in all patients with HLH and argues for cytogenetic analysis for even nondysmorphic females with HLH.
3714	Characterization of a bioeffective monoclonal antibody against thyrotropin beta-subunit.	2433128	To describe a region of the TSH molecule participating in binding to receptor, a monoclonal antibody specific for a TSH epitope shared by beta-subunits of bovine (b), ovine (o), and human (h) TSH was obtained by immunization with mixtures of purified bTSH and hTSH. RIAs showed that the antibody also bound the beta-subunits of bLH, oLH, hLH, and hCG, but not the beta-subunits of porcine LH and TSH. Preincubation of [125I]iodo-bTSH with the antibody completely inhibited binding of the hormone to the TSH receptor of bovine thyroid membrane preparations at pH 7.4 in 50 mM NaCl (ED50 = 10 nM). The antibody also inhibited TSH-induced mitogenesis of FRTL-5 cells (ED50 = 50 nM). We conclude that the antibody binds to a site on the bTSH molecule that participates in high affinity binding of hormone to physiological TSH receptor. The target epitope includes a conserved structural determinant in beta-subunits of the glycoprotein hormones as well as a feature that allows discrimination of porcine hormones from those of bovine, ovine, and human origin.
3715	Plasma amino acids in interrupted aortic arch and the hypoplastic left heart syndrome.	3580424	Plasma amino acids were measured in 8 babies with left-sided congenital heart disease, 3 interrupted aortic arch (IAA) and 5 hypoplastic left heart syndrome (HLH), at 1-6 days of age. There was an increase in total amino acid levels when compared with 40 control infants of similar gestational age and birth weight. Both branched-chain and aromatic amino acids were raised although there were differences between the babies with IAA and HLH. Babies with HLH had higher total amino acid levels than babies with IAA, the difference being due to raised branched-chain amino acids. Apart from the anatomical differences babies with HLH had metabolic acidosis at the time of sampling whereas babies with IAA had normal pH. It is possible that the increase in amino acid levels in babies with obstructed left-sided congenital heart disease is due to hepatic congestion and hypoperfusion, although altered acid base states may also be important.
3716	Comparison of the ability of seven gonadotropin preparations from different mammalian sources to interact with the adenylyl cyclase system in corpora lutea from rabbits and rats.	2881686	The effects of guanine nucleotides and magnesium (Mg2+) on the interaction of seven different gonadotropin preparations with their rabbit and rat luteal receptors were studied and compared to the ability of these gonadotropins to stimulate luteal adenylyl cyclase activity. In both the rabbit and rat, human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) were less efficacious than the other gonadotropin preparations in stimulating luteal adenylyl cyclase activity and thus behaved as partial agonists. Addition of 2 mM MgCl2 increased the affinity of the rat luteal receptors for all seven gonadotropins tested, while in the rabbit Mg2+ increased the affinities for porcine, bovine, ovine, rat and rabbit LH but did not significantly alter the affinities for hCG or hLH. In no instance did the addition of 100 microM GTP alter the affinity of the receptor from that observed in the absence or presence of Mg2+. A positive correlation existed for both species between the Kd values calculated from binding experiments and the Kact values obtained in adenylyl cyclase assays suggesting that the specific gonadotropin-binding sites present in rabbit and rat luteal membranes represent receptors which mediate the stimulatory effect of LH. The magnitude of the Mg2+-induced increase in affinity of a given gonadotropin preparation for its receptor was correlated with the efficacy with which that gonadotropin stimulated luteal adenylyl cyclase activity in both the rabbit and rat.
3717	Development of monoclonal antibodies directed against different epitopes of human chorionic gonadotropin.	2438177	Human chorionic gonadotropin (HCG) is a 40,000 dalton glycoprotein composed of two non-identical alpha- and beta-subunits. HCG and other related pituitary hormones, such as human luteinizing hormones (HLH), human follicle stimulating hormone (HFSH), and human thyroid stimulating hormone (HTSH), consist of nearly identical alpha-chains. However, their beta-chains show a variable degree of amino acid sequence homology. Detection and subsequent quantitative determination of HCG in human biological fluids is useful in early diagnosis of pregnancy and in monitoring of tumor patients. For these applications monoclonal antibodies (McAbs) with defined specificity are required. Several hybridomas secreting McAbs to HCG have been isolated. The hybridoma cells have been developed by fusion of NS1 myeloma cells with spleen cells of Balb/C mice immunized with HCG. With the aid of an enzyme linked immunosorbent assay, six McAbs were characterized. McAbs A-73, A-76 and A-112 recognize an epitope present on the alpha-HCG subunit. McAbs B-68, B-69 and B-106 recognize an antigenic determinant associated with the beta-HCG subunit. Two of these McAbs: B-68 and B-69 are directed against an epitope on the B subunit specific to the HCG molecule and B-106 McAb towards an epitope common to HCG, TSH, LH and FSH molecule. In a hemagglutination test, only the A-73 McAb is capable of inducing agglutination of sheep red blood cells coated with HCG, thus suggesting that this McAb recognizes a repeating epitope on the alpha-HCG molecule.
3718	Characteristics of radioimmunoassays for the alpha- and beta-subunits of human luteinizing hormone.	2435761	The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-alpha and hLH-beta) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-alpha and anti-hLH-beta antisera were calculated at 157 +/- 8.4 nM-1 and 109 +/- 7.4 nM-1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-alpha RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-alpha, was equipotent with the hLH-alpha standard in this assay. In the homologous hLH-beta RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-beta, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-alpha and hLH-beta while the hFSH RIA can be used without correction for the presence of the three other components.
3719	Differences in the carbohydrate moieties of the common alpha-subunits of human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyrotropin: preliminary structural inferences from direct methylation analysis.	3096695	The carbohydrate components of combined alpha-subunits of urinary hCG and human pituitary LH (hLH), FSH (hFSH), and TSH (hTSH), each derived from the intact hormone, were studied by direct sugar analysis and methylation analysis. The methods provide a complete survey of the structural elements contained in the complex sugars associated with these glycoproteins, but do not establish the sugar sequences or anomeric configurations of glycosidic bonds. By analogy to N-linked oligosaccharides that occur in many glycoproteins, the data suggest distinct structural features for carbohydrates of alpha-subunits combined with beta-subunits. hCG alpha contains biantennary asparagine-linked chains terminated by either NeuAc alpha 2-3Gal beta 1- or GlcNAc beta 1-2 Man alpha 1- and lacks fucose. hTSH alpha contains biantennary chains with the same termini as hCG alpha plus terminal R-O-4GalNAc and a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hLH alpha may contain some high mannose chains, but primarily contains biantennary chains terminated by NeuAc alpha 2-3(6)Gal beta 1-, GlcNAc beta 1-, GalNac-1-, R'-O-6GlcNAc-1-, and R"-0-2Man-1-plus a fucosyl residue linked alpha 1-6 to the inner GlcNAc residue of the N-linked chitobiosyl core. hFSH alpha contains more complicated structures that probably include a bisecting GlcNAc residue linked beta 1-4 to a 3,6-di-O-substituted core mannosyl residue, and terminal NeuAc alpha 2-3Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1, Gal beta 1-4(+/- Fuc alpha 1-3)GlcNAc-1-, R"'-O-GalNAc-1-, and GalNAc-1. In addition, the presence of 2,4-di-O-substituted mannose in hFSH alpha indicates that it contains triantennary chains. The identities of the R; R', R", and R"' groups were not determined, but recent studies of glycoprotein hormones suggest that they may be sulfate groups. Our results demonstrate differential glycosylation of virtually identical polypeptide hormone alpha-subunits produced in the same organ or perhaps even in the same cell.
3720	Ovarian responses in macaques to pulsatile infusion of follicle-stimulating hormone (FSH) and luteinizing hormone: increased sensitivity of the maturing follicle to FSH.	3095100	This study investigated the relationship between plasma gonadotropin concentrations and the initiation and maintenance of preovulatory follicular growth in macaques. Eight adult cynomolgus monkeys were treated with a GnRH antagonist [AcD2Nal1-4ClDPhe2, DTrp3, DArg6, DAla10]GnRH X HOAc to block endogenous gonadotropin secretion. In four animals, a pulsatile infusion of human FSH and human LH (hLH) (one 3-min pulse/h) was initiated, and the amount of hFSH delivered per pulse was increased every 3-4 days until serum estradiol concentrations rose. Thereafter, the amount of FSH delivered per pulse was reduced by 12.5%/day for 5 days, whereas the amount of LH delivered per pulse was not altered. Results indicated that plasma FSH concentrations in the range of 15-20 mIU/ml were associated with the initiation of estrogen production; in addition, a progressive reduction in plasma FSH concentration to 8-10 mI/ml over the subsequent 5 days was accompanied by continued rises in estradiol concentrations and preovulatory follicular growth. In contrast, in four control animals, maintenance of plasma FSH concentrations at 8-10 mIU/ml for 13 days did not result in elevation in serum estradiol concentrations or antral follicular growth. These observations demonstrate that after stimulation by elevated FSH concentration, follicles can continue to mature in the presence of FSH concentrations which are unable to support the growth of less mature follicles. Thus, this may account for the mechanisms by which the maturing follicle continues to develop during the mid-through late follicular phase of the menstrual cycle, whereas other less mature follicles undergo atresia.
3721	A new radioimmunoassay for human chorionic gonadotropin using monoclonal antibody.	2435539	A new radioimmunoassay (RIA) for human Chorionic Gonadotropin (hCG) was developed using murine monoclonal antibody to the beta-subunit of hCG (beta-hCG). The IgG fraction of the monoclonal antibody which did not react with 125I-beta-hCG was purified from hybridoma ascites, and covalently coupled to Sepharose 4B. This solid-phase antibody was incubated with standard hCG or serum sampled for 48 hours. The reaction medium was then removed by centrifugation and 125I-beta-hCG and anti-beta-hCG rabbit polyclonal antibody were added to the precipitate. The alcohol precipitation method was used for separating "bound" and "free" forms in the second reaction. The sensitivity for hCG in this assay system was 0.5 mIU/ml serum and the cross-reactivity with human Luteinizing Hormone (hLH) was 0.4%. This assay system was shown to be clinically applicable. Serial serum samples from two patients with trophoblastic disease were assayed and minute amounts of hCG, which could not be determined by conventional assay methods, could be assayed by this new RIA.
3722	Enzyme immunoassay for human chorionic gonadotropin using monoclonal antibodies elicited with a synthetic peptide.	3745924	We recently described the generation of mouse monoclonal antibodies directed against hCG using a synthetic peptide immunogen: the carboxy-terminal peptide (CTP) of beta-hCG. The CTP, residues 109-145, is a portion unique to hCG and is not present in other glycoprotein hormones. These monoclonal antibodies react with beta-hCG-CTP and whole hCG but do not react with hLH. In the present study, a two-site sandwich ELISA specific for hCG has been developed using these CTP-induced monoclonal antibodies. Two monoclonal antibodies were paired: beta-hCG-CTP-a6 was immobilized to give a hCG-specific solid phase, and a biotinylated monoclonal antibody against beta-hCG was used as the indicator antibody. This second antibody was introduced during the hCG-capture step because of its agonist effect on hCG-capture by beta-hCG-CTP-a6 solid phase. This ELISA system can detect 2.2 IU/l hCG in a serum-free medium and 10.4 IU/l in a medium containing 20% human serum. We did not observe any cross-reactivity with hLH up to concentrations of 5000 IU/l. The hCG-ELISA correlated well (r = 0.99) with a control RIA in an assay of 78 human sera from healthy males, pregnant or non-pregnant females and from patients with choriocarcinoma, hydatidiform mole, or teratocarcinoma.
3723	Glycoprotein hormone alpha-subunit in human stomach.	3525666	To demonstrate the immunoreactive alpha-subunit of human chorionic gonadotropin (hCG) or glycoprotein hormones in non-neoplastic gastric mucosa, and to clarify the nature and significance of alpha-subunit-immunoreactive cells, immunohistochemical studies were performed on gastric mucosa using polyclonal antibodies for hCG alpha and beta, hLH beta, hFSH beta, hTSH beta, and gastrin, and a monoclonal antibody for hCG alpha. Surgically resected stomachs were classified as follows: nearly normal (Group A); antral gastritis (Group B); fundic gastritis with pseudopyloric glands (Group C); and intestinal metaplasia (Group D). Cells immunoreactive for the alpha-subunit were present in the pyloric glands and to a lesser extent in the fundic glands (Groups A and B). Almost all alpha-subunit-immunoreactive cells were nonreactive for the beta-subunits of the four glycoprotein hormones. alpha-subunit-immunoreactive cells corresponded to gastrin-containing cells in the pyloric glands, but were unrelated to gastrin in the fundic glands. In fundic gastritis, alpha-subunit-immunoreactive cells appeared to increase (Group C), and many hyperplastic foci were observed in atrophic glands with hyperplasia of the argyrophilic cells (Groups C and D). Isolated hCG alpha or the alpha-subunit of glycoprotein hormones may be present in the endocrine cells of gastric mucosa, and alpha-subunit-immunoreactive cells in the fundic glands seem to proliferate in fundic gastritis.
3724	Luteinizing hormone receptors in human ovarian follicles and corpora lutea during the menstrual cycle.	3016623	The binding of 125I-labeled human luteinizing hormone (hLH) to the 2000-g fraction of human ovarian follicles and corpora lutea during the entire menstrual cycle was examined. Specific high affinity, low capacity receptors for hLH were demonstrated in the 2000-g fraction of both follicles and corpora lutea. Specific binding of 125I-labeled hLH to follicular tissue increased from the early follicular phase to the ovulatory phase. Specific binding of 125I-labeled hLH to luteal tissue increased from the early luteal phase to the midluteal phase and decreased towards the late luteal phase. The results of the present study indicate that the increase and decrease in receptors for hLH during the menstrual cycle might play an important role in the regulation of the ovarian cycle.
3725	Rat ovarian subcellular compartmentalization of luteinizing hormone.	3721059	To determine if human luteinizing hormone (hLH) follows the same subcellular route in the pseudo-pregnant rat ovary as human chorionic gonadotropin (hCG), the distribution of immunoreactive and bioactive hLH within cytosol, lysosomes, and a combined plasma membrane/prelysosomal vesicle fraction was examined. Immunoreactive levels were determined using a specific radioimmunoassay and bioactive levels were determined in an in vitro Leydig cell bioassay. Low cytosolic hLH levels were apparent the first few hours after hLH administration and may reflect at least some contamination by serum and interstitial fluid. Plasma membrane/prelysosomal vesicles attained the highest hLH concentration 1 h after hLH injection, after which hLH levels declined rapidly until barely detectable levels were observed at 18 h. The hormone in this fraction was receptor bound and exhibited the highest bioactivity of the three fractions examined. Lysosomal hLH concentrations were highest at 12 after hLH injection and were maintained at high levels through 18 h. Substantial amounts of lysosomal hLH appeared to be receptor bound but this hormone was not bioactive. In situ degradation of the oligosaccharides may have occurred while lysosomal hLH was receptor bound. Lysosomal degradation is the major pathway for hLH inactivation as has been described for hCG. However, hLH may be degraded within lysosomes more quickly than hCG. The differential subcellular distribution of immunoreactive and bioactive hLH provides strong support for hLH internalization and degradation within ovarian luteal cells.
3726	Induction of ovulation in the postpartum rhesus monkey: factors determining success in obtaining primate luteal tissue.	3086133	Ovulation induction in the postpartum rhesus monkey was attempted with the use of purified human pituitary gonadotropins for assessment of (1) whether this procedure could be used to provide a source of luteal tissue; (2) the extent of ovarian responsiveness to gonadotropic stimulation; (3) factors that might facilitate ovulation induction during the peurperium; and (4) factors that might be a contributory cause of induction failure. Twenty rhesus monkeys were treated with purified human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) twice daily, beginning on days 0, 10, or 20 postpartum, with or without prior administration of bromocriptine. Laparotomies were performed during the midluteal phase. Ovaries were examined, and all corpora lutea were removed. Neither the day of beginning the gonadotropin treatment nor bromocriptine administration had a significant effect on the success rate of ovulation induction, which averaged 60% overall. Inductions begun during July had a significantly (P less than 0.025) lower success rate than those started at other times of the year. Antibodies to hFSH and hLH were detected in serum from monkeys that had undergone ovulation induction. Antibodies to hFSH, but not hLH, were associated with significantly reduced induction success (P less than 0.05). Plasma estradiol rose in response to gonadotropin treatment, and the induced follicular phase averaged 13.6 +/- 0.7 days. In all animals judged to have ovulated, corpora lutea were observed at laparotomy, and plasma concentrations of progesterone were significantly elevated (13.8 +/- 3.8 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)
3727	Effect of insulin-like growth factors on human foetal, adult normal and tumour pituitary function in tissue culture.	3012926	To determine the direct effects of insulin-like growth factors (IGFs) on hormone release by the human pituitary gland, human foetal, adult normal and tumour pituitary tissues were maintained in culture for 2 to 4 weeks and tested with acute (3 h) exposures to different preparations of IGF peptides. Adult normal pituitaries and adenomas were tested with a semipurified preparation of IGFs, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. Human foetal pituitaries were tested with the semipurified IGFs as well as more purified preparations of IGF-I and IGF-II. Culture media were assayed for hGH, hPrl, hACTH and hLH using specific radioimmunoassays. Both foetal (n = 16 (No. of pituitaries), 33 (No. of observations] and normal adult (n = 3, 16) human pituitaries cultures responded to the semipurified IGFs (2-25 ngEq/ml for foetal and 2-4 ngEq/ml for adult pituitaries) with a significant decrease in hGH release compared to basal (P less than 0.01) whereas the GH-secreting pituitary tumours showed no effect when tested with from 2 to 25 ngEq/ml (n = 8, 129, NS). The effect of IGFs on human foetal somatotrope activity was dose-related for both the semipurified IGFs (2-25 ngEq/ml, n = 16, 33) and IGF-I or IGF-II (10-100 ng/ml; n = 3, 18).(ABSTRACT TRUNCATED AT 250 WORDS)
3728	Characterization of corpora lutea in monkeys after superovulation with human menopausal gonadotropin or follicle-stimulating hormone.	3007556	The objective of this study was to characterize the corpora lutea (CL) of superovulatory follicles, which form in nonhuman primates after treatment with exogenous gonadotropins. Adult female rhesus monkeys (n = 15) with amenorrhea or irregular menstrual cycles received im injections of either human menopausal gonadotropin [hMG; equivalent amounts (37.5 IU) of hFSH and hLH] or human FSH (37.5 IU) twice daily for 6 or 9 days. One day later, hCG (1000 IU) was administered to induce ovulation. Serum estradiol levels rose rapidly in hMG-treated monkeys. In contrast, estradiol levels did not rise in FSH-treated animals for 3-4 days, but ultimately reached concentrations comparable to or greater than those in hMG-treated monkeys. Serum progesterone levels were low in all groups before hCG injection, but rose thereafter. Peak progesterone levels were greater (P less than 0.05) in 9- vs. 6-day treatment groups. Serum concentrations of hCG peaked within 24 h of injection and declined to undetectable levels 6-7 days later. The mass of luteinized tissue removed 7 days after hCG injection was markedly (P less than 0.01) increased in hMG- and FSH-treated monkeys compared to that of the active CL of the natural menstrual cycle (n = 6). However, the protein content of luteal tissue from FSH-treated monkeys was less (P less than 0.05) than that in hMG-treated groups or in the CL of the natural cycle. Luteal particulate fractions from all treatment groups had [125I]human LH binding sites, with the Kd for LH interaction comparable to that in the CL of the natural cycle. However, the LH-binding capacity in hMG-treated groups was less (P less than 0.05) than that in the CL of the cycle, when normalized per mg tissue wt or protein. Notably, the binding capacity in FSH-treated groups was comparable to that in the CL cycle when expressed per mg protein. Nevertheless, only after 6 days (not 9 days) of FSH treatment or 9 days (not 6 days) of hMG treatment did tissues have a LH-sensitive (activation constant) or LH-responsive adenylate cyclase comparable to that in the CL of the cycle. Thus, properties of the primate CL after superovulation varied markedly with the type and length of gonadotropin treatment employed for follicular stimulation. The findings support the concept that gonadotropin-regulated events in the developing follicle(s) are important determinants of the subsequent character of the primate CL.
3729	Comparison of the analytical characteristics of ten urinary hCG tests for early pregnancy diagnosis.	3532914	A comparison of assay design, sensitivity and specificity is given for 10 urinary human chorionic gonadotrophin (hCG) test kits for early pregnancy diagnosis. One slide test applies a polyclonal anti-hCG serum, while nine, more recently developed, tube and slide tests use one or more monoclonal antibodies toward intact hCG or its free subunits. Colour development for five immunoenzymetric (IEMA) 'sandwich' assays, or (haem/latex) agglutination (HA, LA)-inhibition (HAI, LAI) for the other systems provide assay sensitivities from 1000-50 IU/L within 3-120 min. The 'best' slide-(i.e., LA, LAI), haem-(i.e., HA, HAI) and IEMA-tests detect respectively 500, 75 and 50 IU/L within 2.5, 120 and 3 min. Assay specificities (i.e., as false-positive and false-negative test results) are studied using intact hCG, hLH and their free alpha- and beta-subunits. The presented assays are improved, perform more sensitively at shorter reaction times with increased specificity, and may, therefore, establish pregnancy at an earlier stage with greater reliability.
3730	Lack of immunological analogy between the beta-subunits of cholera toxin and human choriogonadotropin.	2422725	A chemical relatedness has been described between the beta-subunit of cholera toxin and that of the four dimeric glycoprotein hormones (hCG, hLH, hFSH and hTSH). However, antibodies induced by cholera toxin did not crossreact, when tested by labeled hCG binding and immunocytochemistry, with the beta-subunit of hCG. It appears that differences in the tertiary structures, as shown in this study, account for distinct epitopes. Similarities in biological activity between these two compounds, such as induction of adenyl cyclase or a protective effect against some tumors, are not based on immunological mechanisms.
3731	Cell-free sulfation of human and bovine pituitary hormones. Comparison of the sulfated oligosaccharides of lutropin, follitropin, and thyrotropin.	3934161	Lutropin (LH), follitropin (FSH), and thyrotropin (TSH) from pituitary and human chorionic gonadotropin (hCG) from placenta are a family of glycoprotein hormones, each with an alpha and beta subunit. The alpha subunits of all four hormones have the same amino acid sequence, whereas biological specificity is determined by their unique beta subunits. The carbohydrate compositions of these hormones indicate the structures of their Asn-linked oligosaccharides are not identical. Sulfate is present on most, but not all, of these hormones, and for bovine LH is attached to GalNAc (Green, E.D., van Halbeek, H., Boime, I., and Baenziger, J.U. (1985) J. Biol. Chem. 260, 15623-15630). We used a reconstituted cell-free system to study sulfation of bovine (b) and human (h) glycoprotein hormones and its relationship to glycosylation. Exogenously added bLH, bTSH, bFSH, hLH, and hTSH are sulfated exclusively on the oligosaccharides of both alpha and beta subunits. The distribution of sulfated oligosaccharide structures varies among the hormones and appears to result from differences in the extent and/or pathway of oligosaccharide processing. Significant amounts of disulfated, dibranched complex oligosaccharides are present on all the sulfated hormones. Human FSH is not susceptible to sulfation unless first treated with neuraminidase. The sulfated oligosaccharides obtained from bovine FSH and desialylated human FSH are unlike those of the other hormones. Therefore, there is differential processing of the oligosaccharides on pituitary hormones. For FSH and LH, which are believed to be synthesized in the same cell, we would suggest that the unique beta subunits may regulate processing of all oligosaccharides present on the alpha-beta dimers.
3732	Monoclonal antibodies against human follicle-stimulating hormone.	2415347	Five monoclonal antibodies to human (h) FSH have been prepared, isolated, and characterized. They were produced by hybridomas derived from FO myeloma cells and spleen cells from mice immunized with hFSH or its beta-subunit (hFSH beta). Two of the antibodies (A101 and A102) which recognized the alpha-subunit of hFSH bound much better when alpha was associated with the beta-subunit forming the intact hFSH molecule than when alpha was in free form. These antibodies showed 9-10%, 2-3%, and 1-3% cross-reactions with alpha-subunits in hTSH, hCG, and hLH, respectively. Two antibodies (B201 and B202) recognized only free beta-subunit. One antibody (B305) recognized free beta-subunit and native hFSH. There was no significant cross-reaction of these antibodies to FSH beta with hTSH, hLH, and hCG. Using solid phase competitive binding and sandwich assays, we compared the epitopes for these antibodies. Antibodies A101 and A102 recognize the same epitope on hFSH alpha. Antibodies B201 and B202 recognize different epitopes, but they seemed to be adjacent. Antibody B305 bound a different epitope than B201 and B202. Such characteristics of these antibodies can be useful for sensitive and specific assay of hFSH or hFSH beta and also may be helpful in studying FSH interaction with its receptor.
3733	Qualitative and quantitative differences in hLH species in the first and second LH release induced by continuous stimulation with synthetic LHRH in normal menstrual cycle as assessed by isoelectrofocusing.	3912159	It is known that continuous stimulation with LHRH induces a biphasic release of LH. The two pools theory is generally accepted in interpreting this phenomenon. The present study was conducted in order to elicit qualitative differences in LH included in the two pools. Five hundred micrograms synthetic LHRH was infused for 2h in 10 women in various phases of the menstrual cycle and 5 h blood sampling was performed during and after the infusion. Biphasic release of LH was demonstrated in all the cases investigated. The responsiveness to LHRH was most predominant in the midcycle, followed by the luteal phase. Pools of plasma samples at 30-105 min and 150-225 min of the LHRH infusion (plasma pools I and II; corresponding to the initial and the second LH release, respectively) were subjected to isoelectrofocusing (IEF) fractionation, pH range of 3.5-10. LH in plasma was classified in terms of isoelectric point (pI); i.e. A(pI = 9.13), B(8.60), C(8.16), D(7.67), E(7.24), and F(LH species migrating in the acidic pH area). Percentage of high alkaline LH species was significantly elevated in plasma pool I of the midcycle. No phasic difference was observed in the IEF profiles in plasma pool II. Consistent and significant increase and decrease in the percentage of acidic and high alkaline LH species, respectively, were observed throughout the menstrual cycle when the IEF profiles of plasma pool II were compared with those of pool I. The ratio of biological to immunological LH activities (B/I ratio) was markedly depressed in acidic LH species. The highest B/I ratios were obtained in the LH species migrating in the mid-alkaline region. The acidic LH species might represent the young generation of LH molecules prior to acquisition of biological potency. LH species migrating in the mid-alkaline region might be mature and bio-potent forms of the hormone. High alkaline LH species might represent LH molecules over-processed and having lost a degree of biological potency during a prolonged period of storage.
3734	A radioimmunoassay system for LH based on the antigenic similitude of LH with HCG: methodology and applications.	3841409	A double antibody radioimmunoassay (RIA) system for LH (LH-IEP Kit) was developed using the antigenic similitude of LH with HCG. The first antibody (Ist Ab) is rabbit anti-HCG serum, initial dilution 1:200 000. The tracer is 125I-HCG (code MJ-14 Swierk Poland). The standard curve is calibrated with the reference preparation hLH-Ist-IRP 68/40 kindly offered by WHO. The IInd Ab is pig anti-rabbit IgG serum. The incubation conditions: volume-0.3 ml; time-24 hrs with Ist Ab and 24 hrs with the IInd Ab at ambient temperature. The sensitivity of the RIA system for LH is 1.5 mIU/ml. To validate our RIA system the LH was measured in the serum samples collected from 9 women during the menstrual cycles, from 2 boys during the GnRH test, from 2 amenorrheic women and from 20 children, adolescents and adults with miscellaneous pathologies. In all these samples, parallel measurements of LH and FSH were performed using DDR commercial RIA Kits-SSW. It is to be mentioned that the LH-RIA Kit-SSW is not completely homologous, the Ist Ab being rabbit serum anti-HCG. The results obtained during 4 menstrual cycles, in which the LH peak is observed around the mid point of the interval are: follicular phase 17.92 +/- 5.58 mIU/ml (means +/- SD) with LH-IEP-Kit and 5.51 +/- 2.77 mIU/ml with the LH-SSW-Kit, peak: 26.07 +/- 22.13 mIU/ml and 9.13 +/- 5.60 mIU/ml respectively; luteal phase: 12.55 +/- 5.46 mIU/ml and 3.4 +/- 2.38 mIU/ml, respectively. The LH values observed by the two kits through all 9 menstrual cycles are well correlated ("r" values in the range 0.7-0.9) but high discrepancies were observed in the remaining 3 cycles ("r" between 0.07 and 0.6). These discrepancies as well as those observed in some adolescents with genetic anomalies and in a patient at climacterium are suggesting that the two LH-RIA systems measure not only a common molecular area but also different areas of the LH circulating molecules.
3735	Adenylate cyclase in the corpus luteum of the rhesus monkey. III. Changes in basal and gonadotropin-sensitive activities during the luteal phase of the menstrual cycle.	2992915	The activity of adenylate cyclase was examined in corpora lutea (CL) obtained from rhesus monkeys at specific stages in the luteal phase of the menstrual cycle [3-5, 6-8, 9-12, 13-15, and 16 days (menses) after the midcycle LH surge]. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. cAMP production in luteal homogenates was assessed in the absence (basal activity) and presence of maximum stimulatory doses of forskolin (100 microM), 5'-guanylylimidodiphosphate [GMP-P(NH)P; 50 microM], GTP (50 microM), and GTP plus increasing doses of hLH and hCG. Basal activity was low in the early luteal phase (days 3-5; mean +/- SE, 1.2 +/- 0.2 pmol cAMP/mg protein X min), increased (P less than 0.05) by the midluteal phase (days 6-8 and 9-12, 2.1 +/- 0.4 and 2.0 +/- 0.3 pmol/mg X min, respectively), and then declined (P less than 0.05) during the late luteal phase (days 13-15 and 16-menses, 1.6 +/- 0.3 and 1.2 +/- 0.5 pmol/mg X min, respectively). Activity stimulated by GTP and GMP-P(NH)P [e.g. GMP-P(NH)P approximately 12 times basal level] followed the same pattern as basal activity during the luteal phase. In contrast, cAMP production in the presence of forskolin did not change significantly throughout the luteal phase. In the midluteal phase (days 6-8 and 9-12; n = 12), hCG and human LH (hLH) stimulated adenylate cyclase in a similar dose-dependent manner. Maximal stimulation of cAMP production by hCG was about 10% greater (P less than 0.05) than that by hLH; the activation constant was 12.3 nM for hCG and 28.3 nM for hLH. The maximal response to hLH and hCG as well as the sensitivity of adenylate cyclase to activation by hLH were greater (P less than 0.05) in the midluteal phase than in the early or late luteal phase. Decreased basal, gonadotropin-stimulated, and guanine nucleotide-stimulated cAMP production and diminished sensitivity of adenylate cyclase to hLH correlated with a decline (P less than 0.05) in circulating progesterone and luteal weight during the late luteal phase. Thus, the adenylate cyclase system of the rhesus monkey CL undergoes significant changes during the luteal phase which are associated with the development and regression of the CL of the menstrual cycle. Mechanisms that modulate gonadotropin and nucleotide activation of adenylate cyclase without interfering directly with the catalytic unit are implicated in the changes that accompany luteolysis.
3736	The changing ratio of bioactive to immunoreactive luteinizing hormone (LH) through puberty principally reflects changing LH radioimmunoassay dose-response characteristics.	3894406	The ratio (B/I) of bioactive to immunoreactive LH in plasma varies during pubertal maturation. To elucidate the basis for these changes, we compared the dose-response characteristics of LH standards to those of plasma LH before and after GnRH infusion in normal males at various pubertal stages and girls with Turner's syndrome. We used a human LH (hLH) RIA modified to optimize specificity for LH bioactivity by employing a hLH tracer maximally bioactive in the rat interstitial cell testosterone production (RICT) bioassay. The pituitary hLH standards LER-907, NHPP I-1, and NHPP I-2 were not parallel to one another in the RIA, but were parallel in the RICT. Their relative slopes in the RIA were 1:1.35:1.49, respectively. The B/I for immunoreactive LH in these standards were 1 (LER-907):3 (I-1):5 (I-2). Dose-response characteristics varied greatly by patient category in the RIA. In contrast to the RICT, in which plasma from all subject groups, except the prepubertal basal group, gave parallel dose-response slopes, the groups differed in the steepness of their plasma LH RIA dose-response curves in the following order: adult post-GnRH congruent to adult basal congruent to pubertal post-GnRH greater than pubertal basal congruent to prepubertal post-GnRH congruent to prepubertal basal greater than Turner's syndrome. Prepubertal basal samples most closely resembled LER-907 in dose-response characteristics, while adult samples were most similar to NHPP I-1 and I-2. These characteristics were not related to the absolute LH concentration. Although the RIA dose-response characteristics of plasma LH changed during puberty, the B/I of basal LH did not increase through puberty using the modified hLH RIA, although B/I still rose in GnRH-stimulated samples during puberty. Our data demonstrate that the principal cause of variability in B/I is the changing dose-response characteristics of plasma LH in the RIA. We suggest that the source of this variability is a change in the molecular characteristics of LH in different states of pubertal maturation and gonadal function. The results imply that better ways of assaying LH are needed.
3737	Absence of chorionic gonadotropin in sera of women who use intrauterine devices.	4018277	A controlled study was undertaken to determine whether unnoticed pregnancies routinely occur in users of the intrauterine contraceptive device (IUD). Starting on day 10 of the menstrual cycle and continuing through the onset of menstruation or until the diagnosis of pregnancy, we collected daily blood samples from three groups of normally menstruating young women. The study groups were (A) IUD users (n = 30), (B) women with tubal ligation (n = 30), and (C) women trying to become pregnant (n = 15). The sequential serum samples were analyzed by radioimmunoassay for progesterone (P), human luteinizing hormone (hLH), and human chorionic gonadotropin (hCG). No positive hCG assays in luteal phase blood sera of IUD users were observed. The only positive hCG determinations of IUD users coincided with the preovulatory surge of hLH. Two subjects who became pregnant, as judged by progressive increases in hCG and P levels in the luteal phase, belonged to the group planning pregnancy. The finding of two pregnancies in 15 months of exposure is consistent with the assumption of natural fertility. The probability of no pregnancies in 30 months at risk, as observed among the IUD users, is between 1 in 200 and 1 in 100,000, depending on the assumption made for natural fertility. The study demonstrates that IUD users do not retain their natural fertility, and that IUDs do not exert their antifertility effect as abortifacient agents. If a confirmed pregnancy is detected in an IUD user, it may be assumed to represent an isolated case of contraceptive failure.A controlled study was undertaken to determine whether unnoticed pregnancies routinely occur in users of the IUD. Beginning on day 10 of the menstrual cycle and continuing through the onset of menstruation or until the diagnosis of pregnancy, the authors collected daily blood samples from 3 groups of normally menstruating young women. The study groups were: a) IUD users (n=30); b) women with tubal ligation (n=30); and c) women trying to become pregnant (n=15). The sequential serum samples were analyzed by radioimmunoassay for progesterone (P), human luteinizing hormone (hLH), and human chorionic gonadotropin (hCG). No positive hCG assays in luteal phase blood sera of IUD users were observed. The only positive hCG determinations of IUD users coincided with the preovulatory surge of hLH. 2 subjects who become pregnant as judged by progressive increases in hCG and P levels in the luteal phase belonged to the group planning pregnancy. The finding of 2 pregnancies in 15 months of exposure is consistent with the assumption of natural fertility. The probability of no pregnancies in 30 months at risk, as observed among the IUD users, is between 1 in 200 and 1 in 100,000, depending on the assumption made for natural fertility. The study demonstrates that IUD users do not retain their natural fertility, and that IUDs do not exert their antifertility effect as abortifacient agents. If a confirmed pregnancy is detected in an IUD user, it may be assumed to represent an isolated case of contraceptive failure.
3738	Studies of the heterogeneity of human pituitary LH by fast protein liquid chromatography.	3998654	The Pharmacia fast protein liquid chromatography system was employed to fractionate a purified preparation of human LH (hLH) on the anion exchanger Mono Q at pH 7 X 8 into 14 sub-fractions. Each of the sub-fractions was characterized by its behaviour on polyacrylamide gel electrophoresis, sodium dodecyl sulphate (SDS) gel electrophoresis, LH receptor binding activity and sialic acid content. All sub-fractions contained sialic acid, were active in binding to LH receptors, and exhibited components typical of hLH subunits on SDS gel electrophoresis. None of the subfractions was homogeneous with respect to charge. There is evidence that part of the heterogeneity results from the presence in some molecules of an internal proteolytic cleavage within the beta-subunit, and fractions enriched in species containing such cleavages were prepared by this method.
3739	Effects of FSH and LH on adenylate cyclase activity in rat granulosa cell membranes during follicular maturation.	3925676	Adenylate cyclase activity was measured in membranes prepared of granulosa cells isolated from PMSG-treated immature rats and the effects of ovine, highly purified human and rat gonadotrophins were compared. Furthermore, a comparison of the effects of the human preparations (hLH, hFSH) on the adenylate cyclase activity in membranes prepared from granulosa cells isolated at different stages of follicular maturation, was performed. The adenylate cyclase in membranes of immature granulosa cells was stimulable with FSH but not with LH, while in pre-ovulatory granulosa cell membranes, both gonadotrophins were stimulatory with FSH generally being more effective than LH. Surprisingly, the dose-response curve for ovine LH (oLH) was biphasic with a plateau at a level of adenylate cyclase activity corresponding to the maximal stimulatory effect of hCG. With increasing oLH concentrations the response resumed and the maximal stimulation corresponded to that of oFSH. With highly purified rat gonadotrophins the FSH response was significantly higher than the response to LH at all concentrations tested. Using highly purified human gonadotrophins the maximal FSH response was 50% higher than the maximal LH response and by adding increasing concentrations of hFSH to a maximally stimulatory concentration of hLH it was possible to mimic the biphasic dose-response curve for oLH. When the membranes were prepared from granulosa cells isolated after the pro-oestrus LH/FSH surge there was clear increase in the sensitivity of the adenylate cyclase to stimulation with LH although the maximal response was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)
3740	Stimulation of sperm production by human chorionic gonadotropin after prolonged gonadotropin suppression in normal men.	3922931	The precise hormonal milieu required for quantitatively normal spermatogenesis in man is unclear. The authors previously have shown that both supraphysiologic dosages of human chorionic gonadotropin (hCG) and physiologic dosages of human luteinizing hormone (hLH) can reinitiate sperm production in short-term (four months) gonadotropin-suppressed normal men who have prepubertal FSH levels. To determine whether normal FSH levels were necessary to stimulate sperm production after a prolonged period of gonadotropin and testicular suppression, the authors administered hCG to four normal men whose endogenous gonadotropin levels and sperm production were suppressed by prolonged exogenous testosterone (T) administration. After a 3-month control period, all subjects received 200 mg of T enanthate intramuscularly (im) each week to suppress LH and FSH for a total of 9 months and until successive sperm concentrations (performed twice monthly) revealed azoospermia or severe oligozoospermia (mean sperm concentration less than 3 X 10(6) spermatozoa/ml) for 6 months. Then, while continuing the same dosage of T enanthate, all four men simultaneously received 5000 IU of hCG im three times weekly for 6 months, replacing LH-like activity and leaving FSH activity suppressed. The effect on sperm production of the selective FSH deficiency produced by hCG plus T administration after the period of prolonged gonadotropin suppression was determined. Exogenous T administration resulted in severe suppression of sperm concentrations from 79 +/- 7 X 10(6) spermatozoa/ml (mean +/- SEM) during the control period to 0.8 +/- 0.5 X 10(6)/ml after 12 weeks of T treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
3741	[Biological and immunological characterization of human luteinizing hormone in plasma discharged by LH-RH infusion in normal adult women].	3889181	Five hundred micrograms synthetic LH-RH was infused for 2 hours in women having a normal menstrual cycle. Measurement of the plasma hLH level revealed two phases of hLH discharge; i.e. an acute and a delayed discharge (plasma pools I and II, respectively). These plasma pools were subjected to a column isoelectrofocusing (IEF) fractionation. The IEF fractionation separated plasma LH in both plasma pools into 6 distinct species; A (pI: 9.03 +/- 0.11), B (8.60 +/- 0.10), C (8.12 +/- 0.13), D (7.67 +/- 0.09), E (7.24 +/- 0.10), and F (acidic LH). The ratio of biological to immunological LH activities in each species was determined after concentration by ultrafiltration and found to be 1.12, 2.09, 2.21, 1.87, 1.29, and 0.49, respectively. The acidic LH was more enriched in plasma pool II throughout the menstrual cycle than in its counterpart. The majority of LH activity was found in B, C and D. The relative amount of LH migrating in the high alkaline region (A) was greater in plasma pool I of the late follicular phase where the secretion of the hormone was depressed. In conclusion, LH subpopulations separated by IEF may represent the stage of molecular maturation of the hormone. The acidic LH is probably the LH newly synthesized and not well processed. The LH species possessing alkaline pIs (B, C and D) are mature types of the hormone. The LH migrating in the high alkaline region (A) may be the LH of over-maturation.
3742	Comparison of the analytical characteristics and clinical usefulness in tumour monitoring of fifteen hCG(-beta) immunoassay kits.	2411185	Fifteen immunoassay kits for serum hCG(-beta) measurements have been compared for their analytical characteristics and clinical usefulness in tumour monitoring. Three out of 13 radioimmunoassays and two sandwich hCG immunoassays represent the 'one-component' techniques for intact hCG detection since they are based on antiserum raised against intact hCG. Within this group, method-comparison slopes ranged between 0 X 149 and 0 X 374. The remaining 10 radioimmunoassays all apply antiserum against hCG-beta and are designated 'two (or more)-component' assays since they detect both intact hCG and the free beta-subunit ('total hCG'). For this reason these assays are of potential value for tumour monitoring. Additional criteria for this are: (1) the expression of assay results in terms of 1st IRP-hCG instead of the unstructured use of the 2nd IS or the 1st IRP-hCG-beta; (2) documentation of cross-reactivities especially for the free beta-subunits of hCG and hLH which are almost completely lacking; (3) establishment of the minimum detectable dose in the presence of normo-to-hypergonadotrophic hLH levels in serum (the 'clinical sensitivity') to allow the follow-up of tumour regression especially in the low-dose region. Method-comparison analysis for these assays revealed regression slopes between 0 X 001 and 0 X 873.
3743	Applications of monoclonal antibodies in solid-phase immunoassays of human luteinizing hormone.	4055552	Monoclonal antibodies to human luteinizing hormone (hLH) were generated by a modified hybridoma technique. Out of forty hybrid cell lines that were shown to secrete antibodies reacting with hLH, LH35 and LH40 were further characterized biochemically and immunologically. LH35 was found to secrete immunoglobulin G1 antibody specific to the alpha-subunit of LH, whereas that of LH40 was beta-subunit specific. Association constants between the antibodies and LH were determined to be 2 X 10(8) and 1 X 10(9) M-1, respectively, by using competitive radioimmunoassay and Scatchard plots. Monoclonal antibodies from LH35 and LH40 were purified from the respective ascites fluids by ammonium sulfate fractionations and DEAE ion-exchange chromatography. The purified alpha-subunit-specific antibody of LH35 was immobilized on polystyrene balls (6 mm in diameter), whereas purified LH40 antibody was conjugated with horseradish peroxidase or labeled with iodine-125. Solid-phase radio- and enzyme immunoassays were designed to measure relatively low concentrations of LH (2-100 mIU/ml). The LH surge during the midcycles of women with normal menstrual cycles could easily be detected from daily urine or serum specimens by a 1-h assay procedure. It is proposed that this new LH immunoassay procedure can be routinely used for predicting ovulation of women with normal menstrual cycles.
3744	Comparison between the effects of FSH and LH on the steroidogenic pattern in isolated pre-ovulatory rat follicles.	3922190	The effects of purified FSH preparations from three different species (ovine, human, rat) on the steroidogenic pattern in isolated pre-ovulatory follicles of PMSG-treated immature rats were examined and compared to the effects of LH (ovine, human). Steroids were analyzed by RIA. During 6 h incubation, all three FSH preparations in a concentration range from 10-100 ng/ml caused a significant increase in progesterone (P), testosterone (T), and oestradiol (E2) accumulation. With LH a significant increase in steroid accumulation was seen in a concentration of 1 ng/ml. The time-course of stimulation by hFSH and hLH in a concentration of 10 ng/ml showed no difference in steroidogenic response between the two hormones, with a significant stimulation of steroids within 2 h. During prolonged incubation (6-8 h), accumulation of T was measured in the presence or absence of unlabelled 17 alpha-hydroxyprogesterone. Follicles exposed to LH did not increase their T formation, while follicles incubated in plain medium or in the presence of oFSH increased their T formation. In a concentration of 100 ng/ml hFSH showed a tendency to reduce maximal T production. These results suggest that FSH alone in physiological doses may stimulate T and E2 production in the preovulatory follicle.
3745	Adenylate cyclase in the corpus luteum of the rhesus monkey. II. Sensitivity to nucleotides, gonadotropins, catecholamines, and nonhormonal activators.	2982589	The sensitivity of the adenylate cyclase of the primate corpus luteum to various nucleotides, gonadotropins, catecholamines, and nonhormonal activators was assessed in homogenates of luteal tissue obtained from rhesus monkeys at the midluteal phase of the menstrual cycle. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. GTP, the GTP analog 5'-guanylyl-imidodiphosphate, and ITP stimulated adenylate cyclase activity in the presence or absence of exogenous hormone; however CTP, UTP, GMP, and guanosine did not. The gonadotropins, human (h) LH and hCG, stimulated cAMP production in a dose-dependent manner. Maximal stimulation of adenylate cyclase was achieved at 100 nM hLH and hCG, and the activation constant was 20 nM for both hormones. The addition of GTP increased maximal activation of adenylate cyclase by hLH or hCG, but did not alter sensitivity to the hormones. Neither hFSH nor the isolated subunits of hCG stimulated cAMP production. Deglycosylated hCG (native hCG with 70% of the carbohydrate moieties removed) did not stimulate adenylate cyclase activity. However, hLH and intact hCG failed to enhance cAMP production in the presence of an equimolar amount of deglycosylated hCG. The adenylate cyclase of macaque luteal tissue did not respond to the addition of isoproterenol, epinephrine, or phenylephrine. Furthermore, these catecholamines did not affect hCG stimulation of adenylate cyclase. The nonhormonal activators of adenylate cyclase, forskolin and fluoride, stimulated cAMP production in a dose-dependent manner, with maximal stimulation at 100 microM and 10 mM, respectively. Thus, the macaque corpus luteum at the midluteal phase of the menstrual cycle contains a guanine nucleotide-regulated adenylate cyclase which is equally sensitive to the pituitary and placental gonadotropins, hLH and hCG. However, removal of carbohydrate moieties from hCG endows the molecule with gonadotropin-antagonistic properties in the primate. The adenylate cyclase system of the macaque corpus luteum was not responsive to catecholamines; thus, the primate may lack a potential mechanism for control of luteal function that is available to many nonprimate species.
3746	Effects of oestradiol and prolactin on progesterone production by rhesus monkey luteal cells in vitro.	3918401	The effects of oestradiol and prolactin (Prl) on progesterone production by dispersed monkey luteal cells were examined. Corpora lutea were recovered from monkeys 5-7 days following ovulation induction during the puerperium. The tissue was dispersed by collagenase and mechanical disruption. The resulting cells were incubated in Dulbecco's modified Eagle's medium, containing the hormones to be tested, for 3 h at 37 degrees C. The medium was removed and assayed for progesterone by RIA. Human luteinizing hormone (hLH) produced a significant, dose-related increase in progesterone secretion that was comparable to that produced by dibutyryl cyclic adenosine monophosphate. Human follicle stimulating hormone (hFSH) had no effect upon progesterone production by the luteal cells. Oestradiol (100-10 000 pg/ml) produced a significant, dose-related decrease in both basal and hLH-stimulated progesterone production. Ovine Prl (oPrl) had neither a stimulatory nor an inhibitory effect upon basal progesterone secretion at doses up to 1000 ng/ml. Further, oPrl did not affect hLH-stimulated progesterone production. We conclude that oestradiol is a potent inhibitor of luteal progesterone secretion in vitro and that Prl does not inhibit progesterone production in the primate corpus luteum under these experimental conditions.
3747	Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide.	2578164	Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.
3748	Chemical synthesis of peptide fragments of the hormone-specific beta-subunit of human follicle-stimulating hormone.	2581605	In order to determine the specific antigenic determinants of human follicle-stimulating hormone (hFSH), hFSH-beta peptides with amino acid residues 33-49 (V2), 95-118 (V3), 76-118 (V3 + 1/2 C2), 1-33 (V1 + C1), 22-33 (1/2C1), and 95-107 (V3 + 1/4C2) according to the nomenclature of Stewart and Stewart [Stewart, M., & Stewart, F. (1977) J. Mol. Biol. 116, 175] as well as additional peptides with the residues 93-107, 91-107, 89-107, 87-107, and 85-107 were chemically synthesized. The peptides were examined in radioimmunoassay systems of FSH, luteinizing hormone (LH), or human chorionic gonadotropin (hCG). V3 + 1/2C2 and V1 + C1 showed immunological activity, whereas the other peptides did not. Antibodies were raised in rabbits against these peptides and examined for specific binding with hFSH, LH, thyroid-stimulating hormone (TSH), and hCG. V3 + 1/2C2 as well as V1 + C1 produced antisera, which specifically bound hFSH, hLH, and hTSH, indicating that the amino acid sequences contained in hFSH-beta peptides V3 + 1/2C2 and V1 + C1 share common antigenic sites with hLH and hTSH. Antisera were produced in rabbits against hFSH-beta, against reduced and S-aminoethylated hFSH-beta (AE-FSH-beta), and against AE-FSH-beta coupled to hemocyanin. Reduced and S-aminoethylated beta-subunit of FSH-beta coupled with hemocyanin produced antisera in rabbits that specifically bound only hFSH and not hLH, hTSH, or hCG.
3749	[Ultrastructural localization of pituitary hormones by immunocytochemical reactions in ultrathin sections of adenomas].	3002569	Human pituitary adenomas were fixed in glutaraldehyde and embedded in epon. Ultrathin sections were incubated either with anti-hGH, anti-hPRL or anti-hLH. They were incubated with second step goat anti-rabbit immunoglobulins linked to gold particles. Two PRL secreting adenomas, one GH and PRL secreting adenoma, one ACTH secreting adenoma and two non secreting adenomas were studied. The specificity and the limits of the method were discussed in relation with the results obtained in light microscopy with the PAP method.
3750	Prolactin-mediated progesterone secretion by cultured rat granulosa cells: regulation by purified human glycoprotein hormones and their subunits.	2578034	The effects of purified alpha- and beta-subunits of human glycoprotein hormones on initial luteinization and subsequent prolactin-mediated progesterone responses of cultured rat granulosa cells were studied. Granulosa cells, obtained from immature female rats 50 h after PMSG treatment, were incubated for 24 h in control medium lacking added hormones or in medium containing hCG or the alpha- or beta-subunit of human (h) FSH, LH, CG, or TSH at 0.1, 0.5, and 1.0 microgram/ml. Cultures were maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine PRL (bPRL), with medium changes every 48 h. Indices of luteotropic stimulation in response to bPRL were provided by 1) elevated progesterone concentrations determined by RIA of spent media samples, and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation of monolayers after 7 days of culture. Progesterone concentrations in media from cultures incubated in 0.5 or 1.0 microgram/ml hCG were 6-fold higher than in cultures incubated in control medium, while those in media from cultures incubated in 0.5 or 1.0 microgram/ml hFSH alpha, hLH alpha, hCG alpha, hTSH alpha, hLH beta, or hCG beta (but not in hFSH beta or hTSH beta) were from 2- to 4-fold higher than those in control cultures. This enhancement was not evident when subunits were added to the incubation media at the lowest concentration. Progesterone secretion corresponded directly with the degree of cytoplasmic osmiophilia. These results suggest that the alpha-subunit of each of the glycoprotein hormones as well as the beta-subunit of hLH and hCG have the ability to promote progesterone secretion during initial luteinization and to regulate subsequent PRL-mediated steroidogenesis by rat granulosa cells in vitro. Furthermore, these effects are greater than can be accounted for by potential contamination of subunit preparations with undissociated hormones, as demonstrated by dose-response curves.
3751	High correlation between prolactinemia, 125-I hLH binding and progesterone secretion by an experimental luteoma.	6094939	Autoimplantation of an ovary, containing fresh corpora lutea, into the spleen of an ovariectomized rat is followed by strong luteinization and size increase of the grafted gonad. Thus, large amounts of luteal tissue for biochemical studies, and their histological controls are available. Furthermore, progesterone secretion can be easily determined in samples collected from the portal vein. Since prolactin has been implicated in the control of luteal tissue, the role of this hormone on hLH binding and progesterone secretion was determined. Different levels of endogenous serum prolactin were achieved by pharmacological treatments with neurotropic agents. Scatchard plots of 125-I hLH binding data derived from luteoma particulate fractions revealed the presence of one type of binding site with high affinity. At the same time as binding increased, prolactinemia augmented, with a high correlation (R:0.99) between prolactinemia and LH binding. Moreover, progesterone secreted by the luteoma increased as LH binding sites augmented (R:0,97). It is concluded that a high correlation between prolactinemia and LH binding, as well as between this last parameter and progesterone output exists in the experimental luteoma.
3752	In vitro studies of prolactin inhibition of luteinizing hormone action on Leydig cells of rats and mice.	6514721	Previous in vivo studies have shown that in male rabbits prolactin inhibits the testosterone production stimulated by luteinizing hormone (LH) or human chorionic gonadotropin (hCG). This inhibition has now been studied in vitro using both mouse and rat testicular interstitial cells. First, the dose response of human LH (hLH) stimulation of testosterone was studied in detail using testicular interstitial cells from both species. Next, a small but stimulatory dose of hLH was selected and extensive prolactin doses were studied in vitro. NIH B-6 (bovine) prolactin in varying doses was added to the interstitial cells 30 min prior to the addition of a constant dose of hLH. Under these circumstances prolactin inhibited LH action over a wide range of doses. In both species a biphasic dose-response curve existed: large doses of 100 to 1000 ng/ml produced less inhibition or augmented LH action, compared to smaller doses. Next, entire hLH dose-response curves were produced in the presence of three doses of prolactin (0.33, 33, and 1000 ng/ml) as well as in the absence of prolactin. The addition of prolactin shifted the hLH dose-response curve to the right and depressed the maximal response in comparison to the curve without prolactin. Finally, inhibitory doses of prolactin resulted in no detectable change in LH receptor number as estimated from Scatchard plots. It is concluded that prolactin inhibits LH action on interstitial cells as determined by rate of testosterone production except at very large doses of prolactin where LH action is less inhibited or augmented. The inhibitory action of prolactin in this in vitro interstitial cell assay was not accompanied by a decrease in LH receptor number. Thus, a postreceptor action is likely to be involved.
3753	Prostaglandin F2 alpha inhibits luteinizing hormone (LH)-induced increase in LH receptor binding to isolated rat luteal cells.	6094153	The effect of the luteolytic hormone prostaglandin F2 alpha (PGF2 alpha) on parameters of LH receptor binding to isolated rat luteal cells was examined. The equilibrium binding constant (0.55 X 10(10) M-1), the association rate constant (0.89 X 10(8) M min-1), and the dissociation rate constant (1.70 X 10(-2) M min-1) were not significantly altered by PGF2 alpha. However, as [125I]iodohCG binding approached equilibrium (less than 2 h) and at equilibrium (greater than 3 h), PGF2 alpha, but not PGE2, consistently reduced LH binding by 10-20%. The LH receptor-binding capacity, determined by Scatchard analysis of [125I]iodo-hCG or [125I]iodo-hLH binding, was reduced from 7.5 +/- 0.1 to 6.4 +/- 0.1 X 10(4) receptors/cell in the presence of PGF2 alpha. This reduction of total cell-bound radioactivity by PGF2 alpha was not due to a change in the rate of hormone internalization and degradation. However, when cells were briefly treated with a pulse of LH (5 ng/ml; 5-10 min), [125I] iodo-hCG (LH) binding increased 20-30% within 2 h, and PGF2 alpha prevented this LH-induced increase. A similar pulse of cAMP analogs did not alter LH binding. We conclude that while the initial binding of LH to its receptor is not altered by PGF2 alpha, it does reduce the final equilibrium level of LH binding by a mechanism that involves a block in the appearance of LH-induced cryptic receptors.
3754	Use of a highly sensitive and specific immunoradiometric assay for detection of human chorionic gonadotropin in urine of normal, nonpregnant, and pregnant individuals.	6480810	A highly sensitive and specific two-site immunoradiometric assay (IRMA) for hCG has been developed and applied to the detection of the hormone in the urine of normal nonpregnant and pregnant individuals. The IRMA uses a solid phase coupled monoclonal antibody to the hCG beta-subunit for extraction of hormone from urine. The hCG extracted is then directly quantified by the binding of an affinity purified and radiolabeled rabbit antibody that reacts with the unique COOH-terminal peptide region of the hCG beta-subunit. The assay is capable of reliably and accurately measuring as little as 0.01 ng hCG/ml urine without interference from hLH. Assays of urine from normal men and nonpregnant women of reproductive age indicated that most individuals did not have detectable levels of hCG immunoreactivity, although a minority had minute amounts, with a mean value of approximately 0.01 ng hCG/mg creatinine. In contrast, all normal menopausal women studied had easily detectable levels of hCG immunoreactivity in their urine, with a mean value of 0.123 ng hCG/mg creatinine. A study of the excretion of hCG from three men injected with hormone for treatment of infertility indicated that after the first 24 h, hCG was cleared with a single exponential rate and was detectable to a level of 0.01 ng/ml. Application of the IRMA to measurements of hCG in the urine of two artificially inseminated patients indicated that the method was capable of detecting pregnancy as early as 9 days postovulation. The extreme sensitivity and specificity of the IRMA for urinary hCG in conjunction with the simplicity of assay performance and specimen collection should provide a substantial advantage over currently available methods for detection of early pregnancy and tumor monitoring.
3755	Stimulation of sperm production by human luteinizing hormone in gonadotropin-suppressed normal men.	6434586	The relative roles of FSH and LH in the control of human spermatogenesis are not well established. We previously reported that supraphysiological doses of hCG can stimulate sperm production in gonadotropin-suppressed normal men despite prepubertal FSH levels. To determine whether more nearly physiological levels of human LH (hLH) also can stimulate spermatogenesis when FSH levels are suppressed, we administered hLH to normal men whose endogenous gonadotropin levels and sperm production were suppressed by exogenous testosterone enanthate (T). After a 3-month control period, 11 normal men received 200 mg T, im, weekly to suppress LH and FSH. T administration alone was continued for 3-4 months until 3 successive sperm concentrations (performed twice monthly) revealed azoospermia or severe oligospermia (sperm concentrations, less than 4 million/ml). Then, while continuing T, 4 of the 11 men (experimental subjects) simultaneously received 1100 IU hLH, sc, daily for 4-6 months to replace LH activity, leaving FSH activity suppressed. The effect on sperm production of the selective FSH deficiency produced by hLH plus T administration was determined. The remaining 7 men (control subjects) continued to receive T alone at the same dosage, without gonadotropin replacement, for an additional 6 months. In the four experimental subjects, sperm concentrations increased significantly from 0.7 +/- 0.7 million/ml (mean +/- SEM) during T treatment alone to 19 +/- 4 million/ml during hLH plus T administration (P less than 0.001). However, none of the men achieved sperm concentrations consistently in their own pretreatment range. Sperm motilities and morphologies were normal in all four subjects by the end of hLH plus T administration. In contrast, sperm concentrations in the seven control subjects remained suppressed (less than 3 million/ml) throughout the entire period of prolonged T administration alone. Serum LH bioactivity, determined monthly by in vitro mouse Leydig cell bioassay in all four experimental subjects, was markedly suppressed during T administration alone (120 +/- 10 ng/ml) compared to that during the control period (390 +/- 20 ng/ml; P less than 0.001). With the addition of hLH to T, LH bioactivity returned to control levels (400 +/- 40 ng/ml; P = NS compared to control value). Serum FSH levels determined monthly by RIA were reduced from 98 +/- 12 ng/ml during the control period to undetectable levels (less than 25 ng/ml) during the T alone and the hLH plus T periods (P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)
3756	Metabolism of luteinizing hormone by the pseudopregnant rat ovary.	6745197	Ovarian metabolism of highly purified human LH (hLH) was studied in pseudopregnant rats. The animals were injected with 1.0 microgram (11.1 IU) hLH, iv, and groups of animals were killed between 15 and 300 min. The hLH present in ovarian cytosol and sera and eluted from ovarian membrane was determined by RIA. Ovarian membrane-bound LH increased rapidly for 1 h, after which it plateaued and then decreased 4 h after the injection. Cytosolic LH peaked at 1 h, but concentrations declined thereafter. When ovarian cytosol obtained from rats injected with 10 micrograms hLH was chromatographed on a Sephadex G-100 column, the major immunoreactive fraction cochromatographed with the LH used for injection. Cytosol harvested 15 min after LH stimulation contained a small peak which coeluted with LH alpha. No LH beta was detected. No evidence for extensive dissociation of the LH subunits was found. When hLH present in serum and ovarian cytosol or bound to ovarian membrane was chromatographed on Concanavalin A-Sepharose, different patterns of binding and elution with that lectin were observed and were consistent with differential modification of the oligosaccharide side chains of LH present in serum and associated with the ovary. The biological to immunological ratio of cytosolic LH decreased from 1.7 15 min after LH injection to 0.8 at 5 h. Compared to our previous studies with hCG, LH was metabolized more rapidly. Whether that difference in the rates of ovarian metabolism of the two gonadotropins contributes to the marked disparity in their quantitative biochemical effects induced by those hormones should be examined.
3757	[The preparation of an hCG-specific immunoadsorbent using hCG beta-carboxyl-terminal peptide antibody and its application to isolate HCG-like substances from human pituitary glands].	6207055	It has been difficult to distinguish hCG from hLH because the two are structurally similar. However, hCG beta subunit has a unique carboxyl terminal peptide, which is not present in hLH and other gonadotropins. Taking advantage of this unique structural feature of hCG beta subunit, hCG-specific antisera have been produced. In order to isolate hCG-like substances from human pituitary glands, the specific immunoadsorbent for hCG was prepared using the isolated monospecific antibody from these antisera. Specific IgG fraction was isolated from an antiserum by affinity chromatography using synthetic carboxyl-terminal peptides as ligands. The purified IgG was conjugated to Sepharose 4B to prepare a specific immunoadsorbent. Immunoadsorbent thus prepared was fully specific to hCG without any crossreactivity with hLH. Attempts to isolate hCG-like substances from two different human pituitary extracts were made by affinity chromatography using this hCG-specific immunoadsorbent. Elution conditions for hCG-like substances from the immunoadsorbent were studied. We found that 1 M NH4OH was a highly effective eluent in this affinity chromatographic system. An advantage of 1M NH4OH is its volatile nature, which permits further steps of purification to be performed directly or after lyophilization without extensive dialysis. The immunoaffinity procedures described in this paper may provide a convenient approach to purify hCG-like substances from both endocrine glands and biological fluids.
3758	Autoregulatory control of thyrotropin in rabbits.	6723584	Studies were designed to determine whether an autoregulation system exists for TSH in the rabbit. For this purpose, a species-specific RIA for rabbit TSH that does not cross-react with human (h) TSH was developed. Hypothyroid animals were studied at varying time periods up to 3 months after either surgical thyroidectomy or propylthiouracil (PTU) treatment. Highly purified hTSH was injected iv at doses of 0 (saline control), 0.1, 0.3, 1,3, and 10 micrograms into unanesthetized rabbits bearing chronically implanted Silastic catheters. Blood samples were obtained at -30, 0, 10, 20, 30, 60, 120, 180, 240, and 300 min and 24 h. Doses between 0.3 and 10 micrograms hTSH produced a prompt fall (10 min) in rabbit TSH in hypothyroid rabbits studied 8-21 days after thyroidectomy. The minimum dose of hTSH that significantly suppressed rabbit TSH was 0.3 micrograms. This dose produced a peak value of hTSH in rabbit serum of 1.3 +/- 0.1 (+/- SEM) ng/ml 10 min after injection, which translates into a bioassay potency of 2.0 microU/ml (close to the physiological level in humans). A dose-response relationship existed between the hTSH dose injected and the duration and magnitude of suppression of rabbit TSH. This response to TSH was specific; 10 micrograms hTSH produced no change in endogenous rabbit serum LH and, conversely, 10 IU hLH produced no change in rabbit serum TSH. In contrast to these striking effects in acute hypothyroid animals, hTSH produced no detectable suppression of rabbit TSH in animals that were hypothyroid for 2-3 months. The sensitivity of the autoregulatory system to the suppressive effects of exogenous hTSH decreased with increasing duration of hypothyroidism; a time-response relationship existed. We conclude that: 1) a sensitive and specific autoregulatory control system for TSH exists in the rabbit; and 2) as the duration of hypothyroidism increases, the sensitivity of the autoregulatory system to the suppressive effects of endogenous TSH changes.
3759	Serum prolactin profiles of normal human conceptual cycles.	6148279	Commencing on day 10 of the menstrual cycle through onset of subsequent menses, or confirmation of pregnancy, daily sera collected from 15 women planning pregnancy were analyzed by radioimmunoassays (RIA) for prolactin (hPRL), estradiol-17 beta and luteinizing hormone (hLH). Two of the observed subjects became pregnant in the single cycles studied. The profiles of these hormones during the early gestation following spontaneous ovulation were established. No distinct midcycle peaks of hPRL were observed in either subject. Enormous spikes were observed in daily prolactin values, with wide variations between subjects.Commencing on day 10 of the menstrual cycle through onset of subsequent menses, or confirmation of pregnancy, daily sera collected from 15 women planning pregnancy were analyzed by radioimmunoassays for prolactin (hPRL), estradiol-17beta, and luteinizing hormone (hLH). Two of the subjects became pregnant in the single cycles studied. The profiles of these hormones during the early gestation following spontaneous ovulation were established. No distinct midcycle peaks of hPRL were observed in either subject. Enormous spikes were observed in daily hPRL values, with wide variations between subjects.
3760	[Study on human pituitary gonadotropin synthesis and release in amenorrhea and anovulation by LH-RH two step test].	6429259	The pituitary gonadotropin response of synthesis and release to two step stimulation at a 60 minute interval with 100 micrograms of luteinizing hormone-releasing hormone (LH-RH) was studied by radioimmunoassay of hLH and hFSH in the normal menstrual cycle, amenorrhea and anovulation. The definition of delta 1 and delta 2 was as follows: delta 1 = 30 (or 60) min. value-0 min. value. delta 2 = 90 (or 120, 180) min. value-60 min. value. delta 1, delta 2 ratio = delta 2/delta 1. Serum mean levels of hLH in follicular and luteal phase increased after the first injection of LH-RH. The second injection of the hormone induced an increase to above the first peak, indicating the priming effect of the first LH-RH on the second peak. The first peak is presumed to be the pituitary gonadotropin release due to the first injection of LH-RH, and the second peak can be composed in part of hLH newly synthesized by the first LH-RH stimulation. The response of hLH in the ovulatory phase increased after the first administration, but showed no significant peak after the second. The LH-RH two step test in normoestrogenic and PCO type amenorrhea produced two peaks of the mean levels of hLH. In ovarian anovulation, an hLH value higher than the others was detected before the first LH-RH stimulation. The first injection caused an increase in serum hLH but after the second there is no significant peak. In contrast, serum hLH and hFSH values were lower than in the normal menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
3761	Hypogonadism and Leydig cell hypoplasia unresponsive to human luteinizing hormone (hLH).	6428228	A 22-year-old man with hypogonadism, Leydig cell hypoplasia, high plasma LH, and normal plasma FSH was given human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) in order to assess testicular testosterone responses to exogenously administered gonadotropins. The maximal testosterone level detected after six doses of hCG, 5000 IU per dose given intramuscularly (IM) was 256 ng/dl. After eight doses of hLH, 550-1100 IU per dose given IM, the plasma testosterone level was unchanged, 100-200 ng/dl. The lack of a response to exogenously administered gonadotropins in our patient suggests that there is a primary testicular lesion causing the hypogonadism. Our patient's own LH is probably biologically active since there was no response to human LH, a pituitary extract with a known biopotency (LER 1549). Our patient's endocrine abnormality is apparently another facet in the spectrum of findings described in patients with Leydig cell hypoplasia or aplasia. Other features of this entity can include female pseudohermaphroditism, ambiguous genitalia, hypogonadism, delayed puberty, and the presence of biologically inactive LH.
3762	Constitution and behavior of follicular structures in the human anterior pituitary gland.	6326578	The follicular structures present in the human pituitary gland were studied, at the light-microscopic level, using histochemical and immunocytochemical techniques. The antisera applied in the peroxidase-antiperoxidase procedure were anti-hFSH beta, anti-hLH beta, anti-hPRL, anti-hGH, anti-hTSH beta, anti-hLPH beta, anti-pACTH, and anti-hACTH. In the 10 normal pituitaries examined, follicles were always found in the three areas of the adenohypophysis. The wall of the pars distalis follicles showed the seven immunoreactive cell types studied, while follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were the only ones present in the wall of the pars tuberalis follicles. Most of the cell types studied were also present in the wall of the intermediate area follicles, but these follicles had characteristics not found in the other two areas. They were very large, with frequent interconnections forming a three-dimensional network of anastomotic cavities, and the colloid had different histochemical affinity. None of the hormones studied could be detected by immunocytochemistry within the follicular colloid. Three of the ten pituitary adenomas examined showed numerous follicular structures. Some of the follicles in the adenomatous pituitaries were similar to those found in the normal adenohypophysis, but there were also follicles filled with only traces of colloid and numerous blood cells in the cavity, and follicles filled with neoformed connective tissue. In one of these cases, FSH/LH immunoreactive adenoma cells were seen in the wall of the follicles. The results obtained suggest that the finding of pituitary adenomas with follicular structures is not uncommon and that the follicles originate from the tumor cells. In addition, the follicles seem to have several functional stages, explaining the finding of different types of follicular formation.
3763	Internalization of 125I-human choriogonadotropin in bovine luteal slices. A biochemical study.	6705837	Various intracellular organelles as well as outer cell membranes of bovine corpora lutea intrinsically contain gonadotropin receptors (Rao et al., J biol chem 256 (1981) 2628 [5]). In order to investigate whether exogenously added human choriogonadotropin (hCG) can internalize and bind to the intracellular sites, bovine luteal slices that had been carefully checked with respect to structural and functional integrity were incubated with 0.1 nM 125I-hCG. Following incubation, specific radioactivity was found to be associated with various intracellular organelles, but not with cytosol. The order of radioactivity uptake by subcellular organelles following a 2-h incubation was: Golgi medium greater than Golgi heavy greater than Golgi light greater than plasma membranes = rough endoplasmic reticulum greater than mitochondria-lysosomes- greater than nuclei. The 5'-nucleotidase activity and electron microscopic examination of the fractions revealed that the presence of radioactivity in the intracellular organelles cannot be attributed solely to plasma membrane contamination. The internalization and intracellular binding of 125I-hCG was time and temperature-dependent. Only excess unlabeled hCG and hLH (but not hCG subunits, FSH and PRL) competed with 125I-hCG for internalization in luteal slices. Very little or no 125I-hCG added was internalized in liver or kidney slices; luteal, liver and kidney slices accumulated neither 125I-BSA nor 125I. The radioactivity eluted from various luteal subcellular organelles was able to rebind to fresh corresponding organelles and came off Sepharose 6B columns in a position corresponding to native 125I-hCG. The gel filtration profile of detergent-solubilized radioactivity revealed that 125I-hCG was macromolecular bound. The degraded and altered 125I-hCG was found in the incubation media.
3764	The secretion of human chorionic gonadotropin-like substance in women employing contraceptive measures.	6699130	To study the secretion pattern of a hCG-like substance (hCG') in normal women and those employing contraceptive measures, we assayed daily urine concentrates by RIAs using an anti-hCG beta-COOH-peptide serum and an anti-hCG serum to monitor hCG' and human LH (hLH), respectively. In eight cycles of four normal women, urinary hCG' was within the normal range (less than 100 ng/day), except that four samples in two women had marginal elevations of hCG' during the menstrual or premenstrual period, but not at the time of the midcycle hLH surge. Among seven cycles from three intrauterine device (IUD) users, six showed midcycle hLH surges, and in five, hCG' was prominent (0.17-3.6 micrograms/day) in the late luteal phase. In eight cycles of women ingesting steroid contraceptives on a conventional schedule, hCG' was found during the premenstrual and menstrual intervals. The pattern of hCG' secretion was episodic, and hCG' levels were in the range of 0.3-1.8 microgram/day. It was notable that hCG' was excreted at the highest levels during intervals when ingestion of steroids was withheld. These findings suggest that contraceptive steroid hormones may modulate pituitary hCG' secretion. From gel high pressure liquid chromatographic studies, the immunoactive hCG' detected in urinary extracts from normally cycling women and IUD users had a molecular size larger than hCG beta and hLH, but was slightly smaller than hCG (lot CR119). The hCG' identified in these eluents is unlikely to be a subunit of hCG. On the other hand, the immunoactive hCG' in the urinary extracts from oral contraceptive users was mainly the hCG beta-subunit-like substance. Both hCG alpha and hCG beta immunoactive substances were heterogeneous in size in contrast to those found in normally cycling women and IUD users. In summary, the hCG' excretion pattern was different in menstrual and postmenopausal women, and hCG' identified in these subjects was heterogeneous.To study the secretion pattern of an hCG-like substance (hCG') in normal women and those employing contraception, the authors assayed daily urine concentrates by radioimmunoassays using an anti-hCGbeta-COOH-peptide serum and an anti-hCG serum to monitor hCG' and human luteinizing hormone (hLH), respectively. In 8 cycles of 4 normal women, urinary hCG' was within the normal range (100 ng/day), except that 4 samples in 2 women had marginal elevations of hCG' during the menstrual or premenstrual period, but not at the time of the midcycle hLH surge. Among 7 cycles from 3 IUD users, 6 showed midcycle hLH surges, and in 5, hCG' was prominent (0.17-3.6 mcg/day) in the late luteal phase. In 8 cycles of women ingesting steroid contraceptives on a conventional schedule, hCG' was found during the premenstrual and menstrual intervals. The pattern of hCG' secretion was episodic and hCG' levels were in the range of 0.3-1.8 mcg/day. It was notable that hCG' was excreted at the highest levels during intervals when steroid ingestion was withheld. These findings suggest that contraceptive steroids may modulate pituitary hCG' secretion. From gel high pressure liquid chromatographic studies, the immunoactive hCG' detected in urinary extracts from normally cycling women and IUD users had a molecular size larger than hCGbeta and hLH, but was slightly smaller than hCG (lot CR119). The hCG' identified in these eluents is unlikely to be a subunit of hCG. On the other hand, the immunoactive hCG' in the urinary extracts from OC users was mainly the hCG beta-subunit-like substance. Both hCGalpha and hCGbeta immunoactive substances were heterogeneous in size in contrast to those found in normally cycling women and IUD users. In summary, the hCG' excretion pattern was different in menstrual and postmenopausal women, and hCG' identified in these subjects was heterogeneous.
3765	Comparisons of gonadotropin binding sites and progesterone concentrations among the corpora lutea of individual pigs.	6322875	In polyovular species, it is unclear whether the characteristics of each individual corpus luteum (CL), such as mass, progesterone concentration and receptors for luteinizing hormone (LH), are representative of those of its cohorts during the ovarian cycle. The current study was performed 1) to characterize the conditions for estimation of binding parameters for LH receptors in porcine CL, and 2) to compare LH binding sites, luteal progesterone concentrations and luteal masses among CL of ovaries within individual pigs. Gonadotropin binding sites in porcine CL were characterized via specific binding of 125I-human (h) LH to 20,000 X g particulate fractions of luteal tissue. Specific binding was directly proportional to tissue content and was detectable at the lowest content tested (0.5 mg tissue equivalents/tube). Specific uptake of 0.25 ng LH by 5.0 mg tissue equivalents was time- and temperature-dependent; steady-state binding was achieved within 20 h at 37 and 25 degrees C. Binding of LH after 20 h incubation at 37 degrees C (4718 +/- 192 cpm, means +/- SEM) and 25 degrees C (4112 +/- 340 cpm) was greater than that at 4 degrees C (1930 +/- 5 cpm, P less than 0.01). Luteal particulates from individual CL of ovaries collected from four mature nonpregnant pigs (13-23 CL/pig) were incubated with eight concentrations of 125I-hLH. Steady-state binding depended upon hormone concentration until reaching saturation at 2.5 ng 125I-hLH/tube. Scatchard analyses yielded linear plots. Binding capacities for LH ranged among pigs from 0.71 +/- 0.03 to 3.69 +/- 0.13 fmol/mg CL equivalents and receptor affinities (Kd) ranged from 0.92 +/- 0.05 to 4.89 +/- 0.41 X 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)
3766	Ectopic production of human chorionic gonadotrophin-like material by breast cancer.	6692294	Human chorionic gonadotrophin (hCG) is a placental protein whose ectopic secretion by nontrophoblast tumors has been claimed to be of clinical relevance. Serum levels of hCG were measured in 570 patients with breast disease. A double-antibody radioimmunoassay (RIA) using antisera to hCG-beta was employed. Approximately 14% of patients with breast cancer were found to have elevated serum hCG levels. Such raised titers were not stage- or tumor-type-related, but occurred only in postmenopausal subjects. Further study showed that those patients with elevated hCG levels also had raised levels of human luteinizing hormone (hLH). Assay cross-reactivity was shown to account for the "spurious" hCG elevations. An immunocytochemical study also failed to find hCG an ectopic breast tumor constituent and/or product. It is concluded that hCG is not produced by breast tumors and has no clinical utility.
3767	Clinical validation of enzymeimmunoassay of human luteinizing hormone (hLH) in the detection of the preovulatory luteinizing hormone (LH) surge in urine.	6365598	The preovulatory luteinizing hormone (LH) surge (mean, 60.7 standard error +/- 4.7 mIU/ml) as determined by a solid-phase enzymeimmunoassay in urine has been correlated with clinical parameters in 24 women. In group A, of seven women, the preovulatory LH surge correlated with basal body temperature and cervical mucus. In one of the women in group A, serum levels of pituitary and gonadal hormones confirmed ovulation. In group B, of 17 women, the urinary estrone-3-glucuronide (E1-3-G) peak was either coincident with or preceded the LH surge. The LH surge in all cases occurred 12 to 24 hours prior to follicular rupture, as visualized by real-time sonography. The enzymeimmunoassay for the detection of the preovulatory LH surge is useful in patients for artificial insemination and for aspiration of mature oocytes for in vitro fertilization.
3768	In vitro effect of danazol on steroidogenesis in developing human follicles. An interim report.	6093423	In order to show whether or not danazol exerts direct effects on the cells of the human ovarian follicle, follicular granulosa and thecal cells of follicles in different stages of development are currently being investigated in short-term incubation experiments and during more prolonged cell culture. Cells are isolated by free-hand dissection from follicles of 8 to 25 mm diameter, corresponding approximately to cycle day 7 to 14. Granulosa and thecal cells have so far been incubated for 2 to 4 hours in the presence or absence of hCG and/or danazol. A small number of cell cultures of granulosa cells have been performed for up to 6 days in the presence or absence of danazol, hFSH, and hLH, in various combinations. The progesterone (P), estradiol-17 beta (E) and androstenedione (A) content of the incubation or culture medium and the tissue content of cyclic AMP (cAMP) were measured. Danazol (10 micrograms/ml) inhibited thecal cAMP production in most short-term experiments but only the hCG-stimulated cAMP production of granulosa cells. Short-term production of P and E by granulosa and of A and E by thecal cells was also inhibited by danazol in certain circumstances. In tissue cultures of granulosa cells lasting up to 4 days, danazol (1 and 10 micrograms/ml) inhibited both the FSH and LH-stimulated P production. Provided that any unspecific effects of the concentrations of danazol used can be ruled out, the results seem to indicate that this substance may act directly upon steroidogenesis at the ovarian follicular level.
3769	Testicular gonadotropin receptors, testicular testosterone, dihydrotestosterone and androstenedione in the developing bull.	6089676	The testis homogenate from Red bull (3-17 months of age) show specific binding of hLH and hFSH with Kd of 1.3 +/- 0.1 X 10(-10)M and 1.5 +/- 0.1 X 10(-9)M, respectively. Prepubertal bulls (3-5 3/4 months) exhibit the highest binding capacity of both LH and FSH, revealing the fact that LH and FSH receptors are present in high concentrations prior to the first rise in plasma T. The testicular T concentration was significantly higher in the four prepubertal bulls than in the four older animals in spite of the fact that circulating T is lower before puberty. Thus, local androgen effects within the testis may occur before peripheral androgenization. The prepubertal bull Leydig cell also displayed steroidogenic capacity. The ratio of biologically active (testosterone, dihydrotestosterone) to inactive androgens (androstenedione) increased as the animals approached puberty. Such qualitative changes in steroid production may be one of the factors responsible for peripheral androgenization.
3770	[Monoclonal antibodies directed against the beta hCG subunit and against a synthetic peptide analog of the carboxyl-terminal end. Clinical use in vivo and in vitro].	6085542	In order to detect specifically the beta-hCG, we have produced monoclonal antibodies (Mabs), using as immunogens hCG, beta hCG or a totally synthetic molecule (109-145 peptide) analogous to the beta-subunit carboxyl terminus. 34 fusion experiments were performed and led to 8 Mabs which presented a high reactivity to 125I beta hCG in the screening test. Mab D1E8 was directed to the 1-115 region of beta hCG and was cross reactive with hLH (greater than 60%). Mabs 702, 1032, and 1211 were directed against three different epitopes located in the 109-145 region and were specific for the beta hCG. The in vivo localization of tumors containing beta hCG ty tomoscintigraphy (SPECT) was evaluated in 5 patients by injecting 131I-Mab D1E8, selected for imaging because of its high affinity to beta hCG (Ka = 1.9 X 10(9)). SPECT was performed 48 h and 96 h after injection: 2 patients with proven tumor sites had positive "immunoscintigraphy" results. One patient, simultaneously injected with 125I non-specific IgG, had a tumor resection: the count ratio between normal and tumoral tissues revealed a low specific uptake. Two and three-site immunoradiometric assays (IRMA) were performed with monoclonal antibodies purified from nude mice ascitic fluids. IRMA were based upon two or three Mabs, with D1E8 on a solid phase and 125I Mab 702 or a mixing of 125I-702 and 125I-1032 as tracer. The specificity of IRMA was demonstrated by the absence of binding with increasing amount of hLH, hFSH and hTSH up to 5000 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)
3771	Monoclonal antibodies to human chorionic gonadotropin and its beta-subunit.	6663149	Using the technique of somatic cell fusion, monoclonal antibodies to human chorionic gonadotropin (hCG) and its beta-subunit were produced. Spleen cells from immunized mice were fused with the myeloma line NS-1 using 50% polyethylene glycol and cultured in a selection medium. A sensitive enzyme immunoassay was performed for antibody screening using immobilized solid-phase antigen and anti-mouse IgG Fab' (rabbit)-beta-D-galactosidase complex. Three double-cloned hybridomas (named 4H10, 4G2 and 1D12) were obtained. Anti-hCG 4H10 antibody reacted with hCG, the alpha-subunit of hCG (alpha-hCG) and human luteinizing hormone (hLH); and anti-hCG 4G2 antibody reacted with hCG and the beta-subunit of hCG (beta-hCG). It was interesting that anti-beta-hCG 1D12 antibody had the capacity to bind with free beta-hCG but not with intact hCG, which suggests that the 1D12 antibody can recognize a determinant site that is unique to beta-hCG. This unique epitope in beta-hCG might be expressed in or near a part which is hidden in the intact hCG molecule which is composed of alpha- and beta-subunits.
3772	Indomethacin decreases both luteinizing hormone binding and fluidity of testicular microsomal membranes in rat.	6322203	The objective of these studies was to examine the effects of in vivo treatment with indomethacin, a prostaglandin synthesis inhibitor, on luteinizing hormone receptors and fluidity in microsomal membrane preparations of rat testis. Adult male rats were treated with vehicle or either of two doses of indomethacin (3.8 micrograms/g body weight or 7.5 micrograms/g body weight) every 8 h for 1, 2 and 3 days. Indomethacin treatment decreased human luteinizing hormone (hLH) binding in rat testis in a time-dependent fashion. The lower dose of indomethacin decreased hLH binding by 19%, 35% and 50%, respectively, after 1, 2 and 3 days of treatment. The higher dose of indomethacin decreased hLH binding at these respective times by 21%, 44% and 56%. Scatchard analysis of the hLH binding of testicular membrane preparations indicated that indomethacin treatment decreased the number of hLH binding sites rather than the apparent affinity constant. Testicular membrane fluidity was estimated by a fluorescence polarization technique. The effects of indomethacin on apparent fluidity of microsomal membrane paralleled that of LH binding. The values of apparent microviscosity, inversely related to the fluidity, also increased in a time-dependent fashion. These simultaneous effects of indomethacin on testicular LH binding and fluidity suggest that these phenomena are interrelated and that prostaglandins are involved in the maintenance of LH receptors in vivo.
3773	Receptors for luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the human undescended testis and the effect of hCG-treatment.	6142003	Human testis tissue contains specific receptors for luteinizing hormone (LH) and follicle stimulating hormone (FSH) with Ka's of 1.5 and 1.6 X 10(9) M-1, respectively. Specific binding of [125I]hLH and [125I]hFSH was studied in testicular biopsies from 33 boys (4-16 years of age) with undescended testes and in 2 adult men with normally descended testes. Ten of the boys were treated with human chorionic gonadotrophin (hCG) and operated 1-4 days after the last injection. Samples from each testis were taken for histological evaluation. Specific binding of LH in biopsies from pre-pubertal boys (stage I) was low and differentiated Leydig cells could not be identified histologically. In later pubertal stages the LH binding increased and differentiated Leydig cells could now be recognized. hCG-treatment caused a highly significant increase (P less than 0.001) in the binding of LH without affecting FSH binding. The highest binding of FSH occurred during the onset of puberty. Treatment with hCG did not cause premature differentiation of Sertoli cells or germ cells, but appeared to stimulate Leydig cells.
3774	Biosynthetic Met-hGH: lack of immunoreactivity for other pituitary hormones.	6671467	The immunoreactivities of 5 hGH preparations were examined in RIA systems for hPRL, hACTH, hLH, hFSH and hTSH. All preparations derived from pituitary extracts showed distinct displacements in almost all RIA systems when added 10(3) to 10(4) times larger amounts than the proper hormones. On the other hand, biosynthetic methionyl-hGH did not manifest any immunoreactivity in any system with comparable amounts. These results indicate that the displacements caused by hGH preparations in RIA systems for other pituitary hormones are solely due to contamination, and not due to intrinsic cross reaction. In in vitro and in vivo studies using extractive preparations of hGH one should be cautious about contamination of other pituitary hormones.
3775	Characterizations of anti-oLH beta antibodies acting as contraceptives in rhesus monkeys. II. In vivo neutralizing ability for gonadotropic hormones.	6620250	63 female rhesus monkeys were actively immunized against the beta-subunit of ovine luteinizing hormone (oLH beta). 75% of these monkeys developed antibodies which were sufficiently high to induce infertility. We report here the in vivo neutralizing potencies of anti-oLH beta antisera from 14 monkeys and the correlation between in vitro and in vivo properties of the antibodies. Anti-oLH beta sera neutralized the biological activity (stimulation of testosterone production in male mice) of both hCG and rhCG. The correlation between antibody titers and neutralizing potency of hCG action of the antisera was significant. rhCG-induced testosterone production was inhibited more effectively than that induced by hCG. There was no correlation between binding affinity to hCG in vitro and neutralizing activity. Neutralizing activities correlated well with binding capacities, suggesting that the number of binding sites of the antibody population may play an important role in the mechanism of neutralization. In rhesus monkeys that were effectively immunized against pregnancy circulating oLH beta antisera did induce shortened luteal phases and impaired luteal function. Successful application of oLH beta as an antigen for contraception in humans may depend on relative cross-reactions of hLH and hCG with anti-oLH beta sera and on a neutralizing capability very similar to the one found in rhesus monkeys.
3776	Differences in the rates of internalization of 125I-labeled human chorionic gonadotropin, luteinizing hormone, and epidermal growth factor by ovine luteal cells.	6602702	The rate of internalization and degradation of radioiodinated hCG, ovine LH (oLH), human LH (hLH), and mouse epidermal growth factor (mEGF) was studied in suspensions of ovine luteal cells. Hormone bound to the plasma membrane was quantified by treating the cells with acidic buffer (pH 3.0), and the radioactivity removed from the receptor was measured. The radioactivity remaining in the cell pellet was considered to have been internalized. The extent of degradation of the radioactive hormones was determined by subjecting the radioactivity that was released into the medium during the incubation periods to precipitation with 20% trichloroacetic acid. The non-precipitable radioactivity was considered to have been degraded. The required time for loss of half of the radioactivity that had been bound to the cell membrane at time zero was 22.8 +/- 2.3 h for hCG, 23.3 +/- 1.1 h for asialo-hCG, 15.1 +/- 1.4 h for hLH, 0.4 +/- 0.2 h for oLH, and 0.3 +/- 0.1 for mEGF. The quantities of radioactivity that had been internalized (intracellular plus degraded) were greater at earlier times (15-120 min) for oLH or mEGF than for hCG, asialo-hCG, or hLH. These data suggest that ligands bound to different receptors on ovine luteal cells are internalized and degraded at different rates by the same cell and that ligands that bind to the same receptor (hCG, hLH, and oLH) can also be internalized and degraded at very different rates.
3777	Studies on primate gonadotropin receptors: comparison of follitropin and lutropin receptors in human testis.	6313152	The interaction of 125I-labeled human follitropin (hFSH), human lutropin (hLH), and human choriogonadotropin (hCG) with a particulate fraction prepared from the testes of adult men was investigated. The hormone specific binding was maximal at pH 7.5 and 34 degrees C in the presence of 10 mM MgCl2. The FSH receptor was relatively more stable than the LH receptor (t1/2 at 34 degrees C of 14 and 4 h, respectively). The dissociation constants (Kd) calculated for hLH or hCG were very similar (approximately 1.0 X 10(-10) - 1.4 X 10(-10) M). The affinity of FSH and LH to their receptors did not appear to change with age, as shown from analysis of testes from 17- to 80-year-old men. The number of FSH receptors was greater than the number of LH receptors in these tissues. The hFSH receptor did not show any preference to binding of the homologous hormone, because FSH from nonprimates could displace 125I-labeled hFSH, but the LH receptor showed a marked preference for hLH or hCG as the ligand.
3778	Characterization of the thyrotropin receptor-adenylate cyclase system in neoplastic human thyroid tissue.	6304132	Experiments were performed to determine whether the TSH receptor-adenylate cyclase (AC) system in benign and malignant thyroid neoplasms differs from the TSH receptor-AC system in normal thyroid tissue removed from the same patients. TSH binding and AC assays were performed using the same in vitro conditions. TSH binding was rapid, reversible, saturable, and hormone specific in particulate fractions from both normal and neoplastic thyroid tissue. A positive correlation existed between the equilibrium constants for [125I]bovine ([125I]bTSH) TSH binding and the concentration of TSH required to activate AC, suggesting that binding sites were coupled to AC in neoplastic thyroid tissue. Mean values for dissociation constants (Kd1 and Kd2), capacity (site 2), as determined by Scatchard analysis, and nonspecific binding (NSB) for the TSH receptors were lower in neoplastic thyroid. Some normal thyroid tissue appeared to lack a high affinity site, and some tumors lacked a low affinity binding site. Hormone specificities (bTSH, human (h) TSH, hLH, hFSH, hGH, hACTH, and glucagon) in normal thyroid and neoplastic tissue were virtually identical. hFSH, hACTH, hGH, and glucagon failed to inhibit [125I]bTSH binding or stimulate AC in either normal or neoplastic thyroid tissue, whereas hLH inhibited [125I]bTSH binding and stimulated AC, but required 10- to 100-fold higher concentrations than hTSH or bTSH. The specific binding and NSB of [125I]bTSH in both normal and neoplastic thyroid tissue was highest at pH 7.0 and lowest at pH 8.3. In contrast to bTSH binding, TSH stimulation of AC was lowest at pH 7.0 in both normal and neoplastic tissues and highest at pH levels of 7.5-8.0. TSH binding and TSH stimulation of AC activity were highest in the absence of NaCl and decreased progressively as the salt concentration was increased in both normal and neoplastic thyroid tissues. Increasing the sucrose concentration and, thus, the osmolarity of the system had a minimal effect on the binding of [125I]bTSH. Preincubation with ammonium sulfate did not significantly influence binding. Basal AC activity and the AC response to TSH were greater in neoplastic thyroid than in normal tissues. These studies demonstrate that changes in salt concentration and pH affect the TSH receptor-cyclase system in a comparable fashion in normal and neoplastic thyroid tissues. The discriminatory properties of the TSH receptor are also maintained in thyroid neoplasms. Thyroid tumors, however, have a higher affinity for TSH and display a greater AC response to TSH than normal thyroid tissue.
3779	Characterization of a human anti-hCG antiserum: a proposed standard for laboratories involved with the development of hCG vaccines.	6617206	An antiserum (PC-81-1) was obtained from a man who developed antibodies against hCG during treatment for hypogonadism. The antiserum was unique in that its affinity for hCG was high (greater than 10(-10) M(-1] and its cross-reaction with hLH and the hCG-subunits was only 1-12.5% and 0.01%, respectively, of intact hCG. We propose that this human antiserum be used as a laboratory standard by investigators who are developing vaccines directed against hCG. The use of this standard in the proposed protocol will permit comparison of titers between laboratories. Lyophilized samples of antiserum PC-81-1 are available on request from the Population Council.
3780	[Heart catheterization in newborn and young infants. Description of the method].	6865978	The data of 343 newborns and young infants were reviewed who, from 1968 until August 1982, had undergone heart catheterization. Variations in frequency of catheterization and diagnoses as well as lethality were investigated. The first rise of heart catheterization performances could be noted in the years of 1968 and 1969 (by routine application of the Rashkind procedure), another remarkable rise took place in the years from 1971 to 1975, probably due to better feasibilities of modern cardiac surgery. Later on, a new dynamic plateau was developed. With the utilization of echocardiography, a significant decrease of heart catheterization performances in certain diagnostic groups (HLH, PFC) was registered. Nowadays, however, new diagnostic challenges (infants with low gestational age, respiratory problems, PDA) require heart catheterization. Comparison of the different diagnostic groups' lethality rate reveals that children undergoing heart catheterization, in order to rule out TGA, demonstrate a remarkably higher lethality rate (27%) than children suffering from TGA (3.7%). Introduction of echocardiography has decreased this risk significantly.
3781	Developmental pattern and sensitivity to down-regulation of testicular LH receptors in the pig.	6305849	Testicular receptors for luteinizing hormone (LH) were quantified in 40 pigs between the day of birth and 22 weeks of age. The highest specific binding capacity (fmol hLH bound/mg protein) occurred at about 2--3 weeks of age which coincides with the age when the relative volume of Leydig cells is at a maximum. Significantly lower binding capacity was observed at 7 weeks of age which is the time of maximum Leydig cell regression. hCG was injected intramuscularly to immature (3000 IU, 22 days old) and adult boars (10 000 IU, 30--40 weeks old). Twentyfour h after injection the numbers of available LH receptors were reduced to 6 and 18% of control values, respectively. At this time occupied receptors were at a maximum (90% and 67% of controls, respectively). The total number of LH receptors (available + occupied) was increased 1 h after injection after which a gradual decrease was observed. Three days after hCG administration the total receptor quantities were at a minimum in both immature and adult pigs (59% and 31% of controls, respectively) and returned to control levels 5--7 days after treatment. Thus, pig LH receptors appear to be subjected both 'positive' and 'negative' regulation as previously described in the rat.
3782	Ectopic production of human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) by ovarian carcinoma.	6681763	Human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) are placental proteins whose ectopic secretion by non-trophoblast tumours has been claimed to be of clinical relevance. Radioimmunoassays for hCG and hPL, together with human luteinising hormone (hLH), have been established and plasma levels were measured in 61 patients with carcinoma of the ovary. Approximately 51% of the patients were found to have raised plasma hCG levels. Such raised titres were not stage or tumour-type related but occurred only in post-menopausal subjects. The majority of patients with raised hCG levels also had raised plasma hLH levels. Assay cross-reactivity was shown to account for the 'spurious' hCG elevations. However, hCG may be an ectopic product in a minority of tumours; elevated plasma hCG levels were shown to coexist with low hLH levels. Although such lesions did not show morphologically identifiable choriocarcinomatous elements, all were poorly differentiated carcinomas. In some cells hCG was demonstrated by immunocytochemical methods. No patients had a raised plasma hPL level. It is concluded that these placental proteins are of no clinical use in the management of ovarian carcinoma patients.
3783	Use of a radioreceptorassay (RRA) for human luteinizing hormone/chorionic gonadotropin (hLH/CG) for detection of early pregnancy and estimation of time of ovulation in macaques.	31991966	A radioreceptorassay (RRA) for macaque luteinizing hormone (LH)/chorionic gonadotropin (CG) was adapted from the clinical RRA for human LH/CG, Biocept-G™, for the purposes of detection of pregnancy prior to day 20 of gestation and for estimation of the time of ovulation in macaques. The 90-min assay procedure was simple, accurate, and reliable. Seventy-five rhesus monkeys (Macaca mulatta) and 20 crab-eating monkeys (Macaca fascicularis) were tested for the presence of CG in the serum on estimated days 17-20 of pregnancy. Of a total of 160 tests, four false negative and 0 false positive tests were obtained, for an accuracy of 97.5%. The preovulatory LH peak was detected in 19 rhesus monkeys by semiquantitative RRA of LH/CG. Ovulation was confirmed in these 19 animals by the presence of a fresh corpus luteum at laparotomy 2-10 days after ovulation, collection of an embryo, pregnancy, or subsequent cycle history. The short, simple assay procedure and the low inter-and intraassay coefficients of variation (7.3 and 3.7%, respectively) allow use of this assay in an economical, predictive, as well as retrospective, capacity for estimation of the time of ovulation in rhesus monkeys. The sensitivity, reliability, species nonspecificity, simplicity, and rapidity of performance of this RRA for LH/CG are features which add up to a useful new management tool for breeding macaques for research purposes.
3784	Development, screening and quality specifications of hybridomas synthesizing anti-polypeptide hormone antibodies to be used for diagnostic tests and in-process control.	6677529	Hybridomas were developed for the production of monoclonal antibodies against human chorionic gonadotrophin (hCG) and against bovine and porcine (pro)insulins. Screening tests were devised to select those hybridomas synthesizing antibodies with predefined antigen-specificity. Cell lines, which produce anti-hCG antibodies with a low cross-reactivity against luteinizing hormone (hLH) and which are suitable for agglutination tests, were isolated. In addition hybridomas were obtained with various specifications for porcine and bovine (pro)insulins. Their monoclonal antibodies can be used for quantitative enzyme immunoassays of insulin and proinsulin mixtures. Specifications of hybridomas for production purposes have been defined with regard to growth requirements, antibody production capacity and absence of mycoplasma contamination.
3785	Effect of a partially desialylated human chorionic gonadotropin preparation (Desialo hCG) on ovarian function.	6413269	Six patients with unexplained infertility were administered a partially desialylated hCG preparation (Desialo hCG). 5000 and 10000 IU of this preparation were given as a single IM dose, following the administration of hMG-Pergonal. The circulatory half-life (t 1/2) of Desialo hCG was found to be 42.6 hours, which is similar to the t 1/2 of 35.6 hours previously reported for a hybrid hCG preparation. The t 1/2 values were shorter than the t 1/2 of 65.3 hours estimated for commercial hCG administered by the same route. In spite of the similarities in t 1/2 values between the two preparations, serum progesterone levels were significantly increased 48 to 96 hours after the administration of Desialo hCG, and only 8 to 10 hours after the administration of the hCG hybrid. The difference in response may reflect differences in receptor occupancy at the ovarian level. Large scale testing of preparations with half-lives approaching the t 1/2 of the physiological hormone, hLH, should be carried out in order to determine if the risk of overstimulation and/or multiple pregnancies could be reduced thus, allowing for a safer therapeutic modality in patients pre-treated with hMG.
3786	The human genome contains seven genes for the beta-subunit of chorionic gonadotropin but only one gene for the beta-subunit of luteinizing hormone.	6319099	We have used restriction enzyme analysis to sample similarities and differences among eight cloned genes, isolated from two independent libraries, which hybridize to a cDNA for the beta subunit of human chorionic gonadotropin (hCG). We find that one of the cloned genes encodes the beta-subunit of the closely related hormone, human luteinizing hormone (hLH). Using differential hybridization of human DNA with beta hCG- and beta hLH-specific probes, we show that there are seven beta hCG genes but only a single beta hLH gene in the human genome. This single beta hLH gene is linked to at least three beta hCG genes. Our restriction analysis reveals that the seven beta hCG genes are extremely similar but not identical, and that there are no obvious structural rearrangements to indicate which, if any, are pseudogenes. These data also reveal that the beta hLH gene is far more homologous to the seven beta hCG genes than the beta hCG:beta hLH protein sequence homology of 82% might predict. We propose that the beta hCG gene arose by a duplication of an ancestral beta hLH gene and that, once the beta hCG function was established, the ancestral beta hCG gene itself duplicated and rearranged, creating the present organization of multiple beta hCG genes.
3787	Studies of the human chorionic gonadotrophin-like substance of human pituitary glands and its significance.	6185602	Radioimmunoassays utilizing antisera specific for the carboxyl-terminal portion of human chorionic gonadotrophin (hCG) beta-subunit were used to measure the concentration in human pituitary extracts of an immunoactive hCG factor (hCG') which was different from human LH (hLH). The content of hCG' from different human pituitary pools collected between 1966 and 1979 was relatively constant at 0.5-1.1 microgram per gland. The hCG' concentrations observed in acetone-dried powder of individual human pituitary glands (0.4-26 ng/mg) were close to those reported for full-term placental powder. After separation and partial purification of human pituitary glycoprotein hormones, pituitary hCG' was found mainly in the crude human FSH (hFSH) fraction. It was separated from hFSH by diethylaminoethyl-cellulose chromatography at pH 7 and by gel filtration on Sephadex G-100. On gel filtration its molecular size was larger than that of hLH or hFSH and it was strongly bound to Concanavalin A-Sepharose. The most active preparations of pituitary hCG' obtained by these procedures were approximately 5 per cent as potent by specific radioimmunoassay as hCG purified from pregnancy urine. Although the hCG' content in individual pituitary glands was more variable than the hLH content, on average pituitary hCG' was estimated to be around 25- to 50-fold less than the content of hLH, hFSH or human TSH in the human pituitary gland.
3788	The induction of antibodies to human luteinizing hormone by contaminated clinical pituitary hormone preparations.	6183288	Three children with hypopituitarism had elevated LH levels measured by RIA which were incompatible with their stage of sexual maturation. Each of the children had been administered parenteral pituitary hormone preparations: one patient, human (h) GH for 3 2/1 yr; one patient, bovine TSH twice to evaluate thyroid responsiveness; and two patients, posterior pituitary extract by nasal insufflation for 7 5/12 ad 4 9/12 yr to treat diabetes insipidus. Each of these children had developed antibodies of the immunoglobulin G class which bound [125I]hLH in vitro in a displaceable fashion. In two of the patients, the antibody reacting with hLH was found after therapy with pituitary hormones of bovine or porcine origin and before treatment with hGH, while on child had received only hGH therapy. These antibodies interfered in the assay for hLH and were responsible for the spurious elevations of serum immunoreactive hLH. None of these children had undergone spontaneous puberty, including at least one who may not have been gonadotropin deficient. To reduce the risk of generating high potency neutralizing antibodies, only highly purified, monomeric pituitary hormone preparations or pure synthetic hormone preparations should be used for diagnosis and chronic replacement therapy.
3789	Immunohistochemical localization of a human chorionic gonadotropin-like substance in the human pituitary gland.	6813346	A substance with the biological, immunological, and physicochemical properties similar to those of hCG (hCG-like substance) has been found in human pituitary homogenates and urine of nonpregnant subjects. The purpose of the present study was to localize the hCG-like substance in human pituitary gonadotropin producing cells (gonadotrophs) by using an unique antiserum raised against a synthetic hCG beta C-terminal peptide (P75R). Twenty-five pituitary glands were obtained at autopsy from women of various chronological ages. An immunohistochemical method was employed for localizing hLH, hFSH, and an hCG-like substance on the horizontal serial sections of the pituitary glands. The LH and FSH cells were indistinguishable with the antisera employed in the present study. The hCG-like substance was only localized in the cytoplasm of the gonadotrophs, rather commonly when the pituitary glands from postmenopausal women were examined. The P75R serum pretreated with excessive amount of native hCG failed to exhibit the immunostaining. Normal human tissues aside from pituitary glands did not react with the P75R serum. The results obtained in the present study and those reported by others may indicate that the hCG-like substance present in human pituitary glands is the precursor of hLH.
3790	CNR external quality control program for hLH, hFSH, hPRL and hCG: first-semester experience.	6819344	NaN
3791	Biological and immunological characterization of human luteinizing hormone discharged by the stimulation of synthetic luteinizing hormone-releasing hormone (LH-RH) in normal and anovulatory women.	6763561	Plasma samples were obtained by repeated venopunctures immediately before, and at 15, 30, 60, 120 and 180 min after intravenous bolus administration of 100 micrograms synthetic LH-RH in normal and various anovulatory women. Plasma hLH levels were determined by an in vitro bioassay and a radioimmunoassay with improved reagents. The LH-RH stimulation induced an abrupt elevation of both biological and immunological hLH activities in normal and anovulatory women, although the responsiveness to LH-RH differed from case to case. Both elevated biological and immunological hLH activities decreased gradually with a half disappearance time of 122.4 +/- 27.9 min and 112.5 +/- 25.4 min, respectively (Mean +/- S.D.). A transient and significant depression in the ratio of biological to immunological hLH activities (B/I ratio) was observed at 15 min after the LH-RH administration in normal subjects. This depression is attributable to the cross-contamination of an increased amount of hLH subunits induced by LH-RH stimulation. The B/I ratios were significantly elevated throughout the investigation period in the anovulatory patients when compared with those in the normal subjects. This elevation appears to indicate the increased discharge of special type(s) of hLH subpopulations of high biological potency in the anovulatory cases.
3792	Specific enzyme immunoassay for human chorionic gonadotropin using antibody to the synthetic peptide corresponding to the carboxyl-terminal region of the beta-subunit of hCG.	6189707	A sandwich-type enzyme immunoassay for human chorionic gonadotropin (hCG) was developed with anti hCG-beta antibody-enzyme complex and the immobilized solid-phase antibody which was prepared from the antiserum to the synthetic peptide corresponding to the carboxyl-terminal region of the beta-subunit of hCG (hCG-beta). This assay system showed a high sensitivity equal to the radioimmunoassay and did not cross-react with human luteinizing hormone (hLH), human follicle stimulating hormone (hFSH) or alpha-subunit of hCG (hCG-alpha). The coefficients of variation in within-run and between-run were less than 10% and there was a good correlation between the enzyme immunoassay and the radioimmunoassay of serum sample with a regression equation of y = 1.038 x -0.647 and a correlation coefficient of r = 0.953.
3793	[Pulsatile fluctuation of human luteinizing hormone (hLH) in normal menstrual cycle, as assessed by the in vitro bioassay and improved radioimmunoassay].	7130775	Plasma samples obtained by continuous withdrawal for a 10-hour period in normal menstrual cycle were assayed for human luteinizing hormone (hLH) by both the in vitro bioassay and improved radioimmunoassay (RIA). The pulsatile patterns of both biological and immunological hLH levels were demonstrated with high frequency and low amplitude in the follicular phase and with low frequency and high amplitude in the luteal phase in the present study, as seen in the literature, although the assay procedures were different. The basal levels of immunoreactive hLH were 17.99mIU/ml with 95% confidence limits at 14.49 and 22.34mIU/ ml in the follicular phase and 10.79 (6.62 : 17.58) mIU/ml in the luteal phase. These two values were comparable to those in the literature. On the other hand, those of bioreactive hLH in the corresponding phases were 21.68 (15.21 : 30.90) mIU/ml and 6.22 (4.94 : 7.83) mIU/ml, respectively. The differences of the basal levels in these two phases were more pronouncedly assessed by the bioassay than by the RIA. The ratios of biological to immunological hLH activities (B/I ratios) at the trough (base) were over the unity (1.21 (1.03 : 1.41) ) in the follicular phase, whereas those in the luteal phase were decreased down to 0.58 (0.44 : 0.76). This depression is likely due to the poor specificity of the antiserum employed in the RIA. The half disappearance times (t1/2's) of the immunoreactive hLH were shortened significantly in the preovulatory phase(83.7(64.3 : 103.1)min)in comparison with those of the bioreactive hLH(122.9(98.2 : 147.6) min). The B/I ratios at the peaks of the pulses were depressed down in the preovulatory phase. The possible existence of hLH with immunoactivity and no, or little, bioactivity was suggested from these two evidences in this stage of menstrual cycle. The difference in the t1/2's of bioreactive hLH in the follicular and luteal phase (103.6 (94.7 : 112.5) min and 87.2 (72.5 : 101.9) min, respectively) indicated proportional differences of hLH subpopulations with various clearance rates in these two phases.
3794	Prevention of the postcoital luteinizing hormone surge by ultrashortloop feedback control.	6811332	Studies were designed to determine whether the ultrashortloop feedback (USLF) system could prevent the gonadotropin-releasing hormone (GnRH) stimulation and postcoital rise of luteinizing hormone (LH). Unanesthetized castrated female rabbits bearing chronically-implanted venous catheters were subjected to the following: (1) GnRH stimulation. Ten animals received intravenous (IV) bolus injections of 30 microgram GnRH. Two hours prior to GnRH, animals received either 2000 IU human chorionic gonadotropin (hCG) or saline. A second group of ten animals received 20 microgram GnRH. One hour prior to GnRH, an infusion of human luteinizing hormone (hLH) at 2.75 IU/min or of saline was started and continued over 4 hours. (2) Coital stimulation. Ten rabbits were studied with the use of natural coital stimulus. They received either 2000 IU hCG 2 hours before mating or saline as a control group. Both hLH infusion and hCG injection inhibited GnRH stimulation of LH secretion but did not modify the follicle-stimulating hormone (FSH) response. hCG inhibited the coitus-induced LH surge and did not modify coitus-induced FSH secretion. These studies demonstrate that USLF control of LH can modulate LH secretion under both pharmacologic and physiologic conditions.
3795	Characterization of anti oLH beta-antibodies acting as contraceptives in rhesus monkeys. I. In vitro binding properties.	6290656	Female monkeys actively immunized with oLHbeta are infertile when circulation antibody titers become sufficiently high to bind 30% of an iodinated hCG standard. We report here the extensive characterization of the oLHbeta antibodies. Their binding affinity and capacity for human and rhesus gonadotropins were determined. The affinity was high (Ka - 10(9) M-1) and was similar for high, medium and low titer antisera. In contrast, the binding capacity for hCG correlated well with antibody titers (n = 18, r = 0.888, P less than 0.001) and ranged from 0.09 to 8.3 x 10(-7)M. Column chromatographic analysis showed that anti-oLHbeta was mostly IgG. A temporal increase of affinity ('maturation') after booster was absent in the majority of monkeys. Cross-reaction between hCG and hLH as well as rhCG and rhLH was high. The latter, however, did not interfere with regular cycles and ovulation, suggesting that oLHbeta may be a useful antigen for human contraception.
3796	Stimulation in vivo of testicular 7 alpha-hydroxylase activity in immature rats by LH and hCG.	7109596	Testicular C19-steroid 7 alpha-hydroxylase activity rises during puberty in the rat. Hormonal mechanisms responsible for this rise are unknown. The present study is to the best of our knowledge the first to report successful stimulation of testicular 7 alpha-hydroxylase in immature rats. Administration of crude and purified hCG every day for 3 or more days to immature rats in doses of 3 I.U. lead to significant stimulation of testicular 7 alpha-hydroxylase activity. Purified preparations of subunits of hCG did not stimulate 7 alpha-hydroxylase activity. Stimulation of testicular 7 alpha-hydroxylase was, however, recorded when purified subunits of hCG were allowed to recombine and then administered to the animals. Several preparations of LH failed to cause stimulation of 7 alpha-hydroxylase when administered to immature rats. Treatment of immature rats with mixtures of LH and FSH, LH and prolactin or LH together with FSH and prolactin was also ineffective in this respect. Large doses of highly purified ovine-LH, rat LH and human LH were, however, able to stimulate testicular 7 alpha-hydroxylase activity in immature rats. Preparations comprising hybrids of ovine-LH and hCG (oLH alpha + hCG beta, hCG alpha + oLH beta) stimulated testicular 7 alpha-hydroxylase activity in immature rats. Administration of the same amounts of a hybrid preparation of human and ovine-LH (oLH alpha + hLH beta) to immature rats was ineffective in this respect. Administration of testosterone or estradiol to immature rats lead to suppression of testicular 7 alpha-hydroxylase activity. Combined treatment of such rats with hCG and testosterone or with estradiol and hCG augmented testicular 7 alpha-hydroxylase activity to the same degree as that of animals treated with hCG only.
3797	Immunobiological properties of a carboxy-terminal 53-amino acid peptide of the beta subunit of human chorionic gonadotropin.	6181254	Lengthening of the carboxy terminus unique region of beta-hCG from 30 to 45 amino acids was found in previous studies to improve immunogenicity and hormone neutralization capacity. The present study was carried out to determine whether further elongation of the peptide to 53 amino acids enhances to hormone neutralization capacity without loss of specificity characteristics. The peptide 93--145 of beta-hCG with substitution of cysteines at 93, 100 and 110 by alpha-aminobutyric acid was synthesized by solid phase and conjugated to tetanus toxoid by an active ester method. Rabbit antibodies against this conjugate reacted with CTP-53 and CTP-45 with a parallel slope in anti-CTP-53-[125I]Tyr-CTP--53 radioimmunoassay system. Other CTPs, e.g. CTP-26, CTP-31 and CTP-35 competed with lower efficiency; 50% inhibition of binding was obtained with 10-100 pmol/tube with these peptides instead of 0.5 pmol/tube for the homologous CTP-53. Anti-CTP-53 reacted with both beta-hCG and hCG but around twenty times greater amount of hCG was required to give 50% inhibition of binding as compared to CTP-53. The antigen binding capacity of anti-CTP-53 was around 4000 ng/ml for CTP-53 and 25 ng/ml for hCG. The anti-CTP-53 sera retained non-cross-reactivity with hLH as determined by direct binding with [125I]hLH and by competitive inhibition of CTP-53 binding with anti-CTP-53. Anti-CTP-53 neutralized the bioactivity of hCG in the Leydig cell bioassay and in the mouse uterine weight gain assay. Anti-CTP-53 antibodies were about three times more effective than anti-CTP-45 in their capacity to neutralize the bioactivity of hCG, though still substantially poorer than anti-beta-hCG sera in this respect.
3798	Comparative prolactin profiles in IUD users and non-IUD users.	7116848	Starting from the 10th day of the menstrual cycle through onset of the subsequent menstrual bleeding, daily blood samples were collected from 14 women using Copper T IUDs and 10 normal control women. The serum samples were analysed by radioimmunoassay (RIA) for human prolactin (hPRL) and human luteinizing hormone (hLH). hPRL values were subjected to statistics. Episodic patterns of hPRL were observed for all subjects. IUD users show generally lower daily levels of hPRL than non-IUD users, but the apparent differences were statistically insignificant.
3799	Biological and immunological characterization of human luteinizing hormone discharged in a pulsatile fashion in the normal menstrual cycle.	7128536	Biological and immunological measurements of human luteinizing hormone (hLH) were performed with plasma samples obtained by continuous withdrawal over a 10-hour period in various phases of the normal menstrual cycle. A pulsatile pattern of hLH discharge was demonstrated by both the in vitro bioassay and radioimmunoassay throughout the menstrual cycle, although the frequency and the amplitude of the pulse differed according to the menstrual stage. The decreased half disappearance time of immunoreactive hLH (83.7 min with 95% confidence limits at 64.3 and 103.1 min), in comparison with that of bioactive hLH (122.9 (98.2: 147.6) min), and the rapid recovery of the depressed biological to immunological activity ratio (B/I ratio) at the peak of the pulse (0.87 (0.77:0.96) in the periovulatory phase indicated the concomitant discharge of hLH subunits with hLH. The differences in the half disappearance times of bioactive hLH in the follicular and luteal phases (103.6 (94.7:112.5) min and 87.2 (72.5: 101.9) min, respectively) and in the B/I ratios at the peaks of the pulses in these two phases (1.17 (1.05:1.30) and 1.03 (0.94:1.12), respectively) suggested the selective secretion of hLH subpopulations with various biological and immunological potencies and with various clearance rates. The significantly depressed B/I ratios at the trough in the luteal phase (0.58 (0.44:0.76) were sufficient to question the overall reliability of the hLH-RIA procedure employed, especially when applied to low hLH levels, even after several methodological improvements.
3800	Measurement of human FSH by radioreceptor assay.	6290202	We established a sensitive RRA system for human FSH, employing PMS-treated immature rat ovary. The dissociation constant of the binding of the receptor preparation to NIAMDD human FSH-2 was 1.15 x 10(-10) M. The standard curve was obtained with 0.2-25.6 ng/tube of NIAMDD hFSH-2. Purified hLH, hTSH, and HCG had no significant effect on the binding. When the anterior pituitary homogenates obtained from humans were assayed by this system, the intra-assay and inter-assay coefficients of variation were 11.9% and 13.4% respectively, and the assay values correlated well with those obtained by RIA. This assay is applicable for the measurement of FSH in serum, when the non-specific inhibitor effects of serum are compensated for by the addition of merthiolate and when FSH-free serum is used instead of the buffer for the standard curve. The intra-assay and inter-assay coefficients of variation were 9.31% and 19.7% respectively. The assay values correlated with those obtained by RIA under the same physiological state. The ratio of the assay values RRA/RIA, was dependent upon the physiological state, e.g. 6.29 in men, 3.84 and 4.18 in women at follicular and luteal phase respectively and 2.40 in menopausal women. During the menstrual cycle, our results showed that the value of RRA/RIA derived from serum did not change significantly.
3801	In vitro effects of ethanol on primate luteal membranes: exposure of additional gonadotropin receptor sites.	6277608	The presence of 0.8-8.0% (vol/vol) ethanol increased the specific binding of [125I]human luteinizing hormone (hLH) to membrane particulate fractions prepared from the rhesus monkey corpus luteum in a dose-dependent manner. Ethanol increased the number of available gonadotropin binding sites without altering their affinity for LH. These results suggest that (1) there are additional gonadotropin receptors in the macaque corpus luteum which are not detected by conventional in vitro techniques, and (2) ethanol exposes receptors previously unavailable for hormone interaction.
3802	A simple method for the early detection of pregnancy: (A) Comparative study with radioimmunoassay of beta-HCG: and (B) Fallacies compared to other immunological pregnancy tests.	6179714	A simple, sensitive and reliable non-radioactive method for the detection of hCG in concentrated urine for the diagnosis of early pregnancy is reported. Twenty ml of urine were sampled, filtered and concentrated by ultrafiltration with ultra-microporous membrane under reduced pressure (Immersible Molecular Separator, Millipore Co) and the hCG in the concentrate was detected by the ordinary latex agglutination inhibition method using beta-hCG antiserum to avoid cross-reaction with high levels of hLH and hMG. Concentrated and unconcentrated urine samples taken at different periods of amenorrhoea (1, 2 and 3 weeks) were also tested by two-slide pregnancy tests and one-tube pregnancy test. Blood and urine samples taken at one and two weeks amenorrhea were assayed for beta-hCG by a specific RIA. Results were compared to the new method. Retention characteristics and concentration efficacy of the ultra-microporous membrane were checked by performing recovery experiments using commercial hCG and lyophilization procedure. The new test proved to be more sensitive, specific and reliable than other nonradioactive methods in detecting low levels of urinary hCG for diagnosing pregnancy as early as one week after missed period in regularly menstruating women. In addition, it is simpler and safer than the serum RIA presently used to detect low levels of hCG.
3803	Metabolism of testosterone by human granulosa cells in culture: influence of follicle-stimulating hormone and luteinizing hormone.	6801978	Human granulosa cells were isolated from follicles (8 to 15 mm) and cultivated for 24 hours in the presence or absence of follicle-stimulating hormone (NIH-FSH-HS-1, 1 microgram/ml) and luteinizing hormone (NIAMDD-hLH-1, 1 microgram/ml). Testosterone -4-14C was added subsequently to all cultures for 4-, 6-, and 24-hour periods. Of the seven metabolites of testosterone studied, 17 beta-estradiol (E2) and estrone (E1) were the major products. In all patients, levels of E2 were three to ten times higher than those of E1. Production of E2, but not E1, was stimulated by either follicle-stimulating hormone (FSH) or luteinizing hormone (LH). The cells of the largest follicle (15 mm) showed greater response to LH than to FSH. Production of the other C19 and C18 metabolites was very low or negligible. These results further suggest that FSH regulates the aromatization of testosterone in human granulosa cells, and that LH may have the same effect on the matured follicle during the preovulatory period.
3804	Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops).	32192236	Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for 125 I-hCG, (0.9-2.5 × 109 M-1 , and 0.7-1.64 × 109 M-1 respectively, for green and rhesus monkeys) and low binding capacity (0.343-0.682 fmol/mg and 0.198-0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 109 M-1 ) and low binding capacity (1.04 fmol/mg tissue homogenate) for 125 I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.
3805	Immunocytochemical demonstration of somatotropin-like and prolactin-like activity in the brain of Calamoichthys calabaricus (Actinopterygii).	7037193	Cellular binding of anti-bSTH and anti-oPRL IgG is demonstrated in the brain and the pituitary gland of the African freshwater fish Calamoichthys calabaricus by means of the unlabeled antibody enzyme method at the light microscopic level. In the brain, somatotropin and prolactin are demonstrated in separate neurons in the preoptic area. The somatotropinergic and prolactinergic perikarya are distinct from those of the hypothalamic-hypophysial neurosecretory neurons, i.e., those stainable with aldehyde fuchsin presumed to be vasotocinergic and isotocinergic. The somatotropinergic and prolactinergic neuronal perikarya give rise to separate beaded axons which pass either ventroposteriorly into the infundibulum, terminating in the neurohypophysis, or ventro-laterally through the wall of the preoptic recess, terminating near the superficial capillary bed covering this part of the brain surface. Moreover, coarse dendrite-like processes of both kinds of immuno-reactive neurons extend towards, and end in, the third ventricle. Binding sites in the brain to antisera against hLH beta, hFSH beta, hTSH beta and anti-(1-24) ACTH IgG, all reactive in the pituitary, are not observed in the neurons confined to the preoptic area.
3806	Improved in vitro bioassay of follitropin.	6802740	The FSH-dependent aromatase activity of Sertoli cell enriched cultures of testicular cells from immature rats was utilized as a sensitive and specific in vitro bioassay for FSH activity. The conversion of 19-hydroxyandrostenedione to estradiol was used as the end point of the assay. By the introduction of a phosphodiesterase inhibitor into the culture medium, the sensitivity was improved 5-10 times, so that 0.05 mIU of FSH showed a response significantly different from the blank. Other pituitary or placental hormones, such as hLH, hTSH and hCG exhibited less than 0.8% cross-reactivity, whereas hGH, hPRL, ACTH1-24, LH-RH, and prostaglandin E2 alpha did not show any cross-reaction at the doses tested.
3807	Immunocytochemical localization of angiotensin immunoreactivity in gonadotropes and lactotropes of the rat anterior pituitary gland.	6290923	Utilizing an immunocytochemical technique wherein two different antigens can be visualized on the same tissue section, angiotensin-like immunoreactivity was found in rat adenohypophyseal cells that showed staining with antisera to hLH, hFSH or human prolactin. Angiotensin-like immunoreactivity was not found in cells that showed staining with antisera to hTSH, C-terminal ACTH or hGH. The nature and possible physiologic significance of the angiotensin-like immunoreactivity is discussed.
3808	Pituitary-ovarian function after tubal ligation.	7308505	Pituitary-ovarian function was evaluated by measurement of daily serum levels of luteinizing hormone (LH), 17 beta-estradiol, and progesterone in women with a previous history of tubal ligation. Normally menstruating women served as controls. The duration of the proliferative and luteal phase was similar for both groups. The midluteal progesterone level averages did not differ between the two groups. Preovulatory LH and 17 beta-estradiol peaks were significantly lower in the tubal ligation group; average midluteal LH and 17 beta-estradiol levels were also lower. These results reveal that pituitary-ovarian function can be altered following surgical sterilization.Menstrual disorders have been reported following tubal surgery. This study investigates luteinizing hormone (LH), 17beta-estradiol, and progesterone levels in 30 women (age range, 24-38 years; average age, 32 years), who had had elective tubal ligation for fertility control. Elective surgical sterilization was performed between 1 month and 8 years previously. The controls consisted of 15 normally menstruating women (aged 20-32 years; average age, 24), seeking to become pregnant who had not used either oral contraceptives or IUDs for at least 1 month before the study. Daily blood samples were collected starting from day 10 of the menstrual cycle until the onset of the next menstruation. Case selection and specimen collections were done in Santo Domingo, Dominican Republic. Hormone determinations by radioimmunoassays were done on a double-blind basis at the Population Council in New York. Progesterone and 17-b estradiol were determined by radioimmunoassay according to the method of Thorneycroft and Stone, while human luteinizing hormone (hLH) was measured by the double antibody technique of Vaitukaitis et.al. Average duration of the proliferative and luteal phase were 14.4 + or - 3.0 and 13.3 + or - 2.9 days, respectively, for the control group and 13.5 and 2.4 and 14.2 + or - 1.7 days for the women with tubal ligation. The total length of the cycle, 27.7 days, is the same for both groups. The midluteal progesterone level averages did not differ between the 2 groups, although when the controls were compared with tubal ligated women 30 years of age or younger, the difference was significant. Preovulatory LH and 17beta-estradiol peaks were significantly lower in the tubal ligation group, as were the average midluteal LH and 17beta-estradiol levels. These results suggest that surgical sterilization can result in subtle changes in ovarian function, even though ovulation itself is not affected. Bleeding irregularities following tubal ligation may in part be due to the fact that different surgical procedures may have different effects on ovarian blood supply. Further research should be done to compare specific effects of different sterilization procedures on ovarian function.
3809	A synthetic peptide capable of eliciting antibodies that neutralize human chorionic gonadotropin.	6171462	Antisera generated to the human chorionic gonadotropin beta-subunit (hCGbeta) have been shown not only to neutralize the biologic activity of hCG but also to cross-react with human luteinizing hormone (hLH). In an attempt to reduce such cross-reactivity, a peptide fragment analogous to the amino acid sequence of the carboxylterminal 45 residues (101-145) of the hCGbeta-subunit with alpha-aminobutyric acid substituting for cysteine at position 110 was synthesized and tested for ability to produce antibodies interacting with hCG. Antisera were generated in rabbits to a conjugate of this peptide with tetanus toxoid emulsified with Freund's complete adjuvant. Antibody titers and specificity were assessed by the double-antibody technique. The results show that the antisera to the synthetic hCGbeta fragment bound 125I-labeled hCG and did not cross-react with hLH in the radioimmunoassay system. Most importantly, the antisera effectively neutralized the biologic activity of hCG as determined by the rat uterine weight assay.
3810	Development and characterization of a monoclonal antibody which distinguishes the beta-subunit of human chorionic gonadotropin (beta hCG) in the presence of the hCG.	6169521	A monoclonal antibody specific for the beta-subunit of human chorionic gonadotropin (beta hCG) has been developed which has a cross reactivity of 0.23% with intact hCT. There was no cross reactivity with other glycoprotein pituitary hormones in the range in which they might be expected to be present in serum. The beta-subunits of hFSH, hTSH and hLH did not inhibit binding of [125I]iodo-beta hCG to the antibody at up to 250 ng/tube. The dissociation constant (Kd) with [125I]iodo-beta hCG was 3.3 x 10(-10), and the reaction had reached equilibrium within 1 h.
3811	Specific enzyme immunoassay for human chorionic gonadotrophin.	6267861	A sandwich-type enzyme immunoassay system specific for human chorionic gonadotrophin (hCG) was prepared with the antibody Fab'-beta-D-galactosidase complex and the antibody F(ab')2-immobilized silicone rubber solid phase by using a purified antibody to beta subunit of hCG (hCG beta). The assay system cross-reacted less than 4% with human luteinizing hormone (hLH) and human follicular stimulating hormone (hFSH), and proved to be highly sensitive with hCG measurable at levels as low as 0.3 mIU per assay tube. Using 50 microliter of serum sample, 6-600 mIU/ml of hCG in serum could be determined specifically with the same degree of precision as in radioimmunoassay but without sample interference with the assay. The coefficients of variation within-run and between-run were 8.6-8.9%, and 4.9-10.7%, respectively. Values obtained with the enzyme immunoassay correlated well with those of radioimmunoassay (r = 0.98, slope = 0.94, y-intercept = 10.2 mIU/ml for 75 serum samples). Results of the immunoassay of hCG levels in serial samples of serum from healthy women and patients with choriocarcinoma show that this method is useful in the clinical diagnosis of trophoblastic disease.
3812	Whither an anti-hCG approach for the control of fertility.	12263408	2 groups of investigators reported their strategies for developing an anti-hCG vaccine 5 years ago. 1 group used a C-terminal peptide of beta-hCG and the other group proposed immunochemically purified beta-subunit of hCG linked covalently to a highly purified bacterial vaccine tetanus toxoid to elicit anti-hCG response. hCG was chosen as a target for immunointerception because of its known essential role in maintaining early pregnancy. Since that time, research has furthered knowledge on the immunodominant and immunorecessive parts of the beta-subunit of the hCG. The target tissues seem to react optimally with the associated subunits. beta-hCG produces antibodies which have preferential reactions with hCG but variable crossreactions with hLH. Acute and subacute tocicity studies with the vaccine based on immunochemically purified beta-hCG linked to tetanus toxoid have shown no side effects in the recipients. The tests also showed that these vaccines have the same limitations as vaccines of other types: 1) there is a wide variability of antibody titres among individuals and those with low titres were not protected from pregnancy; and 2) there is individual variability of responsiveness to different antigens. Work on the possibility of passive immunization against h-CG continues.
3813	[Study on human pituitary gonadotropin synthesis and release during gestation by two steps LH-RF stimulation (author's transl)].	6795893	The pituitary gonadotropin response of synthesis and release to two step-stimulation at 60 minutes interval by 100 micrograms of luteinizing hormone-releasing factor was studied by radioimmunoassay of hLH-beta, hLH and hFSH in pregnant women. The immunological affinity of hLH-beta antiserum was 7.9% with hCG-beta, 3.4% with hLH-alpha and 0.41% with LER 907 as compared to 100% with hLH-beta. Serum mean levels of hLH in non pregnant women were 248.5 ng/ml serum before the first injection of LH-RF. The first injection caused an increase in the serum hLH level to 2185.0 ng/ml serum. After the second injection of the hormone, serum hLH levels increased further than the first peak, indicating the "self-stimulating" or "priming" effect of the first LH-RF on the second peak. Serum mean values of hLH-beta in normal menstrual women were 2.60 ng/ml before the first injection. The hLH-beta response to the second injection was significantly greater than to the first. The first peak of hLH-beta was 4.30 ng/ml serum and the second peak was 5.07 ng/ml serum. The FSH response to the second injection of LH-RF was not significantly peaked from that after the first injection. The value of the serum mean levels of hLH-beta during pregnancy were between 0.57 and 0.81 ng/ml and showed no significant increase through two LH-RF stimulations, suggesting that pituitary gonadotropins synthesis and release were depressed during gestation. Thus LH-RF two step stimulation was useful to clarify anterior pituitary function in both synthesis and release of gonadotropin.
3814	Progress in immunological fertility control.	12278629	A variety of contraceptive methods is required, for 1 method simply does not meet the requirements of a population that is adopting practices of fertility regulation. None of the existing methods is absolutely safe, free of all side-effects, and universally acceptable. On this basis alone, further research and alternative contraceptive methods is justified. A mode of contraception requiring only periodic administration would make a significant impact in countries faced with rampant population growth. The question that arises against this background is whether immunological methods have any special place in a program of fertility control. Attention is directed to immunization against human chorionic gonadotropin (hCG), the problem of reaction with the luteinizing hormone (LH), and bypassing LH. A major problem in a program involving immunization with hCG is that the chemical structure of this hormone is similar to the glycoprotein hormone produced by the pituitary--LH. Both these hormones are made up of 2 dissimilar subunits designated alpha beta, and held together by non-covalent bonds. The alpha subunits of hCG and LH cannot be distinguished. Of the initial 115 amino acid residues in the beta subunit of hLH, at least 80 are identical with those found in beta hCG. The carboxyl (COOH) terminal end of beta hCG has an additional 30 amino acids constituting a unique polypeptide sequence not present in hLH. All effort is currently being directed towards developing a vaccine against hCG on antigens derived from the COOH-terminal end of beta hCG.
3815	The human chorionic gonadotropin-like substance in the plasma of normal nonpregnant subjects is not modulated by the gonadotropin-releasing hormone.	6785299	To determine whether the hCG-like substance found in the plasma of normal nonpregnant subjects is secreted and regulated like a pituitary gonadotropic hormone or prohormone, we measured its concentration before and 60 min after the acute iv injection of 500 microgram Gonadotropin-releasing hormone (GnRH). The study was performed in 12 normal young men. The hCG-like substance was purified from the other plasma proteins by 2 successive diethylaminoethyl-Sephadex A-50 chromatographies and by gel filtration on Sephadex G-100. Its concentration was measured by a specific RIA, corrected for losses and for possible contamination with human LH (hLH). The median basal concentration in plasma was 19 pg/ml (mean, 41; range less than 5 to 169), and there was no correlation with corresponding hLH concentrations. After the injection of GnRH, the median, mean, and range of concentrations remained constant, whereas the mean hLH concentration increased over 8-fold. This indicates that no significant amounts of the hCG-like substance are acutely releasable from the pituitary gland and that its plasma concentrations are not modulated by GnRH in a fashion similar to hLH. It also constitutes an argument for an extrapituitary origin of the plasma hCG-like substances in normal men.
3816	Gonadotropin therapy failure secondary to human chorionic gonadotropin-induced antibodies.	7228997	A 15-yr-old male with the diagnosis of Kallmann's syndrome manifested secondary gonadal resistance during a third treatment course of hCG. A serum sample obtained 6 months after failure of the patient to respond to hCG bound [125I]hCG 60% at a 1:2 dilution of serum; 50% at 1.5; 28% at 1:50; 21% at 1:100; and less than 5% at a 1:500 dilution. Serial sampling of the patient's serum over a 22-month period demonstrated a progressive decline in binding capacity, decreasing to 32% binding of [125I]hCG at a 1:2 dilution of serum. The data demonstrated that the patient produced a low affinity, high capacity binding substance with a Ka of 5 x 10(7) liters/M and a R0 of 1.8 x 10(16) sites/g gamma-globulin. The binding substance appeared to be a gamma-globulin of the immunoglobulin G class, which bound labeled hCG and human LH (hLH) more avidly than it bound hFSH and interfered with the biological response to hCG therapy. These data further indicated that antibodies produced against hCG may bind other endogenous glycoproteins such as hLH, hFSH, and hTSH. Although binding to hTSH did not occur in this patient, the presence of a common beta-chain in the molecular structures of hLH, hFSH, hCG, and TSH makes such a potential occurrence not implausible. Despite the apparent infrequency of the development of clinically observable interference with the biological activity of hLH/hCG in hCG-treated patients, this report indicates that a potential hazard exists. The possibility should be considered in the decision to treat a patient with normal gonadotropin secretion with exogenous gonadotropins.
3817	A sex difference of the concentrations of gonadotropins, its subunits and sex steroids in cord veins.	6785991	In order to elucidate the sex difference of cord blood hormones, concentration of hCG, hCG-alpha, hCG-beta, hLH, hFSH, estradiol (E2), progesterone (P) and testosterone (T) in cord veins were assayed by radioimmunoassays. Mean concentrations of cord vein hCG, hCG-alpha and hLH were significantly higher in female than in male, but those of hFSH and T were significantly higher in male than in female. Male and female cord vein levels of hCG-beta, E2 and P were not significantly different and hCG-beta and hFSh levels in cord veins at term are low in both sexes. These lower concentrations of hLH in male may be a result of the feedback inhibitory action by the higher concentrations of T in male at term. These data suggest a sex difference in the synthesis and secretion of gonadotropins by the pituitary gland in the human foetus at term.
3818	Age-related changes in [125I]hLH specific binding to rat interstitial cells.	6259866	Enriched Leydig cell suspensions were prepared from rats ranging from 5 to 80 days of age. The cells were incubated with [125I]hLH in vitro, and the proportion of LH binding cells determined by means of autoradiography. The proportion of 3 beta-hydroxy-steroid-dehydrogenase (3 beta-HSD) positive cells was also determined. By means of Scatchard analysis and determination of grain counts of LH positive cells, an increase in LH receptors per LH binding cells with increasing age was found. LH binding cells from animals 5 and 10 days of age had few LH receptors, more than a doubling occurred between day 10 and day 20, a significant increase was seen between day 20 and 40 whereas only a slight increase in LH receptors per LH binding cell was seen thereafter. Scatchard analysis and grain count analysis of labelled cells gave essentially the same results. The grain count distribution showed cells distributed around low grain values in animals of all ages, whereas a subpopulation of cells with high grain counts appeared in maturing and mature animals. Only a small fraction of LH positive cells had detectable levels of 3 beta-HSD activity in animals 5 and 10 days of age. With increasing age the proportion of 3 beta-HSD positive cells approached that of LH binding cells, indicating that rat interstitial cells acquire LH receptors well before any 3 beta-HSD activity can be traced.
3819	Effects of oestrogen and prostaglandin F2 alpha on luteinizing hormone receptors in human corpora lutea.	6260886	The binding of 125I-labelled human LH (hLH) to the 2000 g subcellular fraction of human corpora lutea of the menstrual cycle was examined. Displacement studies demonstrated that 125I-labelled hLH was specifically bound in the 2000 g fraction of human luteal tissue. Specific binding of 125I-labelled hLH was demonstrated in all the corpora lutea examined except for two aged corpora lutea at an early proliferative phase of the cycle. The number of binding sites for hLH increased between the early to mid-luteal phase and decreased towards the late luteal phase. However, the apparent dissociation constant (Kd) in each corpus luteum did not vary throughout the menstrual cycle. In addition, the effects of treatment with diethylstilboestrol diphosphate (DES) and prostaglandin F2 alpha (PGF2 alpha) on the binding of 125I-labelled hLH to the 2000 g fraction of luteal tissue were investigated and the changes in hLH receptors were estimated by Scatchard analysis. The number of binding sites were 1.59 x 10(-14) mol/mg protein in control tissue, 0.86 x 10(-14) mol/mg protein in DES-treated luteal tissue and 2.95 x 10(-14) mol/mg protein in PGF2 alpha-treated luteal tissue. Thus, the binding sites for hLH decreased as a result of treatment with DES and increased by treatment with PGF2 alpha. In contrast, the apparent Kd in each luteal tissue revealed almost the same value (4.24 x 10(-10) mol/1) after treatment with DES or PGF2 alpha. The results of the present study suggest that oestrogen and prostaglandin might have an important role in modulating hLH receptor in human corpora lutea.
3820	Measurements of human luteinizing hormone in plasma by in vitro bioassay and by conventional and improved radioimmunoassays.	7250071	Daily plasma samples obtained from normally menstruating women were measured for human luteinizing hormone (hLH) by in vitro bioassay and by conventional and improved radioimmunoassays (RIA). The improvement of hLH RIA consisted in the choice of standard and the extensive purification of tracer. The profiles of the biological and immunological activity of hLH throughout the cycle showed a qualitative similarity with the mid-cycle hLH surge. The estimated values obtained with the conventional RIA were consistently and significantly higher than those by the improved RIA (p less than 0.001). The elevation of the Y-intercept on RIA (5.22 +/- 3.22 uIU/ml) indicated the possible influences of crossreacting substances on the estimates obtained with the conventional RIA. The Y-intercept values calculated from the bioassay and the improved RIA estimates were indistinguishable from the origin (0.45 +/- 1.81 mIU/ml). The inter-individual variations of the biological/immunological activity ratios indicate the different proportions of multiple sub-populations of hLH molecules with various biological and immunological activities in each case. The improved RIA was concluded to be more preferable for detecting hLH levels in biological fluid than the conventional RIA. The need for more suitable standard preparations for hLH RIA was proposed.
3821	A rapid radioimmunoassay method for serum luteinizing hormone utilizing polyethylene glycol and a double-antibody method of separation.	7461152	A method is described utilizing polyethylene glycol (PEG) combined with a double antibody for separation for rapid radioimmunoassay (RIA) of human luteinizing hormone(hLH). The total assay time is 3 hours with a 1-hour incubation period. Reliable comparisons with the 3-day assay have been shown. The average between-assay coefficient of variation was 11%, whereas the average within-assay coefficient of variation was 6%. The sensitivity of the assay was 7.5 mIU/ml. It is suggested that this reliable and rapid RIA for hLH will prove to be a valuable adjunct in the treatment of patients when timing of ovulation is imperative, such as for artificial insemination and harvesting of maturing human oocytes.
3822	Physical, biological, and immunological characterization of highly purified urinary human chorionic gonadotropin components separated by gel electrofocusing.	7460824	A highly purified urinary hCG preparation was subjected to electrofocusing on polyacrylamide gel. It was shown to fractionate into at least 11 components that can be detected by coomassie blue staining, a radioligand-receptor assay (RRA), and four hCG RIAs using antisera against the purified hCG, its alpha- and beta-subunits, and the hCG beta carboxyl-terminal peptide. Refocusing shows that these components are not artifacts of the electrofocusing technique. Six of the major components were isolated, and their pIs were shown to correlate with their sialic acid content. When these components were assayed by radioligand-radioreceptor assay, the four hCG RIAs, and a rat uterine weight assay, they showed 1) similar in vitro biological activities, 2) identical immunological activities that were indistinguishable from the in vitro biological activities, 3) hCG-like and not hLH-like immunological characteristics, and 4) different in vivo biological activities which increased with sialic acid content. Thus, sialic acid alone and/or differences in subunit assembly seem to be responsible for the electrophoretic heterogeneity of highly purified hCG.
3823	[Bovine epiphyseal lactotropin, biologically and immunochemically related to pituitary prolactin].	7232350	A substance biologically and immunologically related to the hypophysis lactogenic hormone (HLH) was detected in a purified preparation obtained by extracting the bovine pineal glands (epiphyses) with acidified acetonic saline fractionation, precipitation in isoelectric point and gel filtration. Bioactivity of the pineal gland lactogen was evaluated according to its ability for stimulating the pigeon thyroid gland proliferation. Equal doses of the pineal gland hormone and HLH produced the same biological effect. The lactogen immunochemical properties were studied by gel precipitation in agar gel, immunoelectrophoresis and radioimmunoconcurrent technique. Absolute identity of the hormone immunochemical properties was revealed. Molecular weights of both lactogens, determined by chromatography on Sefadex C-75, were equal. The average HLH content in the organ, determined by radioimmunochemistry, was 4.7 X 10(5) micrograms for the hypophysis and 9.6 X 10(2) micrograms for the pineal gland. HLH content, calculated per 1 mg protein, was 3 X 10(3) micrograms for the hypophysis and 24 micrograms for the pineal gland. The hormone content in the epiphysis was 1000 times higher than HLH concentration in the blood serum.
3824	Increase of ionic strength for charcoal separation of B/F fractions in radioimmunoassays.	6765010	The increase in protein adsorption by charcoal as ionic strength increases (salting-out adsorption), was used to separate the bound and free fractions of glucagon, insulin, hGH, hLH and hPRL in the radioimmunoassay. The hormones were labelled with 125I and to express the immunocomplex, gamma-globulin was labelled with 125I. The charcoal used to produce the separation was suspended in magnesium sulfate 3 M (charcoal-SO4Mg). The optimum amount of charcoal and the final concentration of magnesium sulfate determined for each hormone were: glucagon (charcoal 5 mg/tube, 0.125 M); insulin (charcoal 5 mg/tube, 0.131 M); hGH (charcoal 40 mg/tube, 0. 447 M); hLH (charcoal 40 mg/tube, 0.447 M) and hPRL (charcoal 60 mg/tube, 0.321 M). The serum concentration was 1/20 for all hormones, excepting glucagon, where 1/10 was used. The stability of the immunocomplex was studied and it was shown that, under suitable conditions, increased ionic strength does not cause the dissociation of the bound fraction.
3825	Acute steroidogenic responsiveness to human luteinizing hormone in hypogonadotropic hypogonadism.	7440695	The responses of circulating levels of androgens, estrogens, and their C-21 biosynthetic precursors to a 6-h constant infusion of human Lh (hLH; 2000 IU) were studied in four males with hypogonadotropic hypogonadism (HH) and compared with those in normal male controls. Although similar levels of circulating LH were achieved, the initial and secondary increases in testosterone were significantly greater in the hypogonadotropic subjects than in the normal controls. In contrast, the responses of estradiol, estrone, 17 alpha-hydroxyprogesterone, and 17 alpha-hydroxypregnenolone to exogenous hLH were significantly lower in HH than in normal controls. The data demonstrate a different pattern of testicular steroidogenic responsiveness after pharmacological doses of hLH, with increased concentrations of circulating testosterone in subjects with HH compared to a disproportionate increase in estrogen and progestin levels in normal men.
3826	Immunological properties of the beta-subunit of human chorionic gonadotropin. I. Effect of chemical and enzymatic modifications.	6159200	The beta-subunit of hCG (hCG beta) displays immunological cross-reactivity with human LH (hLH). A detailed study was undertaken to investigate the effect of chemical and enzymatic modifications on the immunological behavior of hCG beta, particularly on its cross-reactivity with hLH, with a view to obtaining a highly hCG-specific antigen. hCG beta was modified in both the carbohydrate and protein parts of the molecule. The carbohydrate part was modified enzymatically by sequential cleavage of monosaccharides by specific glycosidases, and the protein portion was modified chemically in the amino and carboxyl groups and in the cystinyl, tyrosyl, histidyl, and arginyl residues. These derivatives were immunologically evaluated by RIAs; their hCG beta activities were measured in the [125I]hCG beta-anti-hCG beta system, and the hLH cross-reactivity in the [125I]hLH-anti-hLH system. The present studies have led to the following conclusions. 1) The carbohydrate does not play a significant role in the immunological activity of hCG beta. 2) The antigenic determinants of hCG beta reside primarily in the polypeptide chain and are conformational rather than sequential in nature. 3) hCG beta has two types of antigenic determinants, those which are unique to hCG and those which are common to both hCG and hLH. 4) Finally, it is possible to destroy preferentially one or the other type of determinants. The controlled reduction and alkylation of hCG beta yielded derivatives which retain significant immunological activity in the hCG beta system, but have no or reduced cross-reactivity in the hLH system. These derivatives are much more specific antigens than hCG beta and, therefore, are of potential importance for the development of a specific RIA for hCG and possibly for use as contraceptive agents.
3827	[Examination of HFSH "Eiken" and HLH "Eiken" kits (author's transl)].	6780249	NaN
3828	Interaction of human chorionic gonadotropin and human luteinizing hormone with human thyroid membranes.	6245856	In an attempt to identify a possible pathogenetic role for the hCG molecule in the mechanism of the hyperthyroidism which occurs in choriocarcinoma, we have looked for evidence that the hCG molecule has a thyrotropic action on the human thyroid. The thyrotropic activity of various hCG preparations on the human thyroid was assessed by measuring the stimulation of adenylate cyclase activity in human thyroid plasma membranes purified by sucrose density gradient centrifugation. The highly purified hCG CR119 preparation stimulated human thyroid adenylate cyclase activity. Its activity was more than 654 times greater than could be accounted for by human TSH (hTSH) contamination of the preparation, as determined by RIA. The thyrotropic activity intrinsic to 1.0 IU hCG was equivalent to roughly 0.27 microU hTSH. Significant saturable binding of the 125I-labeled highly purified hCG preparation to human thyroid membranes was demonstrated, and the bound component was characterized. Its apparent molecular size, subunit composition, and testis receptor-binding characteristics were those of the hCG molecule. Examination of a crude urinary hCG preparation in adenylate cyclase and TSH radioligand assays using human thyroid membranes showed no evidence of any molecule other than hCG with a thyrotropic action on the human thyroid. Given that hCG binds to and stimulates adenylate cyclase activity in human thyroid tissue, as the above data indicate, then human LH (hLH) would be expected to do the same, since hLH and hCG have such strong structural and functional similarities. As anticipated, a highly purified hLH preparation exhibited TSH binding inhibition and adenylate cyclase stimulation. Its activity was more than 1030 times greater than could be accounted for by hTSH contamination of the preparation. The thyrotropic activity intrinsic to 1.0 IU hLH was equivalent to roughly 44 microU hTSH. Thus, in addition to other shared properties, the hLH molecule and the hCG molecule share the ability to interact with human thyroid tissue. These results strongly indicate that the hCG molecule has a thyrotropic action on the human thyroid and support the hypothesis that hCG is the thyrotropic factor that mediates the hyperthyroidism which occurs in patients with hCG-secreting neoplasms.
3829	Development and validation of a choriogonadotropin beta-subunit radioimmunoassay for serum choriogonadotropin using commercially available components.	6156028	A sensitive, specific, accurate and precise radioimmunoassay procedure developed for the beta-subunit of serum choriogonadotropin (hCGbeta) is described. 1. The assay employs an anti-hCGbeta (rabbit) serum generated against hCGbeta, highly purified intact choriogonadotropin (hCG) as standard, and [125I]hCG as the radioactive ligand. The antibody-bound hCG was separated from the free hormone by the addition of goat anti-rabbit gamma-globulin. 2. The detection limit of the assay was approximately 75 microIU hCG which corresponds to a serum concentration of approximately 0.75 mIU/mL. 3. Cross reactivity studies performed with human luteinizing hormone (hLH) and human thyroid-stimulating hormone (hTSH) indicated minimal interferences from these structurally similar glycoproteins. Parallel dose-response curves were demonstrated between dilutions of sera with elevated hCG concentrations and the standard reference preparation. A non-specific binding of less than 2.5% of the total [125I]hCG was routinely observed. 4. The analytical recovery of hCG added to human sera varied from 94 to 110%, with a mean recovery of 101%. 5. The inter-assay variation was determined by assaying (n=30) 3 different quality control pools. The following data were obtained: X1=4.6 +/- 0.5 mIU/mL (CV=10.9%); X2=8.1 +/- 0.9 mIU/mL (CV=11.1%); and X3=36.8 +/- 2.8 mIU/mL (CV=7.6%). 6. The clinical data collected from subjects with trophoblastic disease agreed with previously published studies. All of the reagents are available commercially.
3830	Protein A as an immunoadsorbent in radioimmunoassay: usefulness in a rapid assay for urinary luteinizing hormone.	7353692	A staphylococcal cell wall protein, designated protein A, is known specifically and rapidly to bind to immunoglobulin G. We have utilized this protein as an immunoadsorbent to separate antigen-antibody complexes from free antigen in a rapid 6-hour radioimmunoassay for urinary human luteinizing hormone (hLH). We have further documented the efficacy of this system and the optimal conditions for this assay. The ability of this assay to provide rapid and reliable measurements of hLH in timed urinary specimens provides a useful tool for determination of the preovulatory LH surge and prediction of the time of ovulation in women.
3831	[Radioimmunologic technics for luteinizing hormone determination in cattle and sheep].	7447597	The antigen relationship which exists between luteinising hormone of various animal species, on the one hand, and human luteinising hormone as well as human chorionic gonadotrophin, on the other, was the background against which possible approaches were tested to the preparation of a heterologous in-vitro test for serum LH determination in cattle and sheep. The following conclusions may be drawn from the results so far obtained: --A homologous or heterologous test with specific relevance to the determination of human LH, on the basis of hLH-anti-hLH or hLH-anti-hCG is not suitable for the determination of LH in animal serum (cattle, sheep). --Quantitative and specific measurement of LH concentrations in the blood of cattle (sheep) is possible by means of an ovine-LH-anti-ovine/bovine-LH (or anti-bovine-LH) test system which is based on cross-reactivity between luteinising hormones of cattle and sheep. --Differentiated measurement of various LH concentrations in animal serum will be possible under certain specific assay conditions, with ovine LH as tracer, ovine or bovine LH as standard, and hCG antiserum. Yet, the clinical value of that heterologous radio-immuno-assay method is attributable primarily to its potential to measure peak values within the cycle to determine the time of ovulation rather than to the built-in possibility to determine base values of the cycle.
3832	Alpha and beta subunit immunoreactivity of reference preparations used in the radioimmunoassay of follicle stimulating and luteinizing hormones in serum.	6785315	The 2nd IRP-HMG, the 1st IS, and LER 907 have been widely used in standard FSH and LH RIA systems to measure circulating levels of FSH and LH in human serum. In these systems, the dose-response curves elicited by the preparations were parallel to each other and to that of serum samples tested. When these materials were studied in the homologous alpha and beta-hLH RIA systems, the three hormones and a serum sample used as internal control in all assays, displayed a higher degree of contamination with alpha than with beta-hLH subunits. On weight basis, the alpha/beta ratios were 18.0, 18.7, 6.7, and 13.0, for the 2nd IRP-HMG, 1st IS, LER 907 and serum, respectively. In the homologous alpha and beta hfSH RIA's, the highest contamination with alpha-hfSH was displayed by (alpha/beta ratio:10), the alpha/beta ratios for the 2nd IRP-HMG and 1st IS were 2.2 and 1.1, respectively. The serum sample contained about 6.2 ng equivalent of alpha-hFSH/ml; beta-hFSH was not detected.
3833	Prolactin binding in rat Langerhans islets.	6271957	Membrane preparations of collagenase-dispersed Langerhans islets of female Wistar rats exhibit specific binding sites for 125I-labelled ovine prolactin (125I-oPrl). Almost negligible binding was detected in islets of male animals. The binding is a saturable and time-temperature dependent process, equilibrium being reached after 16 h incubation at 0 degrees C. The bound oPrl is not displaceable by hFSH, hLH, bGH or hGH. In contrast with other cell fractions, the 12,000 g pellet accounts for more than 80% of the specific binding of 125I-oPrl. Scatchard plots of data obtained in saturation studies indicate a single class of binding sites with Ka = 0.21 x 10(10)M-1. Protein and phospholipid moieties are essential for the receptor activity, since after trypsin or phospholipase C digestions marked loss of binding was verified. In islets of streptozotocin diabetic rats a marked reduction in the number of binding sites was observed. These findings may suggest that some of the actions of prolactin on endocrine pancreas could be explained by its specific interaction with islet cell membranes.
3834	Binding of HLH and HCG by human corpus luteum: an in vitro study.	6114053	The ability of human luteal tissue to bind HLH was studied. The tissue was obtained from patients subjected to gynecological operations intended to correct other than ovarian lesions. HLH iodinated with carrier-free 125I according to the Greenwood method was used. This material has a relatively low specific radioactivity (7-20 microCi/micrograms); therefore the biological characteristics of the hormone were expected to remain intact. The 90% binding point was estimated at 37 degrees C after 5 minutes. Inhibition experiments with HLH and HCG had a characteristic effect, even those with FSH and ovine prolactin indicated a significant interference with the bound hormone. Scatchard plots demonstrated large variability in the number of binding sites and relatively modest variability of constant (Kd).
3835	Biologically active luteinizing hormone (LH) in plasma. V. A re-analysis of the differences in the ratio of biological to immunological LH activities during the menstrual cycle.	575264	The relationship between the biological and immunological activities of human luteinizing hormone (hLH) in plasma collected from female subjects was examined. The biological activity was measured by an in vitro bioassay and the immunological activity by an hLH radioimmunoassay (RIA), using improved reagents, such as the 1st IRP for human pituitary LH for immunoassay (code No. 68/40) as standard, a subunit-free biologically active iodinated hLH preparation as tracer and an anti-hLH serum of relatively high specificity. Similar profiles of biological (B) and immunological (I) activity were obtained in the plasma samples collected daily throughout 40 menstrual cycles (5 cycles from each of 8 subjects). However, the B/I ratios were significantly lower during the period of LH surge (P less than 0.001) than throughout the remainder of the cycle. The within- and between-assay variation in B/I ratios was investigated by the simultaneous assay of biological and immunological activities in plasma pools obtained by combining equal aliquots of plasma from each daily sample of the menstrual cycle from each of 5 cycles of each of 4 subjects. The analysis of these 20 pools revealed highly significant individual differences in B/I ratios, ranging from 0.81 to 1.33. The coefficient of variation was 20% between-subjects and 5% within-subjects. There was no seasonal variation in B/I ratios. That the individual differences in plasma B/I ratios were not attributable to the procedure of pooling was ascertained by the simultaneous assay of both activities in parellel in daily plasma samples and in the pools formed from these samples from three complete cycles. Thus the analysis of the differences in B/I ratios obtained throughout the menstrua- cycle revealed three major sources of variation. The first occurs in the form of generally elevated (higher than unity) B/I ratios, the second consists of a significant drop in B/I ratios during the midcycle LH surge, and the third source is represented by the significant between-subject differences. It is concluded that the first source is attributable to the relatively higher levels of "impurity" (i.e. biologically inactive, immunologically active material) in the standard preparation compared to those present in plasma of biologically inactive, immunologically active material of unknown composition and origin. If so, the latter source limits the quantitative significance of the RIA procedures employed. It is suggested that these three sources of variation account for most of the differences in B/I ratios for plasma lLH reported in the literature.
3836	Immunogenicity of three C-terminal synthetic peptides of the beta subunit of human chorionic gonadotropin and properties of the antibodies raised against 45-amino acid C-terminal peptide.	551178	Immunological studies were carried out in rhesus monkeys and rabbits on three C-terminal synthetic peptides of beta-hCG (115-145; 111-145 and 101-145) after conjugating these to tetanus toxoid (TT). The immunogenicity of the peptide conjugates was comparatively poorer with reference to Pr-beta-hCG-TT conjugates at similar doses and immunization schedule. Amongst the three peptides, the best response was obtained with the 45-amino acid c-terminal peptide (45-CTP; 101-145). The anti-45-CTP recognized native hCG and was devoid of cross-reaction with hLH. hCG-induced testosterone production by mouse Leydig cells was inhibited by anti-45-CTP antiserum, although its neutralization capacity decreased more rapidly upon dilution than anti-beta-hCG sera of comparable titres. Immune complexes formed by the anti-45-CTP with hCG had a lower sedimentation value than those formed by anti-beta-hCG antisera with hCG, suggesting the presence of a limited number of immuno-determinant regions in the 45-amino acid C-terminal synthetic peptide.
3837	Pituitary gland: site of short-loop feedback for luteinizing hormone in the rabbit.	479369	The development of a species specific radioimmunoassay for rabbit luteinizing hormone (LH) has permitted the direct demonstration of LH feedback control of LH secretion (short-loop feedback control). In previous studies we showed that small bolus injections of human LH (hLH) intravenously administered to castrate female rabbits suppressed rabbit LH for 20-30 min. Human LH had no effect on rabbit follicle-stimulating hormone secretion. This control system was responsive to amounts of hLH estimated to be present in blood of eugonadal men and women. These studies were designed to determine whether this feedback control was exerted at a pituitary or hypothalamic level. Two groups of studies were carried out: (a) in vivo studies: Rabbit LH was quantified in the blood of castrated female New Zealand White rabbits receiving either constant hLH perfusion (2.75 IU/min) or saline perfusion, plus a bolus injection of 0.5, 6, or 20 mug of gonadotropin-releasing hormone (GnRH). Human LH decreased the response to 6 and 20 mug of GnRH by 31 and 36%, respectively, and abolished the response to 0.5 mug, GnRH. (b) in vitro studies: Rabbit pituitary slices were incubated in the presence of medium alone, medium plus hLH (25 mIU/ml), medium plus GnRH (20 mug/ml), and medium plus both GnRH and hLH. hLH decreased basal rabbit LH release into the medium and abolished GnRH-stimulated rabbit LH release. hLH had no effect on rabbit follicle-stimulating hormone release. From these results we conclude that a direct and specific feedback control of LH on LH exists at a pituitary level.
3838	Serum prolactin in male infertility.	496036	Hyperprolactinemia was found only in two out of 71 patients with male infertility and in none of 53 patients with impotentia coeundi indicating that hyperprolactinemia is of minor significance in these patients. There was only a weak negative correlation between hPRL and testosterone in oligozoospermic men. No correlation was found between hPRL and hLH, hFSH, sperm count and sperm motility. Nevertheless measurement of hPRL should be performed in all cases with hypogonadotropic hypogonadism, loss of libido or impotence since these symptoms may be signs of a prolactin-producing pituitary adenoma.
3839	Immunoreactive alpha and beta subunits of follicle stimulating and luteinizing hormones in peripheral blood throughout the menstrual cycle and following stimulation with synthetic gonadotropin releasing hormone (GnRH).	393752	The concentration of immunoreactive alpha and beta subunits of human LH (hLH) and human FSH (hFSH) were measured in unfractionated serum throughout normal menstrual cycles, and when GnRH was administered at about midcycle. The RIA subunit systems employed were sufficiently sensitive and specific to measure basal levels of free alpha-hLH, beta-hLH, and alpha-hFSH. The beta-hFSH system lacked sufficient sensitivity to measure beta-hFSH in unfractionated serum. In normal cycles, alpha-hLH, beta-hLH and alpha-hFSH were shown to circulate in the free form with highest concentrations occurring at the time of midcycle peak of LH and FSH. The concentration of subunits during the follicular phase were not significantly different from those observed during the luteal phase of the cycle. In response to GnRH administration during the periovulatory phase of the cycle, the increases in circulating free alpha-hLH, beta-hLH, and of alpha-hFSH subunits were directly related to the increases in LH and FSH. Other investigators have shown that increases in circulating levels of free subunits are due to secretion from the pituitary gland rather than from peripheral dissociation of intact hormone. Therefore, it can be assumed that the increase in immunoreactive alpha-hLH, beta-hLH, and of alpha-hFSH subunits observed in this study at midcycle represent release of free subunits in response to endogenous or exogenous GnRH administration.
3840	Evaluation of beta-subunit chorionic gonadotropin as an aid in diagnosis of trophoblastic disease.	225983	The advent of the radioimmunoassay of Beta subunit chorionic gonadotropin (BhCG) has eliminated a major problem in following the patient with gestational trophoblastic neoplasia (GTN). Whole hCG cross reacts with luteinizing hormone (hLH) causing difficulties when GTN patient titers fall to levels coincidental with pituitary levels of hLH. The use of the Beta subunit of hCG in radioimmunoassay has eliminated this problem because there is essentially no cross reaction between the B-subunit of the hCG and hLH. A review of the use of this sensitive assay in the management of patients with GTN is presented.
3841	Characterization of specific antisera to native human luteinizing hormone.	463449	Antisera against highly purified native hLH (10 000 IU/mg = 0.5 mg hLH/mg protein) were raised in 4 rabbits. The antisera were invariably of high titre and high avidity. They were purified by affinity chromatography on a column with hCG-Sepharose 4 B. Before and after adsorption the antisera were tested in a double antibody radioimmunoassay. Before adsorption hCG cross-reacted in all antisera, but in none were the inhibition curves identical. No such cross-reaction remained after the affinity chromatography. These purified antisera made it possible to develop highly specific and sensitive assays for measuring native hLH. We conclude from studies of the effect of native glycoprotein hormones and their subunits that there are probably differences in the antigenic sites of the complete hLH molecule and in the isolated beta-subunit of hLH. The investigation also indicates that even highly purified preparations of hTSH may be contaminated with hLH and that the preparations of hLH-subunits may contain a certain amount of the native hormone.
3842	A sensitive and specific in vitro bioassay method for the measurement of follicle-stimulating hormone activity.	463448	An in vitro bioassay method for hFSH is presented. The method is based on the principles previously described by Dorrington et al. (1976b) and involves the assay of oestradiol produced from 19-hydroxyandrostenedione by dispersed Sertoli cells of 10-day old rats when cultured in the presence of graded doses of FSH. Using the 1st International Reference Preparation for human pituitary gonadotrophins (FSH and LH/ICSH) for bioassay (code no. 69/104) as standard, the useful range of the method is from 0.5 to 32 mIU/chamber (2 to 128 mIU/ml). The sensitivity of the method is 0.5 mIU/chamger. The mean index of precision (lambda) obtained from 16 multiple assays over 2 or 3 dose levels was 0.084. Parallelism was obtained between the 69/104 preparation and all preparations under study. The practicability of the proposed assay method is such that 15 preparations at 3 dose levels can be assayed by one person in 3 days. The specificity of the assay was investigated by determining the FSH activity in the following preparations: hFSH alpha- and beta-subunits, hLH, hCG, hTSH, ACTH, human growth hormone (hGH) human prolactin (hPRL) and luteinizing hormone-releasing hormone (LH-RH). The ACTH, hGH, hPRL and LH-RH preparations studied showed no detectable FSH activity in the assay. In the remaining preparations very low levels of FSH activity were found, corresponding to 0.004 to 0.6% of the weight of these preparations when compared with a highly purified hFSH preparation, suggesting that the method is specific for FSH. The possible synergistic or antagonistic influence of the above preparations when assayed in the presence of the 69/104 preparation was also assessed. No evidence of a synergistic or antogonistic effect was found. The assay of the hFSH potencies of a limited number of hFSH preparations of varying purity by the proposed in vitro bioassay, an hFSH radioreceptor method and an hFSH specific radioimmunoassay technique revealed that - although the relationship of the various potencies obtained with each method showed a close agreement - the bioassays yielded the highest potency estimates, and the radioimmunoassays the lowest ones. Since the proposed bioassay method is sensitive and considered to be specific for hFSH activity, it provides a suitable basis for the assessment of the specificity of other in vitro methods (radioreceptor and radioimmunoassay) currently used for detecting low levels of FSH activity.
3843	Studies on the stability of rat testis membrane receptors for hLH, hCG and hFSH.	226576	Receptor preparations from rat testes were stored at 4 C with various protease inhibitors and chemicals, at -20 C and -80 C, in freeze-dried form as well as at 4 C and 37 C after reconstitution in various media. Each preparation was tested for stability in terms of binding capacity of human luteinizing hormone (hLH), human chorionic gonadotropin (hCG) and human follicle-stimulating hormone (hFSH). The freeze-dried samples were stable for at least six months, although once reconstituted and stored at 4 C, they were less stable than fresh ones. Furthermore, Na azide and Na azide with diisopropylfluoro-phosphate (DIFP) prolonged the stability of the fresh samples. The samples stored at -20 C or -80 C were stable for at least 90 days and repeated freezing and thawing (15 times) did not change the binding capacity of either the hCG/hLH or hFSH receptors.
3844	Differential properties of human chorionic gonadotrophin and human luteinizing hormone binding to plasma membranes of bovine corpora lutea.	34964	Plasma membranes of bovine corpora lutea contain common receptor sites for [125I]human chorionic gonadotrophin (hCG) and [125I]human luteinizing hormone (hLH) to which hLH binds with 4-fold lower affinity than hCG. The presence of additional sites for hLH was indicated by the lack of saturation of [125I]hLH binding as compared to [125]hCG and lower degree of inhibition of binding by 830 pM of unlabelled hCG, when [125I]hLH instead of [125I]hCG was used. Differences in [125I]hCG and [125I]hLH binding were observed by exposing receptors to increasing temperatures and pHs and by pre-treating membranes with dimethyl sulphoxide, Triton X-100, various enzymes and protein reagents. The above differences can only be reconciled by differential responses of common hCGhLH sites and additional sites for hLH.
3845	A human chorionic gonadotropin-specific antiserum against synthetic peptide analogs to the carboxyl-terminal peptide of its beta-subunit.	446368	A chemically synthesized Na-acetyl-triacontapeptide analogous to the carboxyl-terminal region (residues 116--145) of hCG beta-subunit was conjugated to bovine thyroglobulin. This synthetic antigen was used to immunize five rabbits; one of these rabbits (H-114) generated a useable antiserum. The binding properties of this antiserum were compared extensively to a well characterized antiserum (H-93) against the natural tricosaglycopeptide (residues 123--145) of desialylated hCG beta-subunit conjugated to bovine serum albumin. The two antisera showed nearly identical immunological specificities and sensitivities when examined in a conventional RIA system. These similarities included: 1) antibody recognition sites residing at the carboxyl-terminal pentadecapeptide region; 2) binding neither [125I]iodo-hLH nor [125I]iodo-oLH; and 3) lack of neutralizing capability against the biological activity of hCG in vivo. However, synthetic pentadeca or longer peptides are twice as potent in inhibiting 125I]iodo-hCG binding to H-114 than to H-93. Synthetic peptides may provide a superior means of generating antisera capable of distinguishing hCG from human LH, since they can be obtained more easily in large quantities than analogous purified natural fragments and because peptides of synthetic origin avoid the risk of contamination from strongly antigenic regions of the native molecules. Such hCG-specific antisera may have extensive application in RIA systems and fertility control, where selective binding to hCG but not human LH is desired.
3846	Specificity of short-loop feedback of luteinizing hormone in the rabbit.	471197	The specificity of the gonadotropin short-loop feedback mechanism was investigated in castrated rabbits by blood sampling from an indwelling jugular venous catheter for follicle-stimulating hormone (FSH) after the intravenous administration of purified human luteinizing hormone (hLH). Although we have previously shown that such hLH administration results in a decrease of rabbit LH, no decrease in rabbit FSH was seen in these experiments. This specificity of LH feedback is compatible with the site of feedback being at the (1) pituitary level or (2) at the hypothalamic level provided two separate LH and FSH releasing factors exist.
3847	Effects of cimetidine on the secretion of some pituitary hormones.	118470	Cimetidine infused intravenously into 6 healthy volunteers did not induce significant changes in plasma levels of prolactin (hPRL), thyreotropin (hTSH), gonadotropin (hLH and hFSH) and growth hormone (hGH). Conversely it caused a significant increase in prolactin response to TRH without modifying the hTSH response to thyreotropin-releasing hormone (TRH) and gonadotropin response to gonadotropin-releasing hormone (GnRH). Data obtained in these experiments, together with data reported in the literature, suggest that the amplified prolactin response to TRH observed during cimetidine infusion, is likely to be connected with a blockade of H2-receptors rather than to unspecific actions of cimetidine.
3848	Spontaneous dissociation of human pituitary luteinizing hormone in solution.	748045	Aliquots of a solution of highly purified human pituitary LH (hLH) were incubated with variations in temperature and time. The incubates were chromatographed on Sephadex G-100 as well as on DEAE-Sephadex A25. The column effluents were assayed in radioligand assay and in specific hLH alpha- and hLH beta-RIA systems. The results indicate that there is spontaneous dissociation of hLH at elevated temperatures under otherwise "normal" conditions concerning, for example, pH and ionic strength. The degree of dissociation is both time and temperature dependent. This dissociation is not due to proteolytic enzymes. It is concluded that one should be alert when using hLH subunit RIA systems at elevated temperatures for measuring hLH levels, e.g. in clinical samples, because artificial high levels may be obtained.
3849	Studies on a human chorionic gonadotrophin-like material present in non-pregnant subjects.	707007	The presence of an hCG-like material in urinary and pituitary extracts and plasma obtained from non-pregnant subjects was investigated. Two assay methods were used to detect this material following fractionation of pituitary and urinary extracts by gel filtration (Ultrogel AcA 54) and/or isoelectrofocusing: a) a radioimmunoassay employing an antiserum raised against a specific sequence of the carboxy terminal region (residues 115--145) of the beta-subunit of hCG, and b) an in vitro bioassay method which measures both hLH and hCG activities. The fractionation procedures employed provide a satisfactory separation of highly purified hCG and hLH preparations. In the pituitary and urinary extracts hCG beta-peptide-like immunoactive (PIA) material was found consistently, which co-eluted with iodinated hCG following gel filtration and possessed pI values similar to those of hCG when subjected to isoelectrofocusing. The PIA material also exhibited in vitro biological activity similar to that shown by hLH and hCG. Detectable levels of immunoactive material were also found in plasma; however, the plasma levels of this PIA material were not influenced by classical endocrine measures such as the stimulation or inhibition of gonadotrophin secretion. The low levels of this material in plasma precluded its further characterization by gel filtration or electrofocusing. Whereas the present data and those reported by other investigators seem to suggest the presence of some hCG-like material in urinary and pituitary extracts and possibly in plasma of non-pregnant subjects, it is emphasized that the available evidence is not sufficiently conclusive to exclude other interpretations as to the nature of this material.
3850	Influence of the purity of the iodinated tracer on the specificity of the radioimmunoassay of human luteinizing hormone.	360753	Human luteinizing hormone (HLH) iodinated with 125I by the use of a lactoperoxidase method, was fractionated by either cellulose adsorption, gel filtration or by the combination of these methods. The products of iodination were characterized by their in vitro biological LH activity and by their binding profiles with antisera to HLH, HLHalpha and HLHbeta subunits. Several radioactive components were obtained after gel filtration with or without an initial cellulose adsorption step. One of these fractions was identified as biologically active HLH and another as the HLHalpha subunit. Radioimmunoassay studies were conducted with different iodinated fractions as tracers, using three well defined and widely available antisera to HLH. The standard used was the HLH International Reference Preparation for immunoassay (68/40). Cross-reactivity was examined with several purified pituitary preparations, such as HFSH, HTSH, HLHalpha and HLHbeta subunit. A significantly higher cross-reactivity with HLHalpha, HFSH and HTSH was obtained with the [125I]HLHalpha fraction as tracer than with biologically active [125I]HLH. Furthermore, in the radioimmunoassay of HLH preparations of varying purity, significantly higher estimates of immunological activity were obtained with the [125I]HLHalpha tracer than with the biologically active [125I]HLH. It is concluded that the presence of [125I]HLHalpha in the [125I]HLH tracer can result in serious overestimates of the immunological activity in the measurement of LH. Therefore [125I]HLHalpha should be separated from [125I]HLH prior to radioimmunoassay. Many of the fractionation methods commonly used (cellulose adsorption and short column gel filtration systems) are inadequate for this purpose. However, an adequate separation can be achieved by the use of high resolution gel filtration systems.
3851	Short-loop feedback of luteinizing hormone: dose-response relationships and specificity.	710618	The short-loop feedback control of rabbit luteinizing hormone (rLH) was studied by using a highly specific radioimmunoassay (RIA) system for rLH which does not react with human LH. Permanent intravenous catheters were placed in adult female New Zealand White rabbits at the time of castration. Highly purified human luteinizing hormone (hLH) was injected intravenously at doses of 0.1, 0.3, 0.5, 1, 2, 3, 4, 10, 50, and 100 IU into unanesthetized animals 1 to 16 days following castration. Blood samples were obtained at -20, 0, 5, 10, 20, 30, 60, 120, and 180 minutes via catheter, and rLH and rabbit follicle-stimulating hormone (rFSH) levels were determined by RIA. Doses between 1 and 100 IU of hLH produced a prompt decrease in rLH (within 5 minutes); the maximal response occurred within 20 to 30 minutes. Calculated as integrated area of response between 0 and 180 minutes, a dose-response relationship existed between 0.5 and 4.0 IU of hLH. Human LH, even at high doses of 50 IU, produced no changes in endogeneous rFSH. This is the first description of an entirely specific control system for LH, separate from FSH. The short-loop feedback control system for LH is sensitive to levels of LH estimated to be present in eugonadal animals.
3852	Gonadotropin receptors of human corpus luteum during menstrual cycle and pregnancy.	210668	Specific high-affinity low-capacity binding of human chorionic gonadotropin (hCG) and lutropin (hLH) was demonstrated in the plasma membrane fractions of human corpora lutea. The number of binding sites for both hormones increased from early to late luteal phase, whereas regressing corpus luteum from proliferative phase did not bind either hormone. On the basis of apparent dissociation constants the affinity of the receptor for hLH is highest during early luteal phase and decreases toward the end of the cycle, which may reflect an increasing insensitivity of the corpus luteum to circulating hLH. By contrast, the affinity of the receptor for hCG is highest in the midluteal phase. There are gonadotropin binding sites in the human corpus luteum also during pregnancy, but they are saturated by endogenous hCG. Evidence for this was obtained by elution of hCG with 0.15M sodium chloride at pH 2.3 from washed plasma membrane fractions of luteal tissue from six to 16 week's gestation. After acid treatment and neutralization these preparations showed specific binding for 125I-labeled hCG, but not for 125I-labeled hLH. Our results demonstrate a shift in the balance of affinity of the gonadotropin receptor from hLH to hCG during the course of luteal phase, and during pregnancy the binding sites appear to be available for hCG only.
3853	Effects of prolactin and prolactin plus luteinizing hormone on plasma testosterone levels in normal adult men.	122406	In an earlier study, normal adult men were shown to have increased plasma testosterone (T) levels over a several-hour period after haloperidol-induced increases in plasma PRL levels. The present study was designed both to replicate our first study and to examine the potential synergism of PRL and LH in influencing T levels on a short term basis in normal men. Eight volunteers received on 4 separate days an in injection of saline or 0.5 mg haloperidol at 1000 h and an iv injection of saline or 88 IU human LH (hLH) at 1100 h in a double blind randomized block design arranged to augment plasma levels of PRL, LH, and PRL and LH together on the different test days as well as to afford a saline control day. Only five of the eight subjects had prompt PRL responses to haloperidol equivalent to those of our earlier study. As the purpose of this study was to examine the effect of increased PRL on plasma T levels, these five subjects were used for the determination of changes in plasma T. After haloperidol administration, their PRL levels rose an average of 19 ng/ml, to the high-normal range, and after the hLH infusions, their LH levels rose an average of 71 ng/ml. On the saline control day, mean T levels showed the normal diurnal decline. After 0.5 mg haloperidol, T levels were maintained for several hours, and after 88 IU hLH, T levels were increased for several hours. Increased PRL levels concomitant with hLH administration did not produce a T response greater than that caused by hLH alone. The results of this study replicate the effect of drug-induced PRL augmentation on plasma T levels found in our earlier study, but they fail to demonstrate a synergistic effect of acutely increased PRL on LH-stimulated T secretion. PRL thus seems to be another pituitary hormone capable of increasing plasma T in adult men, but it clearly is a weaker stimulus than LH.
3854	Passive immunization with an antibody to the beta-subunit of ovine luteinizing hormone as a method of early abortion--a feasibility study in monkeys (Macaca radiata).	98358	Antiserum to the beta-subunit of ovine luteinizing hormone (oLH-beta) raised in monkeys (Macaca radiata) has been tested by a variety of criteria both in vivo and in vitro to establish its ability to neutralize oLH, hLH, and human chorionic gonadotropin (hCG). Passive administration of this antiserum caused inhibition of ovulation and termination of pregnancy in recipient monkeys as indicated by premature vaginal bleeding and a significant reduction in serum progesterone and estrogen levels. The results suggest that antiserum raised in monkeys against oLH-beta can neutralize monkey LH as well as monkey CG.
3855	Immunocytological localization of LH, FSH, TSH and their subunits in the pituitary of normal and anencephalic human fetuses.	354794	Immunostaining with antisera to oLH, hCG, hLH, pLHbeta, hFSH, hFSHbeta, hTSHalpha and bTSH was used to delineate the gonadotropic and thyrotropic cells of the human fetal anterior pituitary. Hypophyses from 29 normal fetuses, 3 newborn infants, and 5 totally ancencephalic fetuses were used. Several controls to check for the specificty of the immunocytological reaction were made. In normal fetuses, observations showed that: 1) the alpha subunit was detected from the eighth week and throughout gestation without sex differences; 2) intact LH was detected during the third month, however, age and sex differences were observed during the fourth and fifth months; 3) intact FSH was detected in female fetuses from the beginning of the fourth month, a sex difference was observed; 4) LH and FSH were detected in the same cells; 5) the thyrotropic cells were detectable from 15 weeks of gestation and their number increased during gestation without sex difference; 6) at birth the gonadotropic cells were scarce and were located in the ventromedian zone of the anterior pituitary, while the thyrotopic cells remained numerous and were located in the dorsomedian zone. In amencephalic fetuses: 1) the alpha subunit existed at each stage studies; 2) the reaction induced by anti-pLHbeta and anti-hFSHbeta sera was alwys very weak regardless of sex or age; 3) the thyrotropic cells were more numerous in comparison to the gonadotropic cells. These data are discussed in terms of the relationship of the hypophysiotropic hypothalamic factors to the appearance and evolution of the glycoprotein hormones and their subunits.
3856	Differential affinity of anti-Pr-beta-hCG-TT antibodies for hCG and hLH.	679684	NaN
3857	Development of anti-hLH antibodies after therapy with posterior pituitary extract.	122394	This report describes the appearance of high affinity antibodies to human LH in a girl who had been treated for diabetes insipidus with injections of pitressin tannate, plus occasional nasal insufflations of posterior pituitary powder. Immunological studies indicated that the antibody was a 7S IgG directed against the beta subunit of LH, which is not species-specific. The demonstration of immunoassayable LH in a commercially available pitressin preparation strongly suggests that this patient was immunized by bovine or porcine LH. Although studies of her urinary LH excretion and serum LH (by an interstitial cell bioassay system) suggest that at least some of her endogenous LH is not bound by the antibody, the possibility remains that this type of immunization may have important implications for the development and maintenance of normal adult pituitary-ovarian relationships.
3858	Progesterone levels in monkeys immunized with Pr-beta-hCG-TT after injection of hLH and hCG during luteal phase.	98288	NaN
3859	Detection of ovulation by a radioreceptor assay for human luteinizing hormone.	122440	A specific, sensitive, and rapid radioreceptor assay (RRA), employing membranes from bovine testes as receptor and [125I]hLH as radioligand, has been developed for measurement of human LH in serum. This RRA was used to determine the time of ovulation in seven women. For comparison, four hourly values around midcycle were measured by RIA. The sensitivity of the RRA was 0.78 ng/ml serum and could be increased by prolonged incubation. The coefficient of within and between assay variation at the 50% inhibition level was 7% and 13%, respectively. The mean index of discrimination (RRA/RIA) was 1.02, expressed by the slope of the regression curve. The coefficient of correlation was 0.97. In all women, the LH surge was detected by RRA, and the subsequent ovulation was verified within 30 h by endoscopic examination of the ovaries, as well as serum progesterone concentrations of more than 5 ng/ml on the fifth day after ovulation. As shown, prospective ovulation timing can be done by this simple and accurate method. The RRA can be useful in infertility therapy such as artificial insemination.A 2-hour solid-phase radioimmunoassay (RIA) is described for determination of luteinizing hormone (LH) concentrations to detect ovulation. Time of ovulation was determined in 7 women. For comparison, 4 hourly values around midcycle were measured by 2-hour RIA. The 2-hour RIA sensitivity was .78 ng/ml of serum and could be increased by prolonged incubation. The coefficients of within and between assay variation at the 50% inhibition level were 7 and 13%, respectively. The mean index of discrimination between standard RIA and 2-hour RIA was 1.02, expressed by the slope of the regression curve. The coefficient of correlation was .97. In all women, the LH surge was detected by 2-hour RIA, and the subsequent ovulation was verified within 30 hours by endoscopic examination of ovaries as well as serum progesterone concentrations of more than 5 ng/ml on the 5th day after ovulation. Prospective ovulation timing can be done by this simple, accurate method. The 2-hour RIA can be useful in infertility therapy such as artificial insemination.
3860	Transitory hCG-like activity in the urine of some IUD users.	755054	Using an antiserum (H-93) specific to the COOH-terminal peptide of hCG beta and gel chromatography, we found the transitory (1-2 days) presence of a gonadotropin having immunological, biological, and physical similarity to hCG, but not typical hLH, in the late luteal phase (days 24-29) urine of some (5 of 26) IUD users. We did not detect hCG in sera from any of the women. Although the source of the hCG-like gonadotropin is unknown, the data suggest that late luteal phase implantation may have occurred transiently.
3861	A receptor-immunoassay for the determination of the specificity of anti-HCG-beta sera.	77119	In the receptor-immunoassay, the binding of anti-HCG-beta sera to 125I-HCG and 125I-HLH is measured in the presence of bovine corpus luteum "receptor". Anti-sera specific for HCG show little binding with 125I-HLH. The receptor-immunoassay permits an evaluation of specificity, affinity and the ability of anti-HCG-beta sera to compete with the "receptor" for binding to 125I-HCG.
3862	Structure of human luteinizing hormone alpha subunit.	710366	Structural studies have substantiated the concept that the glycoprotein hormones consist of a "common" alpha subunit and "hormone-specific" beta subunit. Despite this, consensus is still lacking concerning certain portions of the amino acid sequences, including alignment of residues 81--82 in the alpha subunit. We have carried out sequence analysis of the alpha subunit of human luteinizing hormone (hLH) to clarify the assignment of these residues and to examine further the nature of the amino-terminal heterogeneity found among the different alpha subunits. Our structure showed residues 80--84 to be -His-Cys-Ser-Thy-Cys-, consistent with findings of others for human follicle stimulating hormone (hFSH) and human chorionic gonadotropin (hCG). The extensive degree of heterogeneity found in the amino terminal region of hFSH and hCG is present to only a minor extent in hLH. The differences in the pattern of amino-terminal heterogeneity among the various hormones may result from differences in the nature of cleavage of subunit from a larger intracellular precursor peptide.
3863	Thyroliberin and gonadoliberin tests during pregnancy and the puerperium.	411303	Highly specific and sensitive radioimmunoassays for HTSH, HFSH and HLH have been used in order to investigate pituitary thyrotrophic and gonadotrophic functions of pregnant and puerperal women. The radioimmunoassay system for HFSH, using reagents prepared in our laboratory, is described. Thirty-three women were studied during pregnancy and post-partum. Forty-one TRH tests were performed. Basal levels of TSH, and increase in serum TSH after intravenous injection of TRH were similar in pregnant and puerperal women and in a control group. Thirty-three LH-RH tests were also performed. Basal levels of LH and FSH remained undetectable throughout pregnancy and in early puerperium. Furthermore, after intravenous injection of LH-RH neither LH nor FSH activity could be detected. It has thus been demonstrated that pituitary responsiveness to TRH during pregnancy and the puerperium is similar to that of non-pregnant young women. In contrast, a complete cessation of pituitary gonadotrophic functions is observed during the same period.
3864	Biological and immunological characterization of human luteinizing hormone: II. A comparison of the immunological and biological activities of pituitary extracts after electrofocusing using different standard preparations.	590618	The profile of immunologically active human luteinizing hormone (hLH) was determined in aqueous pituitary extracts after electrofocusing using two radioimmunoassay systems and the estimates for each fraction were compared to those obtained by an in vitro bioassay method. Similar biological and immunological profiles were obtained in the pH 7.0-9.0 region, where most of the biological activity was present. Biological to immunological (B/I) ratios ranging from 0.6 to 1.7 (mean ratio 1.01; n = 21) were found in the major biologically active fractions of this pH region when a highly purified human pituitary LH preparation (68/40) was used as standard in both types of assays. The close proximity of these ratios to unity indicates a similar composition of biological and immunological activities in all these fractions in relation to that of the highly purified standard. However, marked discrepancies were observed in the pH region 3.0-7.0 where the B/I ratios ranged from 0.1 to 0.9, indicating the presence of immunological activity associated with relatively little biological activity. When impure human LH preparations of pituitary (69/104) and urinary (hMG 2nd IRP) origin were used as standards for the bioassay and radioimmunoassay of the hLH present in the major fractions of the pH region 7.0-9.0, significantly higher B/I ratios were obtained than with the use of the highly purified standard (68/40). These elevated B/I ratios are attributed to the presence in the impure standard preparations of immunological activity, which is associated with little or no biological activity. These observations may provide an explanation for the differences in B/I ratios which were reported for hLH in plasma, using different standard preparations.
3865	Preliminary evidence for the intracellular localization of luteinizing hormones in mammalian oocytes.	409802	Preliminary autoradiographic and immunofluorescent evidence is presented which suggests the localization of hLH beta, hCG and oLH within the oocytes of several mammalian species. Specificity of the response is supported by negative observations in the presence of cold hormone or antibodies to other proteins. The mechanism of action of these gonadotrophins in such a site remains to be defined.
3866	[Development of a radioreceptor assay for hLH; its application in prospective ovulation determination (proceedings)].	579887	NaN
3867	Gonadotropin receptors in human corpora lutea of the menstrual cycle and pregnancy.	16492	Binding of 125I-human choriogonadotropin (hCG) to homogenates of corpora lutea of the menstrual cycle and pregnancy was examined. While corpora lutea of the menstrual cycle bound 125I-hCG, most of the corpora lutea of pregnancy from six to 34 weeks' gestation bound little or none of the added 125 I-hCG. Further exploration into various possibilities for the above findings led to the conclusion that these corpora lutea either contain very few gonadotropin receptors or lack them altogether. The selected properties of gonadotropin receptors in corpora lutea of the cycle were studied. The apparent dissociation constant for binding was about the same (2.6 to 3.8 x 10-10M) in all but one corpus luteum. In this one sample the constant was three- to five-fold higher (1.3 x 10-9M). The binding was quite rapid and reversible, and it exhibited dependence on the amount of 125I-hCG, homogenate protein, pH of the incubation media, and duration of incubation. hCG and human lutropin (hLH') competed for 125I-hCG binding, but hCG subunits (alpha and beta), human follitropin, and ovine prolactin did not. Although hCG and hLH were parallel in inhibiting 125I-hCG binding, hLH was found to be relatively less effective. The studies with various enzymes revealed that gonadotropin receptors are protein in nature.
3868	Isolated hypogonadotropic hypogonadism: induction of ovulation with exogenous gonadotropins.	856636	Six patients with all of the clinical and laboratory characteristics of isolated hypogonadotropic hypogonadism (bihormonal gonadotropin deficiency) were treated with human menopausal gonadotropins (hMG, Pergonal) and human chorionic gonadotropin (hCG). Of a total of 37 courses of treatment administered, there was evidence of ovulation in 32 (29%). The first four subjects had a total of six pregnancies in 13 treatment cycles. In the remaining two subjects, there were abnormalities in the seminal fluid of both husbands and, as a consequence, only one patient conceived. The average dose and duration of hMG therapy was 3310 IU (44 ampules), administered for 13 days. A wide range of hCG dosage was used to induce ovulation, but three subjects conceived following 5000 IU of hCG on 2 successive days. None of our subjects developed any evidence of the hyperstimulation syndrome, and there was only one instance of twin birth. Serial determinations of hLH performed during hMG therapy were lower than those noted during the normal follicular phase. In contrast, hFSH levels were in the normal follicular phase range. the high success rate in these subjects indicates that the prognosis for inducing ovulation in patients with hypogonadotropic hypogonadism is good.
3869	Ultracentrifugal studies of human luteinizing hormone and its subunits: dependence on protein concentration and ionic strength.	856258	Purified human pituitary luteinizing hormone (hLH) and its alpha and beta subunits have been examined by physical methods. Freshly prepared hLH showed three closely adjacent bands on electrophoresis in polyacrylamide gels under alkaline conditions, but on standing even in the freeze-dried condition additional bands appeared. alpha and beta subunits gave bands which were quite different from the main hLH bands but comparable with the additional bands, as well as traces of hLH bands. hLH was investigated at three ionic strengths (0.1, 0.2, 0.5) at pH 5.9 +/- 0.1. Sedimentation velocity experiments demonstrated a complex system of association-dissociation which was further investigated by sedimentation equilibrium. Association occurred at the higher protein concentrations at each ionic strength, but to a significantly higher level at I = 0.5. Only at I = 0.1, pH 5.8, was there a clear indication over a range of protein concentration of the occurrence of a species of molecular weight 32 000 +/- 2000, in fair agreement only with the sum of the molecular weights of the alpha and beta subunits. At higher ionic strengths, there were indications of dissociation at low protein concentration (less than 0.10 g/100 mL) as well as association at higher values (greater than 0.20 g/100 mL). In view of the occurrence of molecular weights less than 28 000 at I = 0.2, HLH was treated in terms of a monomer of molecular weight 14 000, and some evidence was obtained for tetramer formation (4M = M4). At higher ionic strength this model does not apply and it is thought that indefinite association may be occurring to some extent also. The alpha subunit gave indications of association from its sedimentation coefficient vs. concentration plot, and sedimentation equilibrium (at pH 5.9 I = 0.1) demonstrated molecular weights increasing with increasing concentration. Evidence for tetramer formation also was obtained. The beta subunit, in spite of an apparently simple sedimentation coefficient-concentration curve, showed molecular weights varying from well below 14 000 to beyond 20 000. There is evidence to suggest that the isolated alpha and beta subunits, even on standing as dry solid, are not stable but give rise to lower molecular weight products. Aged freeze-dried hLH did not show such impurity.
3870	Biologically active luteinizing hormone (LH) in plasma: II. Comparison with immunologically active LH levels throughout the human menstrual cycle.	576756	The levels of biologically active luteinizing hormone were determined by an in vitro bioassay method in plasma samples collected daily over a complete menstrual cycle from 12 menstruating women. These cycles were normal according to a number of criteria, including daily plasma levels of oestradiol, 17-hydroxyprogesterone and progesterone. Immunoreactive LH was estimated in the same 12 cycles by a radio-immunoassay (RIA) procedure (HCG-RIA) using an HCG antiserum and iodinated HCG. The 2nd IRP of HMG was selected as standard although significant deviations from parallelism were found with 7 out of the 12 plasma pools studied. The use of the 1st IRP of human pituitary gonadotrophins (FSH and LH (ICSH)) for bioassay (herafter HPG-1st IRP) as standard in this system resulted invariably in invalid assays, due to lack of parallelism. Immunoreactive LH was also measured in 8 of the 12 cycles by a RIA procedure (HLA-RIA) using a human LH antiserum and iodinated human LH of pituitary origin. Results are expressed in terms of the HPG-1st IRP. The plasma levels of biologically and immunologically active LH were qualitatively similar throughout the menstrual cycle. However, the LH levels measured by the bioassay invariably exceeded those estimated by the RIA procedures. The biological to immunological (B/I) ratio over the entire menstrual cycle (312 comparisons) was 5.5 with 95% confidence limits at 5.2 and 5.8 when the HCG-RIA system was employed. Using the HLH-RIA system (208 comparisons), the corresponding ratio was 6.4 (6.0:6.9). When regression lines were calculated using the bioassay results as the independent variable and the RIA results as the dependent variable, the 95% confidence limits of the regression lines did not include the origin. Furthermore,, in keeping with the high B/I ratios, the slopes of the two regression lines and their conficence limits differed markedly from unity. It is concluded that although qualitatively similar profiles were observed between the biological and immunological activities throughtout the menstrual cycle, two aspects require further attention. Firstly, the elevated B/I ratios together with the behaviour of the dose-effect lines obtained with different standards in the various RIA systems suggest that presently available reference standard preparations of pituitary and/or urinary origin are not suitable for the assay of LH in human plasms. Secondly, from the regression analyses of the biological and immunological activities it is infered that the RIA methods detect immunological activity which is not associated with biological activity. If so, the validity of these RIA procedures for specifically measuring low levels of biologically active LH in plasma may be in question.
3871	[Biological criteria for the surveillance of hydatidiform moles and choriocarcinomas].	886170	Secretion of choriogonadotropin (hCG) remains until now the more accurate biochemical label of trophoblastic disease and a close follow-up is possible by means of sensitive and specific radioimmunoassays. After hydatidiform moles, drawing the decreasing curve of hCG concentrations by weekly measurements in plasma or urine, would generally allow distinguishing within a few weeks cases with and without retention of active trophoblastic tissue, although in only 30 p. 100 of moles hCG spontaneously disappears within a month. In some choriocarcinoma under therapy, low persisting hCG levels (lower than 100 or 50 I.U./24 hours) could only be evidenced by repeated radioimmunoassays. Even with apparently physiological hLH/hCG concentrations, the use of anti-beta hCG antiserum could demonstrate, if any, a secretion of choriogonadotropin. In castrated women the same technique would elude the interference of hLH and make easier the diagnostic of relapses.
3872	Gonadotropin-sensitive progesterone production by rhesus monkey luteal cells in vitro: a function of age of the corpus luteum during the menstrual cycle.	401732	Progesterone production in vitro, in the presence and absence of exogenous gonadotropin, was examined in suspensions of luteal cells, isolated by collagenase digestion of rhesus monkeys corpus luteum at various stages of the menstrual cycle. Cells isolated during mid-luteal phase (days 15-19) of the cycle secreted progesterone for up to 6 h in vitro. Mid-luteal phase cells were responsive to physiologic concentrations of human chorionic gonadotropin (hCG), with progesterone production significantly (P less than 0.05) enhanced by as little as 0.1 ng hCG/ml. Maximal stimulation was obtained with 100 ng hCG/ml. Both macaque chorionic gonadotropin (mCG) and human luteinizing hormone (hLH) significantly (P less than 0.01) increased progesterone production, while human follicle stimulating hormone (hFSH) did not. Under control conditions, in the presence of nutrient medium alone (no exogenous gonadotropin), the progesterone synthetic activity of mid-luteal phase cells was significantly (P less than 0.01) greater than that of cells from late luteal phase (days 22-28) of the cycle. Moreover, progesterone production by mid-luteal phase cells was consistently stimulated (P less than 0.01) by the presence of 100 ng hCG/ml, whereas late luteal phase cells were less sensitive or unresponsive to exogenous gonadotropin. The progesterone synthetic activity of luteal cells in vitro correlated positively with both the wet weight of the excised corpus luteum (r = 0.82, P less than 0.01) and the peripheral serum progesterone concentration immediately preceding luteectomy (r = 0.66, P less than 0.01). These findings suggest that freshly isolated luteal cells reflect the functional capability of the corpus luteum in vivo. It is apparent that the age of the rhesus monkey corpus luteum of the non-fertile menstrual cycle is an important factor governing luteal cell progesterone synthetic capability and luteal cell responsiveness to gonadotropin in vitro.
3873	Laser light scattering spectroscopic immunoassay in the agglutination-inhibition mode for human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH).	1017853	NaN
3874	Primate chorionic gonadotropins: antigenic similarities to the unique carboxyl-terminal peptide of HCGbeta subunit.	826547	Antigenic similarities of chorionic gonadotropin from human (hCG), chimpanzee (chCG), gorilla (gCG), orangutan (orCG), baboon (paCG), macaque (mCG), and marmoset (maCG) were investigated in a radioimmunoassay system using an antiserum (rabbit H93) with antigenic determinants known to reside in the terminal 15 amino acid residues of the unique carboxyl-terminal peptide of hCGbeta, which makes it highly specific for hCG without crossreactivity to hLH. Our findings indicate that the extreme antigenic similarity between chCG and hCG at the unique carboxyl-terminal region of hCGbeta, as well as the availability of these apes for laboratory research, make the chimpanzee the optimal nonhuman primate model for assessing potential risks of fertility control in women by immunization against hCGbeta.
3875	Ontogenesis, distribution and relative sensitivity of hCG receptors to hCG and LH in goat ovaries.	187505	The binding of [125I]hCG to immature and mature follicles and corpora lutea of goat ovaries has been studied. The hormone is bound maximally by corpora lutea although mature follicles also exhibit some binding. Immature follicles are practically devoid of receptors for this hormone. In the corpus luteum, the receptors for the hormone are present in thecal and luteal cells. Autoradiographic studies show the location of the bound radioactivity grains primarily along the plasma membranes of these cells, although some radioactivity grains were also seen in the cytoplasm of luteal but not thecal cells. On a mole to mole basis, hCG was found to displace [125I]hCG from binding to receptors on corpus luteum better than hLH and oLH.
3876	Nature of cross-reaction between hCG and anti-oLH serum and development of a radioimmunoassay to measure hLH specifically in the presence of hCG.	63368	Immunological cross-reaction between hCG and anti-oLH sera has been demonstrated using radioimmunoassay techniques. The results indicate that this cross-reaction is incomplete and that the anti-oLH sera used have the ability to distinguish between LH and hCG. Following absorption with purified hCG, anti-oLH serum was used to develop a heterologous radioimmunoassay "[125I]iodo-hLH + anti-oLH serum" (H-O, RIA) which specifically and selectively measures hLH in serum samples containing both hLH and hCG. In this radioimmunoassay hCG and subunits of hCG do not cross-react with hLH, in the range in which these hormones are present in human serum under physiological conditions. Other hormones such as hPL, hPRL, hGH, hFSH, hTSH, and GnRH do not interfere with the measurement of LH by radioimmunoassay. The sensitivity of the assay was 1.5 mIU (25 ng) per ml (LER 907 standard), and the inter- and intra-assay coefficients of variations for samples were 10.83% and 8.4%, respectively. The recoveries of hLH added to pregnancy serum containing an hCG concentration of 8.55 IU/ml were in the range 95-108%. Determination of LH content of human pituitary extracts by H-O RIA gave values which were in close agreement with those derived by bioassay (indices of discrimination 0.72-1.12). Serum LH patterns in women during normal menstrual cycles as well as in amenorrheic patients who received GnRH treatment are comparable to those reported by other investigators using other radioimmunoassay systems. Serum samples obtained during the first trimester of pregnancy, when analyzed by H-O RIA, showed basal LH levels.
3877	Pituitary gonadotropin response in women with idiopathic hyperprolactinemia.	786745	Inappropriate lactation and idiopathic hyperprolactinemia are frequently associated with amenorrhea. In these individuals, peripheral levels of follicle-stimulating hormone (hFSH) are usually normal, and luteinizing hormone (hLH) levels are often found in the low-normal range. The present study was undertaken to evaluate the functional capacity of the pituitary by the response of hFSH and hLH to synthetic gonadotropin-releasing hormone (Gn-RH). Six women with amenorrhea, inappropriate breast secretion, and idiopathic hyperprolactinemia (prolactin levels ranged from 45 to 355 ng/ml) were given 100 mug of Gn-RH intramuscularly. Serum hFSH and hLH levels were assessed in samples obtained at 15-minute intervals over the next 2-hour period. Initial hFSH levels were normal in all women, with a mean of 242 +/- 72 ng/ml. The absolute increase after Gn-RH administration averaged 486 +/- 193 ng/ml. Serum hLH was below normal in three of the six women, and normal in the remaining three women initially. The absolute increase averaged 1308 +/- 315 ng/ml. The greatest percentage increase in hLH was found in the women with the subnormal basal titers. In these women, hLH rose from a mean of 22 ng/ml to a mean of 1092 ng/ml. These data demonstate an exaggerated increase in hFSH and hLH levels after exogenous Gn-RH administration. This suggests that the amenorrhea associated with elevated serum prolactin levels is principally of hypothalamic origin.
3878	[A study on serum luteinizing hormone response to luteinizing hormone-releasing hormone iny hyper- and hypothyroidism (author's transl)].	798699	In order to study the mechanism of gonadal dysfunction in patients with abnormal thyroid function, the pituitary luteinizing hormone (LH) response to 200 mug of luteinizing hormone-releasing hormone (LH-RH) was investigated in 20 untreated and 18 treated hyperthyroid patients and in 21 untreated and 8 treated hypothyroid patients in addition to 29 matched control subjects. Serum LH levels were measured by double-antibody radioimmunoassay technique with HLH kit Daiichi. In untreated hyperthyroid men, the slightly elevated mean basal level and the exaggerated mean response to LH-RH of LH were observed, and in treated hyperthyroid men, both values were found to be similar to those of matched control men. Furthermore, in these patients, the maximal increments and the net increments of responses to LH-RH of LH were correlated with serum thyroxine levels and Thyopac-3 values. In most untreated and treated hyperthyroid premenopausal women with and without menstrual disturbances, the basal levels and the responses to LH-RH of LH were similar to those of matched control premenopausal women. In untreated hyperthyroid postmenopausal women, the mean basal level of LH was elevated, although the mean LH response to LH-RH was similar to that of matched control postmenopausal women, and in treated hyperthyroid postmenopausal women, the mean basal level and the mean response to LH-RH of LH were similar to those of matched control postmenopausal women. Furthermore, in these patients, the basal levels were correlated with serum thyroxine levels. In untreated and treated hypothyroid men, the mean basal level and the mean response to LH-RH of LH were similar to those of untreated and treated hyperthyroid postmenopausal women. In most untreated and treated hypothyroid premenopausal women with and without menstrual disturbances, the basal levels were observed to be inconsistent, and the LH responses to LH-RH were similar to those of matched control premenopausal women. In untreated and treated hypothyroid postmenopausal women, the mean basal level and the mean response to LH-RH of LH were similar to those of matched control postmenopausal women. These data indicate that the ability of the pituitary to secrete LH in patients with abnormal thyroid function was augmented in hyper- and hypothyroid men, and augmented in hyper-, but normal in hypothyroid postmenopausal women. Therefore, it is concluded that the cause of gonadal dysfunction at least in the premenopausal women with abnormal thyroid function lies not inside but outside the pituitary gland.
3879	Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.	182687	Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...
3880	Pituitary hormone levels in plasma of the human fetus after administration of LRH.	780370	The concentrations of hFSH, hLH/hCG, hGH, and hTSH1 were measured in maternal and fetal blood during the second trimester of pregnancy before and after the administration of 10 mug LRH to 9 fetuses. A comparison was performed with 9 control fetuses not receiving LRH. The initial value of hFSH was significantly higher in female than in male fetuses (P less than 0.01) and in both sexes exceeded the low maternal hFSH level. A similar fetomaternal difference existed also for plasma hGH, in contrast to plasma hLH/hCG, which concentration was higher in maternal than in fetal blood LRH did not cause any change of circulating hFSH, hLH/hCG, and hTSH levels in the fetus 10 min after its administration. A rise of fetal hGH levels occurred during the observation time irrespective of LRH administration. In amniotic fluid, the hFSH concentration accorded with that in fetal blood, and the hLH/hCG concentration with that in maternal blood. The fetal plasma levels of these pituitary hormones suggest selective release mechanisms for the various peptides. The causes of the absent response of blood gonadotropin levels to LRH in the fetus are discussed.
3881	The specificity of gonadotropin binding by the human corpus luteum.	182556	The specificity of gonadotropin binding was studied in fresh and frozen human corpora lutea. Ovine, bovine, and porcine luteinizing hormone (LH) competed with 125I-labeled human LH (125I-hLH) and 125I-labeled human chorionic gonadotropin (125I-hCG) for binding to tissue receptors in homogenates of human corpora lutea frozen for 3 to 12 months. In contrast, oLH, bLH, and pLH competed minimally for 125I-hLH and 125I-hCG binding sites in homogenates of fresh human corpora lutea. Ovine follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) did not compete in homogenates of fresh or frozen tissue. Competition of oLH and hCG for 125I-hCG binding sites at several dose levels in a homogenate of a fresh corpus luteum was studied. One hundred micrograms of oLH and ten nanograms of hCG gave an equivalent competition--a 10,000-fold difference in competitive potency. Only hCG competed with 125I-hCG for binding when the competition of oLH, bLH, pLH, oFSH, oTSH, hCG and hCG subunits, and hCG were compared at the 10-mug level in a homogenate of fresh human corpus luteum. The binding of 125I-labeled homologous human hormones by the corpus luteum was examined in a limited fashion. 125I-Prolactin did not bind to preparations of fresh stroma from a patient with polycystic ovaries nor did it bind to three separate preparations of fresh corpora luteum which did bind 125I-hCG. 125I-hTSH did not show significant binding to a fresh human corpus luteum preparation which did bind 125I-hCG. These studies indicate that the gonadotropin receptor of the fresh human corpus luteum possesses a unique species specificity and illustrate the importance of working with human corpora lutea in their most native state.
3882	Evidence for a gonadotropin from nonpregnant subjects that has physical, immunological, and biological similarities to human chorionic gonadotropin.	60763	Substances from urinary extracts of normal, nonpregnant subjects and human pituitary gonadotropin preparations were found to react similarly to human chorionic gonadotropin (hCG) in a radioimmunoassay system that is highly specific for hCG and without crossreactivity to human luteinizing hormone (hLH). The antiserum was produced in a rabbit immunized with a bovine albumin conjugate of the unique carboxyl-terminal peptide (residues 123-145) isolated from a tryptic digest of the reduced, S-carboxymethylated hCGbeta subunit. The antibody recognition site on the peptide was found to reside on the last 15 amino acid residues of the carboxyl-terminal peptide, as evidenced by the competitive binding activities against 125I-labeled hCG of a series of peptides chemically synthesized according to the carboxyl-terminal sequence of HCGbeta. In order to elucidate the nature of the crossreacting substance in urinary extracts, a human postmenopausal urinary preparation (Pergonal) and a kaolin-acetone extract of urine from a patient with Klinefelter's syndrome were subjected to gel chromatography on Sephadex G-100. The results indicate that fractions showing immunocrossreactivity with the antiserum to hCGbeta-carboxyl-terminal peptide coeluted with 125I-labeled hCG which was separated distinctly from hLH. The same fractions from this postmenopausal urinary gonadotropin preparation exhibited in vitro biological activity proportional to the immunocrossreactivity of the hCG-specific antiserum. Concentration of postmenopausal women's urine by acetone precipitation retained approximately five times more immunoreactivity per unit volume than kaolin-acetone extraction, when assayed with the antiserum to hCGbeta-carboxyl-terminal peptide.
3883	Adenylyl cyclase activities in ovarian tissues. IV. Gonadotrophin-induced desensitization of the luteal adenylyl cyclase throughout pregnancy and pseudopregnancy in the rabbit and the rat.	939195	We measured the adenylyl cyclase (AC) activity in dissected CL and the responsiveness of the AC system to LH, FSH, and prostaglandin (PG)E1 at different times following the administration of high doses of hCG or hLH to pseudopregnant and pregnant rats and rabbits. In rabbits, ovulatory doses of hCG promoted desensitization of the AC system in both CL of pregnancy and CL of pseudopregnancy (PSP), but at varying rates. At least a 50% decline in the LH-stimulated AC system was demonstrable 2 h after the hCG injection in CL obtained during PSP and the first 18 days of pregnancy. However, after day 21, AC activity was unaltered at 2 or 24 h after hCG injection, necessitating as much as 72 h for the AC system to become desensitized to LH. It seems that CL in the last third of pregnancy are afforded partial protection from the desensitizing effects of hCG. This protective effect was found not to be conferred upon follicles contained in ovaries after day 21 of pregnancy or upon newly, hCG-induced 3-day-old CL in 24-day pregnant rabbit ovaries. hCG-induced desensitization of CL adenylyl cyclase in rabbits was prevented neither by cauterization of tertiary follicles not by the continued administration of estradiol-17beta (1.5 mug SC twice daily), suggesting that this effect of hCG is due to a direct interaction with the CL, and not due to interruption of the follicular estrogen supply. In rats, the injection of an ovulatory dose of hCG (50 IU SC into prepubertal rats; 50 IU ip plus 50 IU SC into mature rats) also induced desensitization of the AC system in ovaries of superovulated prepubertal rats and in CL of pseudopregnant and pregnant rats. Desensitization of the AC system was not detectable at 2 h, was 30% of total by 6 h, and was complete at 24 h after hCG injection. Both regression of the CL and desensitization of the AC system in CL are induced only by doses of hCG which are ovulatory and not subovulatory. Desensitization of AC appears to precede functional luteolysis, at least in the pseudopreganant rabbit. Thus, the apparent close association between hCG-induced luteolysis and the desensitization of the adenylyl cyclase system in CL would suggest that desensitization may be a marker for luteal regression.
3884	Effect of bromo-ergocryptine on serum hPRL, hLH, hFSH, and estradiol 17-beta in women with galactorrhea-amenorrhea.	934580	Fourteen women with amenorrhea-galactorrhea were treated with bromo-ergocryptine. Serum prolactin hPRL, hLH, hFSH, estradiol 17-beta (E2), and progesterone values were determined before and during treatment. No consistent pretreatment hormonal pattern was found. During treatment hPRL levels fell in 13 of 14 patients, and E2 rose in 12 of 14 patients. Levels of hLH became normal, and amounts of hFSH did not change. Galactorrhea lessened in all cases and ceased in 11. Menses resumed in 11 patients and 3 women became pregnant. Cessation of galactorrhea and resumption of menses occurred irrespective of initial hPRL or hLH levels. It is suggested that improvement of galactorrhea and resumption of menses in some patients with normal hPRL values are the result of increased serum E2 levels and may be due to a direct action of bromo-ergocryptine on the ovary. Bromo-ergocryptine may also be effective in treatment of amenorrhea, unassociated with galactorrhea or elevated hPRL levels.
3885	Gonadotropin binding sites in human postmenopausal ovaries.	181277	Glucose-6-phosphate dehydrogenase (G6PD) activity and gonadotropin binding sites were localized within seven postmenopausal ovaries. G6PD was localized in the cells of cortical stroma and hilus using a histochemical technique for the reduction of the tetrazolium salt, Nitro-Blue tetrazolium. Gonadotropin binding sites were localized by autoradiography following incubation of ovarian sections with (125I-hLH) and 125I-labeled follicle-stimulating hormone (125I-hFSH) were identified in the cortical stroma and hilus cells. Since these cells contain G6PD and other enzymes necessary for steroidogenesis and also have the capacity to bind both hLH and hFSH, steroidogenesis in postmenopausal ovaries appears to be controlled by circulating gonadotropins. Blood vessels within postmenopausal ovaries also bound both gonadotropins, but 125I-hFSH binding was often more intense than 125I-hLH binding.
3886	Ovarian biochemical competence following gonadotrophic deprivation from birth.	179258	Normal ovarian morphogenesis is impaired by the absence of gonadotrophic hormones from birth. In the present study, the following question was asked: Does gonadotrophic deprivation also affect ovarian biochemical competence? Newborn mice were treated daily with an antiserum neutralizing endogenous circulating gonadotrophins. At the age of 14 days, ovaries from these mice and control littermates were incubated in the presence and absence of gonadotrophic preparations; cAMP and lactic acid levels were then measured in tissue and incubation medium. Ovaries from anti-gonadotrophin treated and control mice had the same basal levels, per mg tissue, of cAMP and lactic acid. Moreover, the levels increased to approximately the same extent following in vitro gonadotrophic stimulation: an increase in cAMP of 8-10-fold (using a preparation with an hLH:hFSH ratio of 1:1) or 2 1/2-fold (using a preparation with an hLH:hFSH ratio of 1:5), and about a 2-fold increase in lactic acid (using oLH). The acute effect of gonadotrophins on ovarian glycolysis, reported up till now only in the rat ovary, is the first such demonstration in mice. The results also indicate that despite impaired morphogenesis, the enzymatic systems necessary for ovarian glycolysis (as measured by lactic acid production) and cAMP formation can develop without gonadotrophic participation. Furthermore, ovarian capacity to respond to hormonal stimulation is acquired post-natally, as shown by refractoriness to gonadotrophic stimulation during the first week of life. Finally, post-natal gonadotrophic exposure does not seem an essential requirement, at least as measured by the above parameters, for acquiring ovarian competence to respond to hormonal stimulation.
3887	alpha and beta glycoprotein hormone subunits (hLH, hFSH, hCG) in the serum and pituitary of the human fetus.	1270589	Utilizing specific radioimmunoassay and gel chromatographic separation methods, the concentration of a glycoprotein hormone subunit and the beta subunit of hCG, hLH, and hFSH was determined. The alpha glycoprotein hormone subunit is present throughout gestation in the serum and pituitary of the human fetus; little or no subunit is apparent in contrast to that of intact LH and FSH.
3888	Short-loop feedback control of luteinizing hormone in the rabbit.	1267022	A radioimmunoassay for rabbit luteinizing hormone (rLH) in which rLH shows no significant cross-reaction with human LH (hLH) or human chorionic gonadotropin (HCG) was employed to test for the existence of a short-loop feedback for LH in the rabbit. Two weeks after castration, hCG and hLH were administered intravenously to rabbits, and the effects on circulating rLH were measured. Purified hLH (10 ng or 100 IU) produced significant depression of blood rLH within 30-60 min of intravenous injection. Saline administered to the same animals produced no changes in rLH. Injection of hCG (2,000 IU) under the same conditions also produced a significant fall in rLH. However, hCG administered to rabbits castrated 6 wk prior to study failed to suppress endogenous rLH. These data demonstrate, by direct radioimmunoassay quantification of blood hormones, the existence of a short-loop negative feedback for LH in the rabbit. They also suggest that the sensitivity of the short-loop changes with time after castration.
3889	[Purification of subunits from human chorionic gonadotropin and radioimmunoassay].	987936	A crude hCG with an activity of about 3,000 IU per mg was purifed to 10,000--15,000 IU per mg of dry weight using Amberlite CG-50 chromatography combined with DEAE-Sephadex A-50 and Sephadex G-75. The alpha and beta subunits of hCG were prepared by urea-treatment of the hormone and isolated by DEAE-Sephadex A-50 chromatography. Further purification of the subunits was achieved by gel filtration on a Sephadex G-76 column. For radioimmunoassay hCG was iodinated by the DMSO-chloramine T method. Iodination of hCG with non-radioactive iodine revealed that the addition of DMSO to the iodination mixture seemed to reduce the iodination damage to the antigenic activity of the hormone. Non-radioactive iodine substituted hCG accomplished by the DMSO-chloramine T method showed 1.5 times more immunoreactive in the hCG radioimmunoassay than hCG iodinated by the usual chloramine T method. The radioimmunoassay of the hCG-beta subunit developed in our laboratory was satisfactory with respect to specificity; hLH, hFSH, hTSH and hCG-alpha subunit tested were cross-reacted very poorly in our assay system. Desialylated-hCG and subunits, whose biologic potency was almost zero, exhibited also decreased immunoreactivity, about 30% of the native hormones with grossly unimpaired parallelism in their respective homologous radioimmunoassays. The concentrations of hCG and the subunits were determined on human sera from pregnant patients during the course of pregnancy. The hCG levels reached to the peak at the first trimester of the pregnancy, however, the hCG-beta subunits varied in their concentrations poorly throughout the pregnancy periods. The hCG-alpha levels, on the other hand, depicted two distinct peaks, at the early period and the term of pregnancy.
3890	Processing of the preparations of beta-subunit of human chorionic gonadotropin for minimization of cross-reactivity with human luteinizing hormone.	1245123	The immunological reactivity of various components of beta-human cho rionic gonadotropin (beta-HCG) preparations with antisera developed against HCG and human luteinizing hormone (HLH) was assessed. Anti-beta-HCG reactivity was primarily located in 2 or 3 adjacent fractions. Anti-HLH reactivity was partly found in a fraction co-migrating with 1 of the anti-beta-HCG fractions, and also in a fraction migrating more slowly than the anti-beta-HCG fractions. Preparations with no, or minimal, cross-reactivity with anti-HLH were obtained upon absorption of beta-HCG with anti-ovine LH.
3891	Infusions of hFSH and hLH in normal men. II. Serum testosterone response to infused hLH and hFSH.	946145	Standardized 4 h intravenous infusions of human follicle stimulating hormone (hFSH) and/or human luteinizing hormone (hLH) were given either separately or combined to 7 normal male volunteers. The infusions raised the serum gonadotrophin levels at least 10 (FSH) and 18 (LH) times above the basal ones. Serum testosterone (T) levels were measured serially before, during and after the infusions and, in 4 subjects, during a corresponding period of another normal day. During a normal or basal 24 h period fluctuations were seen and also a circadian rhythm with lower levels in the evening. The infusion of hFSH alone (3 subjects) did not alter the serum T levels. The infusion of hLH alone in 2 subjects raised serum T levels by 17% and 55% over those of the basal day. The combined FSH/LH infusion caused a significant rise (35-68%) in 4 subjects and greater rise in 2 of them than after infusion of the hLH alone. The serum T responses were gradual, reaching a maximum 7-8 h after the end of the infusion.
3892	Infusions of hFSH and hLH in normal men. I. Kinetics of human follicle stimulating hormone.	946144	Serial serum assays of immunoreactive FSH before, during and after a 4 h intravenous infusion of human follicle stimulating hormone (hFSH) in five healthy men revealed two disappearance rate constants with corresponding mean half-lives of 2.9 and 50.6 h. The mean distribution spaces calculated for the fast and slow component were 4.36 1 and 75.9 1 respectively. The average value for the metabolic clearance rate was 17.2 ml/min and for the endogenous production rate 50.9 mU/min. Two of the subjects had human luteinizing hormone (hLH) added in the infusions without the FSH kinetics changing.
3893	Circular dichroism of human pituitary luteinizing hormone and its glycopeptides. Curve resolution and band assignments to the peptide chromophore, aromatic residues, disulfides, and N-acetylated amino sugars.	1270190	The circular dichroic (CS) spectrum of the glycoprotein hormone, human pituitary luteinizing hormone (hLH), has been determined between 195-320 nm and resolved into gaussian constituents. Below 230 nm the CD spectrum is characterized by a negative extremum at 207 nm with a shoulder at 217 nm. Resolution into gaussian constituents of the 200-230 nm CD spectrum resulted in two resolved negative bands, one at 206 nm and the other at 215 nm. The latter band is assigned to beta-structure which is estimated to be about 25%. The 206 nm resolved band is assigned to the N-acetylated carbohydrate groups (e.g. N-acetyl glucosamine, galactosamine, and neuraminic acid). This is based partly on the evidence that the CD spectrum of the hLH glycopeptide fraction (prepared by a pronase digestion of s-carboxymethylated hLH) exhibited a negative extremum at 207.5 nm, which is close to the resolved 206 nm band in hLH. Above 230 nm the CD spectrum is characterized by a negative extremum at about 275 nm. Most of the ellipticity in this region is attributed to the disulfides in hLH. Both strong acid (0.1 N hcl) and concentrated guanidine hydrochloride (4 M) affect the ellipticity in the vicinity of 275 nm, but only the latter (as well as concentrated urea) has a major effect on the CD spectrum below 230 nm indicating extensive conformational changes. There is, however, some loss of beta-structure in 0.1 N hcl. Thus, it appears that the conformation of the hLH subunits in these subunit-dissociating agents is rather different. There was no dramatic change in the magnitude of the 207 nm extremum of native hLH between 10-50C.
3894	Application of a radioligand receptor assay for determination of luteinizing hormone in human serum.	946142	A sensitive, specific and economic radioligand receptor assay is described for measurement of LH in human serum (RRA-LH). By means of ethanol fractionation of (NH4)2SO4-precipitation serum can be prepared for LH-quantitation and tested in the RRA-LH without interference of a non-specific inhibiting substance present in untreated serum from various human sources. Treatment of serum with 8% ethanol separates non-specific inhibiting substances from LH, the latter remaining in the supernatant at this ethanol concentration. The criteria of specificity are examined. The results of experiments designed to produce evidence for or against specificity suggest specificity of the RRA-LH. Recoveries, as estimated by administration of [125I]hLH and unlabelled hLH to untreated serum samples are shown to be between 80 and 95% for the thanol fractionation procedure and between 65 and 75% for the (NH4)2SO4-precipitation method. The ethanol fractionation procedure is preferred for routine serum-LH determination because of its simplicity, speed and higher recoveries. Ethanol-treated sera from post-menopausal women show, on average, higher RRA-LH concentrations than ethanol-treated sera from young women. RRA-LH values are consistently higher than LH-values found by radioimmunoassay (RIA-LH). The LH-concentrations in sera from two menstrual cycles and from two LH-releasing hormone tests are measured by RRA-LH and RIA-LH. Similarities and discrepancies of the LH-profiles found by the two assay systems are described.
3895	A specific FSH receptor in rat granulosa cells: properties of binding in vitro.	174895	The specific binding of [125I]iodoFSH to granulosa cells collected from immature, hypophysectomized, DES-treated rats, was studied in vitro. Specific binding occurred after 5 min and reached maximum after 3 h of incubation at 37 C. Non specific binding was very low (less than 10% of the total binding). The [25I]iodoFSH remained tightly associated with the receptor at pH 7.5, but was rapidly dissociated at pH 5. Unlabeled hFSH competitively inhibited [125I]iodoFSH binding. Kinetic analyses of equilibrium binding experiments gave an apparent association constant (Ka) of 1.34 (+/- 0.31) x 10(10)M-1 [mean (+/- SE)] and a number of binding sites per cell of (NB) 1, 130 +/- 70 (mean +/- SE). Rat prolactin, wheat germ agglutinin, and concanavalin-A did not compete with [125I]iodoFSH, but hLH, hCG, and rTSH competed at doses 300- to 900-fold higher than those of hFSH. Granulosa cells isolated from adult DES-treated rats, as well as cells collected from medium and preovulatory follicles of proestrous rats, gave Ka and NB values similar to those described above. A comparative study of rabbit granulosa cells indicated a much lower binding affinity compared with those from the rat.
3896	Disappearance of biologic activity of synthetic LRH in normal and hypogonadal men.	1107348	The disappearance rates of the biologic activity of synthetic LRH have been measured in normal and hypogonadal men before and during estrogen treatment. Biologic activity was assayed by the change in radioimmunoassayable rat LH levels induced by injection of human plasma into ovariectomized rats pretreated with estrogen and progesterone, and compared with a dose-response curve using synthetic LRH. Following rapid iv injection of 100 micrograms synthetic LRH, peripheral blood peak levels of biologic activity occurred in less than 2-6 minutes, and the disappearance curve contained 2 exponential components. In 3 normal men the initial component had a t1/2 of 2 minutes and the second component a t1/2 of 10 minutes. Six hypogonadal men had first and second components of disappearance no different from normals whether or not they responded to LRH injection with increased endogenous hLH. While diethylstilbestrol 2 mg orally for 7 days suppressed pituitary hLH response to exogenous synthetic LRH (mean max.deltahLH 236 VS 46 NG/ml), it did not alter the disappearance rates in normal or hypogonadal men. These studies show that the biologic activity of synthetic LRH disappears from the blood after iv administration at rates similar to radioimmunoassayable and radioactive-labeled LRH. They further suggest that the lack of pituitary responsiveness to LRH in hypogonadal and estrogen-treated men is not due to more rapid clearance of the releasing hormone.
3897	Ligand-induced self-association of human luteinizing hormone. Negative cooperativity in the binding of 8-anilino-1-naphthalensulfonate.	1182113	The self-association of human luteinizing hormone (hLH) is enhanced in the presence of 8-anilino-1-naphthalenesulfonate (ANS). Sedimentation equilibrium measurements indicate that the hormone exists primarily as a dimer in the presence of excess ANS. It is shown that, for a self-associating protein system in which monomer and dimer have different affinities and/or capacities for ligand, both the shape and the position of the binding curve depend on protein concentration. Gel filtration and fluorescence measurements indicate that the hLH dimer has a single high affintiy site (K greater than 10(6) M-1) for ANS while binding to the monomer is too weak to be observed. This leads to negative cooperativity in the binding and to a shift of the binding curve to lower free ligand concentration with increasing concentration of the hormone.
3898	Selective radioimmunoassays for human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG).	57756	Highly specific radioimmunoassay systems were developed for measurement of hLH and hCG using antisera purified by affinity chromarography or simple adsorption to select the antibodies reacting specifically with either gonadotropin. Such systems permit specific measurement of lLH and hCG in samples containing both. These assays are suitable for various clinical and physiological studies, particularly, study of pituitary functions in the presence of chorionic or trophoblastic secretion.
3899	Gel filtration profile of circulating immunoreactive thyrotropin and subunits of myxedematous sera.	1159062	Five sera from hypothyroid patients were passed through gel-filtration columns and the collected fractions were analyzed in three different radioimmunoassays (hTSH-anti-hTSH; hTSHbeta-anti-pTSH; hLH-alpha-anti hLHalpha). For each immunological system, cross reactions were carefully studied. In the hTSH system one main peak was observed at the elution position of the entire molecule, with variable contributions in the subunits area. In the alpha subunit system, every profile showed an asymmetric peak, corresponding roughly to the position of the entire molecule; only two sera exhibited small amounts of free alpha chain. In the beta subunit system, a high molecular weight (100,000 or more) immunoreactive protein was present in every serum. Moreover, cross-reacting intact molecule was eluted as a second peak and in four sera free beta chain was detected. The significance of the big beta subunit, detectable only in the beta assay, is not elucidated.
3900	Increase in numbers of gonadotropin receptors on granulosa cells during follicle maturation.	169290	The binding of both human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) to a homogeneous population of isolated intact granulosa cells from increasingly mature porcine ovaan follicles has been studied. The number of receptor sites per granulosa cell increases 35-fold as the follicle enlarges, although cell size remains constant. This may explain the increased biologic responsiveness to gonadotropin of mature cells from large follicles. The affinity for both hormones, as determined by equilibrium dissociation constants, is high, and does not appear to change significantly as the cells mature. Comparison of dissociation constants, numbers of binding sites, and competitive inhibition between hCH and hLH, indicates that these two hormones probably interact with the same receptor on the granulosa cells.
3901	Measurement of the production rate of human luteinizing hormone using the urinary excretion technique.	1170478	Labeled and/or unlabeled human luteinizing hormone (hLH) was injected into normal and abnormal males and into hypopituitary patients. In two patients, two phases of disappearance (T 1/2) were found to be 1.2 and 2.4 hr. Labeled and unlabeled hormone were excreted in the same proportion. Of the injected hormone, normal males excreted 10.1% plus or minus 0.9%, hypopituitary patients excreted 11.4%- 17.5% and two other patients excreted 10.3% and 5.3% over 48 hr. The urinary excretion of unlabeled hLH in normal males was 22.1 plus or minus 4.9 IU/24 hr. The production rate of hLH in normal males was 224.3 plus or minus 65.3 IU/24 hr and, in the two abnormal males, was five times higher. The advantages of this methodology are discussed.
3902	Subunits of human chorionic gonadotrophin: immunological and biological studies.	1173971	The alpha and beta subunits of human chorionic gonadotrophin (hCG) were prepared by incubation in 8 M urea, pH 4.5. The separation of the two subunits was obtained by DEAE-Sephadex A-25 chromatography and purification was carried out by gel filtration on Sephadex G-100. The beta subunit obtained was biologically active and was therefore further purified by affinity chromatography using as immuno-adsorbent the alpha antibodies coupled to Sepharose 4B. The beta subunit so purified showed a biological activity less than 1 IU/mg. The immunological and biological properties of the hCG subunits have been studied. It was found that the anti HCG beta serum can discriminate between hCG and hLH and that in the 125I-hCG + anti-beta serum radioimmunoassay, the cross-reactivity of pituitary hLH was lower than that of urinary hLH. Moreover, it was observed that the less purified was the urinary LH preparation, the higher was the cross-reactivity. Therefore we considered the hypothesis that during the purification of human menopausal gonadotrophin (hMG) some LH subunits or smaller immunoreactive fragments could have been discarded with the waste fractions. In order to test the validity of this hypophysis, all the protein fractions obtained during the purification of the hMG were gel-filtered on Sephadex G-100. The immunoreactivity of the effluents from the gel filtration was tested by hCG, hCG-beta, hCG-alpha and hLH radioimmunoassays. While the alpha reactive material was found in some fractions as a peak having the same Ve/Vo value as hCG-alpha, the beta reactive material presenude hMG fractions was not observed in other fractions. The cross-reactivity with the anti beta serum was very low and was found in the LH region of the gel chromatogram. Furthermore, the neutralization of the biological activity of hCG and of urinary and pituitary LH by the anti hCG beta serum was studied by incubating a fixed amount of the three hormones with increasing volumes of antiserum and measuring the LH ACTIVITY AFTER INCUBATION BY THE OADD test. It was observed that the anti hCG beta serum inhibits hCG more than urinary or pituitary LH.
3903	[Genetic studies in hypoplastic left heart syndrome (author's transl)].	1172158	The families of the 18 patients with hypoplastic left heart syndrome (HLH), who died in the Universitätskinderklinik Erlangen between 1967 and January 1974, have been investigated. There was no consanguinity of the parents. Only one sib has died of a cardiac failure which possibly could have been HLH. Therefore it could be excluded that HLH is an autosomal-recessive disorder. The overall incidence of cardiac failure in the sibs of patients was 3. Most of the patients with HLH have been born in June-August and December-January. Environmental factors seem to be of importance in the genesis of HLH. Our results suggest multifactorial inheritance of the hypoplastic left heart syndrome.
3904	Studies on the metabolic clearance rate and production rate of human luteinizing hormone and on the initial half-time of its subunits in man.	1170215	The metabolic clearance rate (MCR) of human luteinizing hormone (hLH) has been determined in 10 normal men, 3 normal women, and in 12 women with ovulatory disorders resulting in oligomenorrhea or amenorrhea. The MCR was determined by the constant infusion technique using either iodinated or unlabeled highly purified hLH, and these results were compared to MCR determined by using crude pituitary preparations containing both follicle-stimulating hormone and hLH. Both preparations produced essentially similar results for the MCR of hLH and virtually identical results were obtained when complete or incomplete immunoprecipitation of the infused material was achieved. The MCR/body surface area of hLH was significantly greater in normal men (25.6 plus or minus 3.6 ml/min-m-2) than in normal premenopausal (19.2 plus or minus 0.9 ml/min-m-2) or postmenopausal women (17.4 plus or minus 1.9 ml/min-m-2). No difference was noted in the MCR of hLH in women with oligomenorrhea or amenorrhea. Production rates (PRs) were calculated by using a pituitary standard, the values being 85.1 plus or minus 21.5 IU/24 h in normal men, 39.9 plus or minus 12.6 IU/24 h in normal premenopausal women, and 294.6 plus or minus 61.9 IU/24 h in normal postmenopausal women. The initial half-times of disappearance of the alpha- and beta-subunits of hLH were measured in two normal men and found to be 15-18 min, respectively. The half-time of intact hLH was twice as great.
3905	Heterogeneity of purified human pituitary luteinizing hormone: effect on hormone measurement by radioimmunoassay.	1170075	Purified preparations of human luteinizing hormone (hLH), Hartree IRC-2, and NIH LER-960, have been examined after gel filtration of unlabelled and iodinated hormone. Several peaks of radioactivity were observed corresponding to dimeric (peak I), monomeric (peak II), and sub-unit (peak III) lLH forms. The immunologic and receptor activities of each fraction have been evaluated, Receptor activity was found in peaks I and II but not in peak III. Within peak II all fractions were not equally active in the receptor assay, and maximum activity appeared to correspond to molecules with elution characteristics similar to the unlabelled molecule. All fractions of peak II were immunologically active when tested against rabbit anti-hCG in excess. Two immunoassay systems with improved specificity for hLH and hLH-alpha, respectively, have been employed to show the presence of alpha subunit in one of the hLH preparations. These data have relevance with respect to testing of immuno and receptor potencies of hLH preparations.
3906	Neuroendocrine dysfunction in galactorrhea-amenorrhea after oral contraceptive use.	1153129	Nonpuerperal alactorrhea and amenorrhea have been reported following the use of oral contraceptives. Treatment of this condition with ergot alkaloids has proved to be of great therapeutic value. Pretreatment plasma hLH and hFSH concentrations in 13 women with postqill galactorrhea-amenorrhea (PPGA) were 6.6 plus or minus 0.6 (SE.) and 5.0 plus or minus 0.8 mlU/ml, respectively. The mean prolactin concentration was 80.7 plus or minus 13.2 ng/ml. After complete evaluation in which diagnostic evidence of pituitary tumor was absent, the patients were treated with ergocryptine (CB-154). The mean hPRL concentration at 14 days of therapy was 7.8 p;us or minus 1.9 ng/ml. Cyclic gonadotropin secretion resumed in all but one instance; ovulation was confirmed on the basis of a biphasic temperature chart and in 5 cases, endometrial biopsy. Measurement of serum dopamine-beta-hydroxylase (DBH) activity indicated a significant decline at the end of 8 weeks of CB-154 therapy. The fall in hPRL was not necessarily associated with a fall in DBH. The majority of women in this study exhibited a consistent personality suggesting varying degrees of anxiety unrelated to the PPGA and usually antedating the use of oral contraceptives. PPGA was found in women without hyperprolactinemia, but altered hPRL secretion was evident in all instances. The data suggest that the disorder of cyclic gonadotropin secretion is related to altered hPRL secretion, but the mechanism is possibly related to a catecholamine abnormality. The data support the presence of an inherent cyclic mechanism for the secretion of gonadotropins. CB-154 therapy does not affect conception, and no teratogenic effects were observed in 2 infants born to women who had received CB-154 during the first 40 days of gestation.
3907	Isolated deficiency of follicle-stimulating hormone: Further studies.	1092710	We record further studies over the past 2 yr on a unique female subject with isolated follicle-stimulating hormone (hFSH) deficiency, who developed human anti-gFSH antibodies after treatment with exogenous urinary gonadotropins. Administration of LRH resulted in a significant rise in serum hLH, but hFSH levels remained undetectable, "alpha Subunit" (the common alpha chain of the glycoprotein hormones) was detectable in basal samples obtained from our patient, and rose sharply after LRH. This is concordant with the hypothesis that the defect in our subject may be in the synthesis of the beta chain of hFSH, but it does not exclude other possibilities. The concentration of hFSH antibodies in the patient's serum has been monitored and her response to a further course of exogenous gonadotropins is recorded. The anti serum exhibits specificity for the hFSH molecule; the alpha and the beta chains of hFSH are virtually inert in competing with tracer 125-I-hFSH for binding to the antibody.
3908	A rapid solid-phase radioimmunoassay specific for human chorionic gonadotropin in gestational trophoblastic disease.	164644	A rapid solid-phase radioimmunoassay (RIA) specific for human chorionic gonadotropin (hCG) has been used for the measurement of serum hCG activity in patients with molar pregnancy and gestational trophoblastic disease (GTD). Serum hCG regression as determined by the specific RIA method after evacuation of uncomplicated molar pregnancy was noted to occur over a longer duration of time than previously reported from this Center using a nonspecific RIA system which measures human luteinizing hormone (hLH) and hCG simultaneously. Therapy for proliferative trophoblastic disease was withheld after evacuation of molar pregnancy while the serum hCG level regressed normally, but was instituted when the serum hCG level rose or plateaued for more than two consecutive weeks. Serum hCG levels in patients requiring chemotherapy for GTD were also more accurately monitored with the specific RIA method than with the nonspecific technic. Therapy was based solely on the hCG titer rather than the subsidence of toxicity, as has been our practice in the past. As a result, the duration of hospitalization, total dose of drug required for remission, and toxic side effects were substantially reduced without sacrificing the effectiveness of chemotherapy.
3909	Dissociation of human follicle-stimulating hormone. Comparison with luteinizing hormone.	235538	Rat testis tissue receptor assays were utilized to study the kinetics of dissociation of human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) under varying conditions of urea concentration and pH. In these competitive protein binding assays, 125I-hFSH and 125I-hLH were the radioligands and hormone dissociation was followed by a decrease in the ability of the dissociating hormone to inhibit uptake of the radioligand by tissue receptors. Rate data for dissociation of the gonadotropins were analyzed for quality of fit to first or second order integrated rate equations by nonlinear regression analysis. Treatment of hFSH with 4 M urea at pH 8 and 25 degrees for 22 hours did not result in significant dissociation, whereas in 8 M urea, over 90% dissociation was observed. The rate of dissociation of hFSH in 8 M urea was increased approximately 4-fold by raising the temperature from 25 to 37 degrees. Similar results were obtained when dissociation of hFSH was followed through use of an accepted whole animal bioassay for FSH, thus confirming the reliability of the tissue receptor assay for such dissociation studies. Kinetic studies showed that hFSH was undissociated by incubation in 6 M urea of pH 8 after 4 hours at 25 degrees. In contrast, hLH was 90% dissociated under similar conditions. This differential rate of inactivation of hLH allowed preparation of hFSH having significant reduced levels of contaminating LH activity, as determined by tissue receptor assays and by whole animal bioassays. Marked differences were noted in the rate of dissociation of hFSH and hLH under acid conditions. hFSH completely dissociated after approximately 2 min of incubation of pH 2 (25 degrees), and over 90% dissociated after 15 min of incubation at pH 3. In contrast, hLH was dissociated 60% after 20 min of incubation at pH 2 (25 degrees) and 40% dissociated after 60 min at pH 3. Neither hormone was significantly dissociated at pH 4.4 after 60 min, but hFSH showed a slightly greater rate of dissociation than did LH in the period between 1 and 23 hours of incubation at that pH. hFSH and hLH were relatively resistant to dissociation after incubation at pH 12 for 1 hour, bu;t dissociated significantly after incubation for 22 hours at that pH. The time course for dissociation of hFSH or hLH under the various conditions described above did not conform clearly to either first or second order kinetics, indicating that the over-all dissociation process represents a mixed order reaction. It appears that urea or acid-induced denaturation of one or both subunits of hLH and hFSH may occur prior to their dissociation. The very rapid rate of dissociation at acid pH values, particularly of hFSH, indicate that ionic interactions contribute importantly to the subunit association phenomenon.
3910	A rapid radioimmunoassay method for plasma luteinizing hormone.	1168551	1. A rapid double antibody radioummunoassay method for the determination of plasma luteinizing hormone (hLH) is described. 2. The method involves preincubation of the anti-LH serum with the precipitating second antibody and incubation of this mixture with unknows and standards at room temperature overnight. The incubation tubes are then centrifuged, supernatants decanted and the precipitates counted. 3. A total of two working days is required to complete the assay and report the results. 4. This method is more rapid than the conventional ones currently used for the estimation of LH in blood.
3911	Lack of correlation between gonadotropin and adrenal androgen levels in agonadal children.	165213	To evaluate the relationship between the secretion of gonadotropins and adrenal androgens, patients with gonadal agenesis were evaluated by (a) administering human luteinizing hormone (hLH) for 5 days with or without estrogen pretreatment to agonadal patients who had prepubertal LH levels; (b) correlating circulating gonadotropin levels with adrenal androgens in 45 patients; and (c) comparing adrenal androgens with gonadotropins after long-term administration of estrogen or androgens. Results are as follows: (a) No alteration in serum concentrations of dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), estrone (E1), testosterone (T), or in excretion of urinary 17-ketosteroid (17 KS) occurred after the administration of hLH. (b) No clearcut relationship between endogenous level of LH or FSH and DHA OR DHAS was demonstrated although a coincident increase of all hormones with age occurred. (c) Administration of estrogen to patients with gonadal agenesis did not affect their levels of DHA and DHAS although those patients given androgen developed higher DHAS, but not DHA, levels. Hence, increasing gonadotropin concentrations would not appear to be a primary etiologic factor in the maturation of the adrenal.
3912	Alpha subunit of glycoprotein hormone: presence in peripheral serum after LHRH.	50235	A method is described for the selective measurement of human luteinizing hormone (hLH) and alpha subunit. The assays employ highly purified tracers of hLH and of subunit of human chorionic gonadotropin (hCGalpha) and a "mixed population" of antibody: Population I (directed against determinants on beta subunit of hCG) and Population II (directed against determinants on alpha subunit of hCG). The former is present in greater concentration than the latter. When the mixed antibody is used at higher dilution (1:1.2x10(6)), Population II is effectively diluted out, and using 125I-labelled hLH as tracer, the assay recognize hLH, hCG and their beta subunits 20-50 times more sensitively than hCGalpha. When the mixed antibody is used at fivefold higher concentration, Population I is present in relative excess and acts as a "sink" for hCG, hLH and their beta subunits. Under these conditions, using 125I-hCGalpha as tracer, the assay recognize the alpha subunit 20 to 50-fold more sensitively than hLH and hCG. These assays have been employed in the study of sera which have been filtered over Sephadex G-100. Alpha subunit was detected in serum within minutes after intravenous injection of luteinizing hormone releasing hormone (LHRH) in four subjects tested.
3913	LH and its subunits in human pituitary, serum and urine.	1172998	Human postmenopausal serum and urine, and saline extracts of human pituitaries were gel-filtered on Sephadex G-100. The eluted fractions were radioimmunoassayed using 3 specific assays for native hLh, hLHalpha and hLHbeta. The elution profile of immunoreactive components in postmenopausal urine exhibited 2 peaks of activity corresponding to those demonstrated for 125I-labeled hLH and hLHalpha. No LHbeta was demonstrated. The immunoreactive components of post-menopausal serum eluted identically to 125I-LH, LHalpha and LHbeta. In addition, a peak of immunoreactive LH material was observed in the pre-LH region of the elution profile, distinctly separate from the void volume. The percent of the total immunoassayable serum LH contained in this peak varied. In elution profiles of all pituitary extracts, LH, LHalpha and LHbeta elution peaks corresponding to the respective labeled preparations were observed. An apparent excess of free alpha subunit was noted. All three antisera demonstrated immunoassayable LH material in the void volume. This large species contained a higher proportion of material reacting in the anti-hLHalpha system than did native hLH. Examination of the void volume material by electron microscopy indicated membrane material, but no ribosomes. Chemical assay failed to detect RNA. When void volume material was subjected to conditions which are known to (a) disrupt membrane continuity and (b) dissociate the noncovalently bonded subunits of native LH, this large species of LH failed to dissociate into smaller molecular species. These studies reveal: (1) an excess of free alpha subunit in the pituitary, serum and urine; (2) a large species of LH in the pituitary which upon analysis does not appear to be simple aggregation of LH, MEMBRANE ENTRAPPED NATIVE LH, OR RIBOSOMAL BOUND LH.
3914	Inhibition of cyclic gonadotropin secretion by endogenous human prolactin.	1115151	The resumption of cyclic uterine bleeding reportedly accompanies the use of human prolactin (HPRL)-suppressing agents in postpill galactorrhea-amenorrhea. In this laboratory, HPRL suppression with L-dopa was variable and short lived. Basal plasma HPRL levels were elevated before and after as much as five months of therapy. Galactorrhea persisted and mean gonadotropin concentrations were subnormal. An immediate and sustained attenuation of HPRL secretion ( less than 200 per cent) followed the use of 2-Br-alpha-ergocryptine (CB-154). Cyclic gonadotropin secretion resumed and was accompanied by ovulation and, in one instance, pregnancy. The cessation of galactorrhea was positively correlated with the rise in the daily concentration of 17 beta-estradiol. Cyclic postovulatory menstruation continued after the cessation of CB-154 treatment. HPRL levels remained normal. The daily patterns of human follicle-stimulating hormone (HFSH) and human tuteinizing hormone (HLH) secretion created by the suppression of HPRL displayed an inherent rhythmicity identical to that observed at the time of menarche. The inhibitory effects of HPRL appeared directed at cyclic rather than tonic gonadotropin secretion. At the same time, diminished ovarian estrogen production seemed to increase mammary gland sensitivity to HPRL, leading to lactation. One may postulate, therefore, that the ingestion of sex steroids is associated with an over-all suppression of endogenous cyclic and, to a lesser extent, tonic gonadotropin secretion secondary to which ovarian function is attenuated. Without physiologic concentration of circulating estrogen, HPRL induces mammary alveolar function with the production of a milklike secretion.Agents used to induce cyclic menstruation following nonpuerperal galactorrhea and amenorrhea, such as l-dopa, appear to decrease the plasma concentration of human prolactin (HPRL). This study presents preliminary data on the clinical activity of such drugs with regard to HPRL and gonadotropin secretion in postpill galactorrhea-amenorrhea. 5 women suffering from this postpill syndrome after using Depo-Provera for various periods of time were given l-dopa (2 gm daily); HPRL suppression by l-dopa was variable and short-lived. However, an immediate and sustained attenuation of HPRL secretion of greater than 200 percent was found after administration of 2-Br-alpha-ergocryptine (CB-154); women so treated resumed cyclic bleeding and ovulation; 1 pregnancy occurred. Cessation of galactorrhea was positively correlated with the rise in daily concentration of 17-beta-estradiol. Cyclic postovulatory menstruation continued after cessation of CB-154. HPRL levels remained normal. Daily patterns of human follicle stimulating hormone and luteinizing hormone created by suppression of HPRL showed an inherent rhythmicity identical to that observed at menarch. HPRL inhibitory effects were directed at cyclic rather than tonic gonadotropin secretion. Diminished ovarian estrogen production also seemed to increase mammary gland sensitivity to HPRL, resulting in lactation. It is postulated that the use of sex steroids is associated with an overall suppression of endogenous cyclic gonadotropin secretion secondary to which ovarian function is attenuated.
3915	The syndromes of isolated gonadotropin deficiency.	1156688	Six theoretically possible syndromes of IGD are shown in Table 1. 1) IBGD is well-substantiated both in males and in females, and appears to be either of pituitary or more frequently of nonpituitary origin. 2) An example of isolated FSH deficiency has been described. The defect appears to reside at the pituitary level and may be localized to the FSH beta subunit. Recently a male patient has been studied with isolated FSH deficiency and a concordant testicular picture viz germinal cell aplasia. However, the syndrome is complicated by an associated chromosomal abnormality (XO/XXY/XY) whose significance is unclear. 3) Several examples of isolated hLH deficiency have been described. Several questions remain about the exact nature of the defect in some of the published reports of this syndrome.
3916	LH--FSH/RH: an equally potent stimulus for the release of HLH and HFSH.	1100453	NaN
3917	Human chorionic gonadotropin as a tumor marker.	4376658	Ectopic production and secretion of hormones by a wide variety of tumors has been known for decades. Initially, ectopic hormone secretion was recognized by signs and symptoms of excess circulating biologically active hormone. With development of more sophisticated assay techniques, biologically inactive fragments and "big" forms of authentic hormones have been identified in these syndromes. Recently, a specific hCG assay has been developed for selectively measuring hCG in plasma samples containing both hLH and hCG. Studies reported to date suggest that hCG may be a good tumor marker. Using that assay system, several hundred sera obtained from patients with a wide variety of tumors were screened. Patients with tumors of the stomach, liver, ovary and testis were found to have the highest incidence of ectopic hCG secretion.
3918	Evaluation of radioimmunoassay kits for the beta subunit of hCG, FSH, and hLH.	4856282	NaN
3919	[Cross reaction components in anti-HFSH serum and HLH antibody].	4797007	NaN
3920	Gonadotropin receptor of a mouse luteoma: interactions with luteinizing hormone (LH) and its and subunits.	4351508	A radioligand-receptor system for luteinizing hormone (LH), USING transplantable mouse luteoma, was used to investigate the interactions of LH, other peptide hormones, and LH subunits. Since tumor size decreased as did production of androgenic hormones following hypophysectomy, the luteoma is believed to have been dependent on pituitary tropic hormones; posthypophysectomy histologic changes supported this conclusion. An homogenate was prepared from 1-4 gm luteomas, which had been borne by mice for 4-10 months. Ovine LH, bovine LH, and human chorionic gonadotrophin reduced the binding of iodine-125 human luteinizing hormone (125-I-hLH). Growth hormone, adrenocorticotrophic hormone, and prolactin had no capacity to interfere with binding of 125-I-hLH. Though follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) reduced the binding somewhat, the reductions were consistent with the known presence of contaminating amounts of LH in the FSH and TSH. The accumulated results of a number of experiments suggest that binding to the luteoma LH receptor requires a particular polypeptide structural conformation, one found in the native hormone but found in neither alpha nor beta subunit alone.
3921	Interaction of 1,8-ANS with human luteinizing hormones: a probe for subunit interactions of hCG and hLH.	4735854	NaN
3922	The primary structure of the hormone-specific, beta subunit of human pituitary luteinizing hormone (hLH).	4685398	NaN
3923	[Radioimmunological determination of human luteinizing hormone (HLH), using a double antibody method].	4720324	NaN
3924	Autoradiographic and spectrometric studies on the localization of 125 I labelled HCG, HLH and FSH in immature rats.	4677768	NaN
3925	[Radioimmunological determination of human luteinizing hormone (HLH) using labelled antibodies].	5040621	NaN
3926	The uptake of human luteinizing hormone (hLH) by slices of luteinized rat ovaries.	5120648	NaN
3927	[Study of human luteinizing hormone (HLH) during the nycthemeron].	4988154	NaN
3928	[Effect of clomiphene on the plasmatic concentration of pituitary luteinizing hormone (HLH) determined with the radioimmunological method].	5535620	NaN
3929	A study of the cross-reaction between human chorionic and pituitary luteinizing hormones (HCG and HLH).	5528405	NaN
3930	Production rates and metabolic clearance rates of human follicle-stimulating hormone in premenopausal and postmenopausal women.	5764014	The production rates (PR) and the metabolic clearance rates (MCR) of human follicle-stimulating hormone (HFSH) were determined in six pre- and five postmenopausal women. Human FSH (PER-780) labeled with (131)I to specific activities of 50-150 muc/mug was used as a tracer. Both double antibody and trichloroacetic acid (TCA) precipitation techniques were used to determine HFSH-(131)I levels in infusate and plasma. In four of the subjects MCRs measured by both constant infusion and single injection techniques were the same. By constant infusion, plasma HFSH-(131)I levels reached equilibrium between 4-5 hr.MCRs in six premenopausal women were 14.2+/-1.1 (mean +/-SE) ml/min. MCRs in five postmenopausal women were 12.6 +/-1.1 ml/min. Simultaneous HFSH and human luteinizing hormone (HLH) MCRs were determined in a single patient using HFSH-(125)I and HLH-(131)I as tracers by both constant infusion and single injection methods. These studies showed that the MCR of HFSH was 10.8-11.1 ml/min, and the MCR of HLH was 18.5-19.4 ml/min. From these data and previous MCR and PR studies of HLH from this laboratory, it appears that the MCR of HFSH is about one-half that of HLH. Endogenous HFSH and HLH levels were measured by radioimmunoassay. The PRs of HFSH, calculated by the product of endogenous level and MCR, were 146 +/-27mU/min in the premenopausal women and 2141 +/-264 mM/min in the postmenopausal women. 24-hr PRs, based on these results, compared with reports of 24-hr urinary excretions of biologically active HFSH indicate that 3-5% of production is found in urine in biologically active form. After our single injections of HFSH-(131)I, 8-29% was recovered in urine over 24 hr.
3931	Metabolic clearance and production rates of human luteinizing hormone in pre- and postmenopausal women.	16695945	Metabolic clearance rates and production rates of human luteinizing hormone (HLH) were determined in pre- and postmenopausal women by the constant infusion technique. Highly purified HLH-(131)I was infused into the fasting subjects at a constant rate. Serial plasma samples were obtained and the radioactive hormone was precipitated by a double antibody technique. Plasma HLH-(131)I levels reached equilibrium by 4 hr after the infusion started. Metabolic clearance rates were: 24.4 +/- 1.8 (mean +/- SE) ml/min in five normal premenopausal women; 23.3 +/- 1.1 ml/min in five normal women taking norethinodrel and mestranol; and 25.6 +/- 4.1 ml/min in four postmenopausal women. Endogenous plasma HLH levels measured in the same subjects by radioimmunoassay immediately before infusion were 32.0 +/- 9.6 mU/ml in the normal women, 16.8 +/- 3.2 mU/ml in the women on oral contraceptives, and 99.2 +/- 23.2 mU/ml in the postmenopausal women. The corresponding HLH production rates were: 734 +/- 170 mU/min in the normal women: 387 +/- 86 mU/min in the women on norethinodrel and mestranol; and 2400 +/- 410 mU/min in the postmenopausal women. The metabolic clearance rate did not change after ovariectomy in one women, but the production rate rose from 583 to 1420 mU/min. Based on previously reported bioassay values for pituitary content and urinary excretion of HLH, the estimated turnover of HLH in the pituitary is about once per day and less than 5% of the total HLH produced appears in the urine in a biologically active form.
3932	Harrie Leslie Hugo Schütze.	14784920	NaN